Reprogramming Nature's Machinery: A Chemoenzymatic Strategy for Lariat Lipopeptide Biosynthesis

Abstract

This article explores a groundbreaking chemoenzymatic strategy for synthesizing lariat lipopeptides, a complex class of antimicrobial agents. For researchers and drug development professionals, we detail how versatile non-ribosomal peptide cyclases—SurE, WolJ, and TycC thioesterase—can be repurposed through innovative substrate design to overcome traditional synthetic hurdles. The content covers the foundational principles of non-ribosomal peptide synthesis, the methodological breakthrough of stereochemical control and one-pot tandem reactions, optimization for yield and selectivity, and a comparative validation of the platform's efficiency and discovered bioactivities. This paradigm shift enables the modular construction of diverse macrocyclic libraries, directly fueling the discovery of new antibiotic candidates against rising drug-resistant pathogens.

Lariat Lipopeptides: Unveiling Complex Architectures and Biosynthetic Hurdles

Lariat lipopeptides represent a fascinating class of naturally occurring antimicrobial agents characterized by their unique topological architecture. These complex molecules feature a carboxy-terminal macrocyclic "head" group and a long acyl chain appended to an amino-terminal "tail," creating a distinctive lasso-like or lariat-shaped structure [1]. This particular configuration is not merely a structural curiosity but is intimately linked to their biological function, particularly their ability to interact with biological membranes and disrupt microbial cell surfaces [2]. Naturally occurring lariat lipopeptides, such as the clinically significant antibiotics daptomycin (used against severe Gram-positive infections including MRSA) and colistin (a last-line defense against multidrug-resistant Gram-negative pathogens), underscore the therapeutic importance of this molecular family [1].

The global rise of antibiotic resistance has intensified the search for novel antimicrobial agents with new modes of action, positioning lariat lipopeptides as promising therapeutic candidates. However, the efficient exploration of their rich chemical space has been persistently hampered by their molecular complexity [1]. The conventional chemical synthesis of these compounds faces substantial challenges, particularly in achieving regioselective macrocyclization, which typically requires orthogonal protecting group strategies and stoichiometric coupling reagents [1]. Furthermore, the dilute conditions needed to suppress intermolecular coupling consume substantial amounts of organic solvents, making production inefficient and environmentally burdensome. It is within this context that innovative biosynthetic approaches, particularly those leveraging non-ribosomal peptide synthetases (NRPS) and their cyclase domains, have emerged as transformative methodologies for accessing and diversifying these valuable natural products [1] [3].

Structural Topology and Biosynthetic Origins

Defining Structural Characteristics

The lariat lipopeptide family exhibits several defining structural features that differentiate them from other peptide natural products. At their core, they are amphiphilic molecules composed of a hydrophobic fatty acid chain and a hydrophilic peptide moiety [2]. This amphiphilic nature enables their interaction with biological membranes, which is fundamental to their mechanism of action. The characteristic lariat shape arises from a macrocyclic peptide core (the "head") and a linear lipid tail, creating a topology that resembles a lasso [3].

The structural diversity within this family is considerable. The fatty acid chain can vary in length, isomeric form, and saturation degree, and may be further modified through β-hydroxylation, β-amination, or guanylation [2]. The peptide moiety consists of variable sequences incorporating both proteinogenic and non-proteinogenic amino acids, including residues in the D-form, which confer resistance to proteolytic degradation [4]. These peptides can range dramatically in size, with fatty acid chains reported from C7 to C43 and peptide moieties containing anywhere from 2 to 25 amino acid residues [2].

Biosynthesis by Non-Ribosomal Peptide Synthetases

Lariat lipopeptides are primarily synthesized by massive multi-enzyme complexes known as non-ribosomal peptide synthetases (NRPSs) [5]. These enzymatic assembly lines operate through a thiotemplate mechanism that does not rely on ribosomal translation, allowing for the incorporation of diverse non-proteinogenic amino acids and the formation of complex structures [4].

The NRPS machinery is modular in organization, with each module typically responsible for incorporating a single amino acid building block into the growing peptide chain [5]. Each module contains several core domains with distinct catalytic functions:

• Adenylation (A) domains select and activate specific amino acid building blocks as acyl adenylates using ATP [5] [4].
• Peptidyl Carrier Protein (PCP) domains shuttle the activated amino acids and peptide intermediates between catalytic sites using a phosphopantetheine cofactor [5].
• Condensation (C) domains catalyze peptide bond formation between the upstream peptidyl-S-PCP and the downstream aminoacyl-S-PCP [5].
• Thioesterase (TE) domains, located in the final module, catalyze the release of the completed peptide from the NRPS machinery, often through cyclization or hydrolysis [5].

The macrocyclic "head" of lariat lipopeptides is typically constructed by specialized thioesterase domains that catalyze cyclization using side-chain nucleophiles [1]. For lariat-shaped structures, this involves a unique cyclization pattern where the nucleophile is not the N-terminus (as in head-to-tail cyclization) but rather an internal residue, creating the characteristic lariat topology [1].

Table 1: Core Catalytic Domains in Non-Ribosomal Peptide Synthetases

Domain	Function	Key Features
Adenylation (A) Domain	Selects and activates amino acid substrates	Recognizes specific amino acids; uses ATP to form acyl-adenylate intermediate [5] [4]
Peptidyl Carrier Protein (PCP) Domain	Shuttles substrates and intermediates	Contains phosphopantetheine cofactor; covalently binds amino acids and peptides via thioester linkage [5]
Condensation (C) Domain	Catalyzes peptide bond formation	Forms amide bonds between upstream peptidyl-S-PCP and downstream aminoacyl-S-PCP [5]
Thioesterase (TE) Domain	Releases and cyclizes final product	Typically located in terminal module; catalyzes macrocyclization or hydrolysis [1] [5]

Revolutionary Chemoenzymatic Synthesis Approach

Overcoming Traditional Synthetic Challenges

Traditional chemical synthesis of lariat lipopeptides faces significant obstacles, particularly in achieving regioselective macrocyclization. Conventional approaches require orthogonal protecting group strategies and stoichiometric amounts of coupling reagents, along with dilute conditions to suppress intermolecular side reactions [1]. These requirements make production inefficient, low-yielding, and environmentally taxing, severely hampering structural diversification and exploration of structure-activity relationships.

A groundbreaking study published in Nature Chemistry in 2025 has introduced a transformative chemoenzymatic approach that bypasses these limitations [1] [6]. This methodology repurposes nature's biosynthetic machinery—specifically, non-ribosomal peptide cyclases—by combining enzymatic precision with synthetic flexibility. The strategy involves preparing tailored peptide substrates chemically, then using highly selective enzymatic catalysts to achieve the macrocyclization under mild conditions [1].

Reprogramming Cyclase Specificity Through Substrate Engineering

The key innovation in this approach lies not in engineering the enzymes themselves, but in strategically redesigning their substrates to redirect cyclization specificity [1] [3]. The researchers focused on penicillin-binding protein-type thioesterases (PBP-type TEs), such as SurE and WolJ, which are naturally proficient head-to-tail macrocyclases [3]. These enzymes typically join a peptide's N- and C-termini, forming conventional cyclic peptides rather than lariat structures.

To redirect this activity toward lariat formation, the researchers engineered branched peptide substrates containing a "pseudo-N-terminus"—a dipeptide unit featuring an additional N-terminus within a peptide side chain [1] [3]. This design creates two potential nucleophilic sites for cyclization: the native N-terminus and the pseudo-N-terminus. When the native N-terminus had an L-configured amino acid, SurE produced both the conventional head-to-tail macrocycle and the lariat-shaped peptide in comparable amounts (60% and 40% respectively), demonstrating that the pseudo-N-terminus could serve effectively as a nucleophile [1].

Stereochemical Control for Exclusive Lariat Formation

To achieve exclusive lariat formation, the researchers implemented a stereochemical switching strategy [1] [3]. They exploited the fact that PBP-type TEs strictly recognize the stereochemical configuration of nucleophiles, accepting only L-configured residues while rejecting D-configured ones. By replacing the native N-terminal L-amino acid with its D-configured counterpart, they effectively blocked the head-to-tail cyclization pathway. This forced the enzyme to use exclusively the pseudo-N-terminus (with its L-configured residue) as the nucleophile, resulting in quantitative production of the lariat-shaped cyclic peptide with complete regiospecificity [1].

This strategy demonstrated remarkable generality, working effectively with multiple macrocyclases including SurE, WolJ (both PBP-type TEs), and TycC-TE (a type-I thioesterase) [1] [3]. The ability to repurpose different cyclases through substrate design alone, without protein engineering, significantly expands the synthetic toolbox available for lariat lipopeptide production.

Diagram 1: Stereochemical Control of Lariat Cyclization

Tandem Cyclization-Aylation Strategy

A particularly innovative aspect of this methodology is the development of a one-pot tandem cyclization-acylation process [1] [3]. After enzymatic macrocyclization, the remaining nucleophile (the one not used in cyclization) serves as a reactive handle for subsequent diversification via site-selective Ser/Thr ligation (STL). This enables the direct attachment of various acyl groups to create the characteristic lipophilic "tail" of lariat lipopeptides.

This sequential one-pot approach eliminates the need for intermediate purification and enables efficient parallel synthesis of diverse lariat lipopeptide libraries [3]. The ability to perform both cyclization and acylation under mild conditions in a single pot represents a significant streamlining of the synthetic workflow, facilitating rapid generation of structural diversity for biological evaluation.

Table 2: Key Enzymes in Chemoenzymatic Lariat Synthesis

Enzyme	Classification	Role in Synthesis	Key Feature
SurE	Penicillin-Binding Protein-type Thioesterase (PBP-type TE)	Macrocyclization catalyst	Broad substrate tolerance; strictly accepts only L-configured nucleophiles [1] [3]
WolJ	Penicillin-Binding Protein-type Thioesterase (PBP-type TE)	Macrocyclization catalyst	Functions similarly to SurE; demonstrates versatility in lariat formation [1]
TycC Thioesterase (TycC-TE)	Type-I Thioesterase	Macrocyclization catalyst	Originally a head-to-tail cyclase; repurposed for lariat synthesis [1]

Experimental Protocols and Methodologies

Substrate Design and Synthesis

The synthesis of branched peptide substrates begins with solid-phase peptide synthesis (SPPS) on a resin functionalized with ethylene glycol (EG), which serves as a simplified surrogate for the natural pantetheine leaving group [1]. This design simplifies substrate synthesis and streamlines the overall process. The peptide main chain is assembled using standard SPPS protocols, with the incorporation of an L-lysine residue at the intended branching point. This lysine side chain is protected with an orthogonal 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) protecting group.

After assembly of the linear sequence, the Dde protecting group is selectively removed using 2% hydrazine, exposing the ε-amino group of lysine. The pseudo-N-terminal dipeptide unit is then installed on this side chain through additional coupling cycles. Finally, resin cleavage and global deprotection yield the ethylene glycol-functionalized branched peptide substrate ready for enzymatic cyclization [1].

Enzymatic Macrocyclization Protocol

The enzymatic macrocyclization is performed under mild, aqueous conditions. The branched peptide substrate (typically at concentrations of 0.5-1.0 mM) is incubated with the selected cyclase enzyme (SurE, WolJ, or TycC-TE) at a catalytic loading of 5 mol% in an appropriate buffer system [1]. The reaction proceeds at 30°C with gentle agitation for several hours. Reaction progress is monitored by LC-MS until complete consumption of the starting material is observed.

For the stereochemically controlled exclusive lariat formation, the substrate features a D-configured amino acid at the native N-terminus and an L-configured amino acid at the pseudo-N-terminus. This configuration ensures that only the pseudo-N-terminus can serve as the nucleophile for cyclization, resulting in quantitative conversion to the lariat product without formation of head-to-tail byproducts [1].

One-Pot Tandem Cyclization-Acylation

The tandem process begins with the enzymatic macrocyclization as described above. Upon completion of cyclization (typically within 3 hours), the reaction mixture is directly subjected to Ser/Thr ligation without intermediate purification [1]. The site-selective acylation targets the free N-terminus not used in the cyclization process, allowing attachment of various lipid tails under mild conditions compatible with the aqueous enzymatic reaction.

This streamlined approach enables the rapid generation of diverse lariat lipopeptide libraries. The resulting compounds can be directly screened for biological activity without extensive purification, significantly accelerating the discovery process [1] [3].

Diagram 2: Chemoenzymatic Synthesis Workflow

Therapeutic Significance and Biological Activity

Antimicrobial Properties and Mechanisms

Lariat lipopeptides exhibit potent antimicrobial activity through diverse mechanisms, primarily targeting bacterial cell surfaces [3]. Their amphiphilic structure enables interaction with biological membranes, leading to disruption of membrane integrity or interference with essential membrane-associated processes [2]. Different lipopeptides may target specific components of microbial membranes, with some acting on cell wall biosynthesis through complex formation with bactoprenol phosphate, while others directly disrupt membrane integrity [7].

The unique lariat topology appears to optimize these interactions, positioning the macrocyclic head for specific molecular recognition while the lipid tail embeds within membrane environments. This structural arrangement often results in selective toxicity toward microbial cells over mammalian cells, though this varies considerably with specific structural features [2].

Antimycobacterial Activity of Synthetic Variants

Biological screening of the lariat lipopeptide library generated through the chemoenzymatic approach revealed significant antimycobacterial activity [1] [7]. Several compounds demonstrated potent inhibition of Mycobacterium intracellulare growth, with half-maximal inhibitory concentrations (IC50) ranging from 8-16 µg/ml [1]. This activity is particularly valuable given the rising concerns about drug-resistant mycobacterial infections, including tuberculosis and non-tuberculous mycobacterial diseases.

The screening identified eight particularly promising compounds that effectively inhibited M. intracellulare growth at these low concentrations [1]. Importantly, the site-selective acylation strategy was found to be crucial for conferring this antimycobacterial activity, as the specific nature and positioning of the lipid tail dramatically influenced both potency and selectivity [1].

Antiviral Activity and Structure-Activity Relationships

Beyond antibacterial applications, lipopeptides also demonstrate significant antiviral potential, particularly against enveloped viruses such as SARS-CoV-2 [2]. Studies have shown that certain lipopeptides can reduce SARS-CoV-2 RNA to undetectable levels at concentrations of 100 µg/ml, with surfactin, WLIP, fengycin, and caspofungin emerging as particularly promising anti-SARS-CoV-2 agents [2].

These lipopeptides appear to target multiple stages of the viral life cycle that involve the viral envelope. Surfactin and WLIP significantly reduce viral RNA levels in replication assays, comparable to neutralizing serum, while surfactin uniquely inhibits viral budding, and fengycin impacts viral binding after pre-infection treatment of cells [2]. Structure-activity relationship studies have identified that lipopeptides with a high number of amino acids, particularly those with charged (preferentially anionic) amino acids, tend to exhibit the most potent anti-SARS-CoV-2 activity with lower cytotoxicity [2].

Table 3: Biological Activities of Representative Lipopeptides

Lipopeptide	Primary Activity	Reported Efficacy	Proposed Mechanism
Daptomycin	Antibacterial (Gram-positive)	Clinical use against MRSA	Disrupts bacterial membrane potential; inhibits cell wall synthesis [1]
Colistin	Antibacterial (Gram-negative)	Last-line defense drug	Binds to LPS, disrupts outer membrane [1]
Surfactin	Antiviral (SARS-CoV-2)	Reduces viral RNA to undetectable levels at 100 µg/mL [2]	Multiple mechanisms: reduces viral RNA, inhibits budding [2]
Chemoenzymatic Variants	Antimycobacterial	IC50 8-16 µg/mL against M. intracellulare [1]	Membrane disruption; precise mechanism under investigation [1]

The Scientist's Toolkit: Essential Research Reagents

The chemoenzymatic synthesis of lariat lipopeptides requires specialized reagents and materials that enable this sophisticated approach. The following table details key research reagent solutions essential for implementing these methodologies.

Table 4: Essential Research Reagents for Lariat Lipopeptide Research

Reagent/Material	Function/Application	Specific Example/Note
PBP-type Thioesterases (SurE, WolJ)	Macrocyclization catalysts	Recombinantly expressed; 5 mol% catalytic loading; strict L-nucleophile specificity [1] [3]
Type-I Thioesterase (TycC-TE)	Alternative macrocyclization catalyst	Broad substrate tolerance; repurposable for lariat synthesis [1]
Ethylene Glycol (EG)-Functionalized Resin	Solid support for SPPS	Simplifies substrate synthesis; surrogate for pantetheine leaving group [1]
Dde-Protected Lysine	Orthogonal protection for branching point	Allows selective deprotection with 2% hydrazine for pseudo-N-term installation [1]
Ser/Thr Ligation (STL) Reagents	Site-selective acylation	Enables lipid tail attachment to free N-terminus in one-pot procedure [1]
H-DL-Glu(Ome)-OMe.HCl	H-DL-Glu(Ome)-OMe.HCl, CAS:23150-65-4, MF:C7H14ClNO4, MW:211.64 g/mol	Chemical Reagent
D,L-Tryptophanamide hydrochloride	D,L-Tryptophanamide hydrochloride, CAS:67607-61-8, MF:C11H14ClN3O, MW:239.7 g/mol	Chemical Reagent

The structural topology of lariat lipopeptides—characterized by their macrocyclic head and linear lipid tail—confers unique biological properties that make them valuable therapeutic candidates, particularly in an era of escalating antimicrobial resistance. The development of innovative chemoenzymatic synthesis methodologies represents a paradigm shift in our ability to access and diversify these complex natural products.

By repurposing versatile non-ribosomal peptide cyclases through strategic substrate design rather than protein engineering, researchers have overcome long-standing synthetic challenges. The implementation of stereochemical control to switch cyclization specificity, coupled with tandem cyclization-acylation in one pot, enables efficient generation of diverse lariat lipopeptide libraries for biological evaluation. These advances have already yielded promising compounds with potent antimycobacterial activity and revealed structure-activity relationships that can guide future design.

Looking forward, this chemoenzymatic platform offers exciting opportunities for further innovation. The directed evolution of peptide cyclases could expand substrate scope and enhance catalytic efficiency, while incorporation of non-natural amino acids or chemically modified lipids could dramatically enrich structural diversity. As the threat of antimicrobial resistance intensifies, such advanced synthetic strategies will become increasingly critical for developing the next generation of therapeutic agents. The integration of nature's catalytic precision with synthetic ingenuity, as demonstrated in the synthesis of lariat lipopeptides, provides a powerful blueprint for future natural product-based drug discovery.

The Role of Non-Ribosomal Peptide Synthetases (NRPS) in Natural Biosynthesis

Non-ribosomal peptide synthetases (NRPSs) represent a class of massive modular enzymes that catalyze the synthesis of structurally and functionally diverse peptide secondary metabolites independently of the ribosomal machinery [8]. These enzymatic assembly lines are primarily found in bacteria and fungi and are responsible for producing numerous pharmacologically active compounds, including antibiotics (e.g., penicillin, vancomycin), immunosuppressants (e.g., cyclosporine), and anticancer agents [5] [9]. Unlike ribosomally synthesized peptides, non-ribosomal peptides (NRPs) often feature complex structures including cyclic, branched, or linear architectures; incorporate non-proteinogenic amino acids (e.g., D-amino acids, N-methylated amino acids); and contain various modifications such as glycosylation, acylation, and halogenation [10] [8]. This chemical diversity, inaccessible to ribosomal synthesis, underpins the broad biological activities of NRPs and their importance in drug discovery [9].

The biosynthesis of lariat lipopeptides represents a particularly sophisticated application of NRPS machinery. These compounds, exemplified by important antimicrobial agents like daptomycin and colistin, feature a carboxy-terminal macrocyclic "head" group and a long acyl chain appended to an amino-terminal "tail" [1]. Their complex lariat-shaped topologies have posed significant challenges for chemical synthesis, hampering efficient structural diversification for drug development [1] [3]. Recent advances in understanding NRPS cyclization mechanisms, particularly through the repurposing of non-ribosomal peptide cyclases, have opened new avenues for the chemoenzymatic synthesis of these valuable compounds, demonstrating the continued relevance of NRPS systems in addressing modern antibiotic resistance challenges [1] [3].

NRPS Modular Architecture and Domain Organization

Core Domains and Their Functions

NRPSs exhibit a modular organization where each module is responsible for incorporating one monomeric building block into the growing peptide chain. Each module contains three core catalytic domains that work in concert to activate, carry, and connect amino acid substrates [5] [9].

Table 1: Core Domains in Non-Ribosomal Peptide Synthetases

Domain	Abbreviation	Primary Function	Key Features
Adenylation Domain	A	Selects and activates amino acid substrates using ATP	Forms aminoacyl-AMP intermediate; determines substrate specificity [5]
Peptidyl Carrier Protein	PCP	Carries activated amino acids and peptide intermediates	Contains 4'-phosphopantetheine prosthetic group; swivels between domains [5]
Condensation Domain	C	Catalyzes peptide bond formation between donor and acceptor substrates	Contains HHxxxDG catalytic motif; exhibits substrate gating function [11] [9]
Thioesterase Domain	TE	Releases full-length peptide from NRPS assembly line	Catalyzes hydrolysis or cyclization; often forms macrocyclic structures [12] [5]

The adenylation (A) domain belongs to the ANL superfamily of adenylating enzymes and performs the critical first step of substrate recognition and activation [5]. Using ATP, it catalyzes the formation of an aminoacyl-adenylate mixed anhydride, which then gets loaded onto the thiol group of the 4'-phosphopantetheine (PPant) arm attached to the peptidyl carrier protein (PCP) domain [5] [9]. The PCP domain serves as a flexible swivel arm, shuttling the covalently tethered substrates between different catalytic sites [5]. The condensation (C) domain catalyzes the central chemical step of peptide bond formation, facilitating nucleophilic attack by the α-amino group of the downstream ("acceptor") aminoacyl-PCP on the upstream ("donor") peptidyl-PCP thioester [11] [9]. In the termination module, the thioesterase (TE) domain mediates product release, often through hydrolysis or intramolecular cyclization, the latter being particularly relevant for generating macrocyclic scaffolds found in lariat lipopeptides [1] [12].

Modular Organization and Assembly Line Logic

NRPSs are organized as sequential modules, with the number and order of modules typically correlating with the sequence of the final peptide product [9]. A minimal elongation module contains C-A-PCP domains, while the initiation module often lacks the C domain, and the termination module contains additional domains like TE for product release [5] [8]. This assembly line logic enables the coordinated elongation of the peptide chain, with each module incorporating one specific monomer before passing the growing chain to the next module [9].

Table 2: Optional Modification Domains in NRPS Systems

Domain	Abbreviation	Function	Structural Outcome
Epimerization Domain	E	Converts L-amino acids to D-amino acids	Incorporates D-configured residues [8]
N-Methyltransferase Domain	NMT	Adds methyl groups to amino groups	Creates N-methylated peptides [8]
Cyclization Domain	Cy	Catalyzes heterocyclization of Cys/Ser/Thr	Forms thiazoline/oxazoline rings [8]
Oxidation Domain	Ox	Oxidizes heterocycles to aromatic forms	Converts thiazolines to thiazoles [8]
Reduction Domain	R	Reduces terminal thioester to aldehyde/alcohol	Creates peptide aldehydes/alcohols [8]

Figure 1: NRPS Modular Organization and Assembly Line Logic

Structural and Mechanistic Insights into NRPS Domains

Condensation Domain: The Peptide Bond Formation Catalyst

The condensation (C) domain serves as the central catalyst for amide bond formation in NRPS systems. Structural studies have revealed that C domains typically consist of approximately 450 amino acids and adopt a pseudo-dimeric chloramphenicol acetyltransferase (CAT) fold, composed of two similar subdomains at the N- and C-termini [11] [9]. Between the interfaces of these subdomains, a cleft forms that accommodates the donor and acceptor Ppant arms, which must access the conserved active site motif HHxxxDG located in the N-terminal subdomain [9].

Structural characterization of C domain complexes has provided crucial insights into their mechanism of action. The structure of the PCP2-C3 didomain from the fuscachelin NRPS of Thermobifida fusca revealed that the interface between the PCP and C domains is dominated by hydrophobic interactions, with key residues from both domains mediating this interaction [11]. Access to the C domain active site appears to be gated by an arginine residue (R2906) that helps position the PCP domain and likely prevents unloaded PCP substrates from accessing the active site [11].

The precise catalytic mechanism of C domains remains partially unresolved. While the second histidine in the HHxxxDG motif was initially proposed to act as a catalytic base that deprotonates the α-amino group of the acceptor substrate, mutation studies have shown that this residue is not always essential for activity [9]. Alternative hypotheses suggest this histidine may instead function in positioning the acceptor substrate or stabilizing the transition state [9]. Beyond catalysis, C domains play a crucial role as secondary gatekeepers for substrate specificity, providing a proofreading function that significantly reduces error rates in monomer incorporation [9].

Thioesterase Domains and Cyclization Mechanisms

Thioesterase (TE) domains perform the critical terminal step in NRPS synthesis by releasing the full-length peptide from the assembly line. These domains typically adopt an α/β-hydrolase fold and contain a distinctive bowl-shaped hydrophobic cavity that hosts the acylpeptide substrate and accommodates its folding into cyclic structures [12]. Two major classes of TEs have been identified in NRPS systems: type I TEs (α/β-hydrolase type) and the more recently discovered penicillin-binding protein (PBP)-type TEs [1] [3].

In lariat lipopeptide biosynthesis, TE domains catalyze intramolecular cyclization to form macrolactam rings. Traditional lariat-forming type I TEs typically utilize side chain nucleophiles for cyclization but are often sensitive to changes in the local environment of the nucleophilic residue, limiting their utility for generating diverse lariat peptides [1]. In contrast, PBP-type TEs such as SurE and WolJ demonstrate broader substrate tolerance and have been repurposed for the chemoenzymatic synthesis of lariat peptides through strategic substrate redesign [1] [3].

Structural studies of TE domains, such as the excised 28 kDa SrfTE domain from surfactin synthetase, have revealed how the hydrophobic cavity hosts the acylpeptide substrate and facilitates its folding into cyclic structures [12]. Docking studies with the peptidyl carrier protein domain immediately preceding SrfTE have shown how the 4'-phosphopantetheinyl prosthetic group positions the nascent acyl-peptide chain for transfer to the TE domain [12].

Experimental Approaches in NRPS Research

Chemoenzymatic Synthesis of Lariat Lipopeptides

Recent groundbreaking work has demonstrated the repurposing of head-to-tail NRP cyclases for lariat peptide synthesis through strategic substrate engineering rather than protein engineering [1] [3]. The key innovation involves introducing an internal dipeptide unit as a "pseudo-N terminus," where an L-Ile residue is attached to the side chain of L-Lys via an isopeptide bond, creating a branched substrate with two potential nucleophiles for cyclization [1]. When incubated with the PBP-type TE SurE, this branched substrate is converted into both canonical head-to-tail cyclic peptides and lariat-shaped cyclic peptides in comparable amounts [1].

To achieve exclusive lariat formation, researchers exploited the stereospecificity of PBP-type TEs, which accept only L-configured residues as nucleophiles [1] [3]. By replacing the native N-terminal L-Ile residue with D-Val, head-to-tail cyclization was suppressed, forcing the enzyme to use the pseudo-N-terminal L-Ile as the nucleophile and quantitatively producing lariat-shaped cyclic peptides [1]. This strategy demonstrated remarkable regioselectivity, as substrates containing three different nucleophiles (N-terminal D-Val, pseudo-N-terminal L-Ile, and ε-NH₂ of L-Lys) cyclized exclusively via the pseudo-N-terminal L-Ile [1].

Table 3: Experimental Results for SurE-Catalyzed Lariat Peptide Synthesis

Substrate	N-terminal Configuration	Products Generated	Yield	Key Finding
Branched substrate 1	L-Ile (native)	Head-to-tail cyclic (2) + Lariat cyclic (3)	60% + 40%	Pseudo-N-terminus as effective nucleophile [1]
Branched substrate 4	D-Val (modified)	Lariat cyclic (5) only	Quantitative	Complete regiospecificity for lariat formation [1]
Substrates 6-11	D-Val with varied pseudo-N terminus positions	Lariat peptides with different ring sizes	Varied	Tolerance for different pseudo-N terminus positions [1]

The tandem cyclization-acylation strategy enables one-pot, modular synthesis of lariat-shaped lipopeptides equipped with various acyl groups [1]. This approach facilitated the creation of a 51-member library of lariat lipopeptides that could be directly screened for antimicrobial activity, leading to the identification of compounds that inhibit Mycobacterium intracellulare growth by 50% at concentrations of 8-16 μg ml⁻¹ [1] [3].

Figure 2: Chemoenzymatic Synthesis Workflow for Lariat Lipopeptides

Structural Biology Techniques for NRPS Characterization

Structural characterization of NRPS domains and complexes has been instrumental in understanding their mechanisms. X-ray crystallography has provided high-resolution structures of individual domains, including adenylation domains, condensation domains, peptidyl carrier proteins, and thioesterase domains [11] [12] [5]. The crystallization of didomain constructs, such as PCP-C and A-PCP complexes, has offered insights into interdomain interactions and conformational dynamics [11] [5].

Molecular dynamics (MD) simulations have complemented experimental structural data by providing dynamic information about substrate-enzyme interactions. In studies of SurE-catalyzed lariat peptide formation, MD simulations of covalent docking models constructed using the crystal structure of apo SurE revealed that L-Ile nucleophiles are retained in the nucleophile binding site with an average distance of 4.5 Ã… from the C-terminal carbonyl carbon, while D-configured nucleophiles exhibit positional variability with much longer distances (>10 Ã…), explaining the observed stereospecificity [1].

Mechanism-based inhibitors have been particularly valuable for trapping transient interactions between catalytic and carrier protein domains [5]. For example, the use of stable analogs of acyl acceptor substrates complexed to C domains has enabled structural characterization of these typically dynamic complexes [11]. Similarly, the formation of crosslinked domains using mechanism-based inhibitors has facilitated crystallization of meta-stable intermediates along the catalytic pathway [5].

Research Reagent Solutions for NRPS Studies

Table 4: Essential Research Reagents for NRPS Experiments

Reagent/Category	Function/Application	Specific Examples	Experimental Notes
EG-functionalized Resins	Solid-phase synthesis of peptide substrates with C-terminal ethylene glycol leaving group	Wang resin derivatives with ethylene glycol linkers	Simplifies enzymatic substrate synthesis; streamlines process [1]
PBP-type Thioesterases	Enzymatic macrocyclization of linear peptide substrates	SurE, WolJ	Broad substrate tolerance; 5 mol% catalyst loading; quantitative yields [1] [3]
Type I Thioesterases	Comparative cyclization studies; lariat formation	TycC thioesterase	Demonstrated adaptability to lariat synthesis via substrate engineering [1]
Stable Acyl-CoA Analogs	Investigation of C domain specificity and PCP interactions	Aminoacyl-coenzyme A analogs	Bypasses A domain selectivity; allows direct C domain specificity studies [9]
Mechanism-based Inhibitors	Trapping domain interactions for structural studies	Crosslinking analogs of aminoacyl-AMP	Enables crystallization of transient complexes [5]
MbtH-like Proteins	Activation of A domains in certain NRPS systems	Various MbtH homologs	Required for activity of some A domains; essential cofactor [8]
Phosphopantetheinyl Transferases	Conversion of apo- to holo-PCP domains	Sfp from B. subtilis	Broad substrate specificity; essential for in vitro reconstitution [9]

Applications and Future Directions in NRPS Engineering

Genome Mining for Novel NRPS Discovery

Bioinformatics approaches have revolutionized the discovery of novel NRPS systems through genome mining. Analysis of 123 complete genomes of Bacillus strains isolated from soil and fermented foods using antiSMASH version 7 revealed that 83% possess biosynthetic gene clusters for siderophore bacillibactin, 61% for surfactins, 37% for fengycins, 23% for iturins, 15% for kurstakins, and 3% for bacitracin [13]. Importantly, this study identified seven novel biosynthetic gene clusters coding NRPSs in various Bacillus strains, demonstrating the significant potential of genome mining strategies for discovering new metabolites [13].

Computational tools have been developed to predict substrate specificity from DNA or protein sequence data, with methods such as SANDPUMA and NRPSpredictor2 enabling researchers to identify the likely products of NRPS clusters directly from genomic information [8]. These approaches have accelerated the discovery of novel nonribosomal peptides and facilitated the prioritization of clusters for experimental characterization.

Engineering Strategies for Novel Peptide Production

NRPS engineering holds tremendous promise for generating novel peptides with tailored properties. The modular architecture of NRPS systems theoretically allows for the creation of hybrid assembly lines through domain or module swapping [10] [9]. Successful examples include the engineering of surfactin and daptomycin synthetases to produce analogs with modified amino acid composition [10].

Rational protein design has yielded methodologies to computationally switch the specificities of A-domains, with ten amino acids identified that control substrate specificity and can be considered the 'codons' of nonribosomal peptide synthesis [8]. Engineering of C domains has proven more challenging due to their complex structure and dual donor-acceptor selectivity, but progress in understanding C domain structure and mechanism is gradually enabling more targeted engineering approaches [9].

The chemoenzymatic approach to lariat lipopeptide synthesis represents a particularly promising direction for NRPS engineering. By combining enzymatic cyclization with chemical diversification, this strategy leverages the efficiency and selectivity of enzymatic transformations while enabling modular access to diverse compound libraries [1] [3]. The ability to perform cyclization and acylation reactions sequentially in a single pot without intermediate purification significantly streamlines the synthesis workflow and enables rapid generation of libraries for biological screening [1].

As structural and mechanistic understanding of NRPS systems continues to advance, and as tools for manipulating these complex enzymes improve, the potential for engineering NRPSs to produce novel therapeutic compounds continues to expand. The integration of computational design, structural biology, and synthetic biology approaches promises to unlock the full potential of these remarkable molecular assembly lines for drug discovery and development.

The Emergence of Non-Ribosomal Peptide Cyclases as Biocatalytic Tools

Non-ribosomal peptide synthetases (NRPSs) represent nature's sophisticated molecular assembly lines for producing structurally complex peptides with valuable biological activities, including antibiotics, immunosuppressants, and anticancer agents [9] [14]. These massive multi-modular enzymes activate, incorporate, and join amino acid building blocks in an assembly-line fashion, with the final product release often catalyzed by specialized thioesterase (TE) domains that can hydrolyze or cyclize the fully assembled peptide chain [5] [12]. For decades, scientists have recognized the potential of harnessing these enzymatic machineries for synthetic biology and drug development, particularly for generating complex cyclic peptide architectures that are challenging to produce using conventional chemical methods [9].

The global health crisis of antimicrobial resistance has intensified the search for new antibiotics with novel mechanisms of action, bringing lariat lipopeptides into sharp focus [1] [3]. These naturally occurring compounds, exemplified by clinically important drugs like daptomycin and colistin, feature a unique topology consisting of a carboxy-terminal macrocyclic "head" group and a long acyl chain appended to an amino-terminal "tail" [1]. This lariat configuration enables diverse modes of action, primarily targeting bacterial cell surfaces, which reduces the likelihood of cross-resistance with conventional antibiotics [1]. However, the efficient exploration of this rich chemical space has been hampered by significant synthetic challenges. Traditional chemical synthesis of lariat-shaped lipopeptides requires orthogonal protecting group strategies, stoichiometric coupling reagents, and dilute conditions to suppress intermolecular side reactions, resulting in substantial organic solvent waste and limited scalability [1].

This technical guide examines the emerging paradigm shift in which non-ribosomal peptide cyclases are being repurposed as versatile biocatalytic tools to overcome these synthetic limitations, with particular emphasis on recent breakthroughs in chemoenzymatic synthesis of lariat lipopeptides.

Fundamental NRPS Architecture and Cyclization Mechanisms

Core Domains and Their Functions

Non-ribosomal peptide synthetases employ a modular architecture where each module is responsible for incorporating a single amino acid residue into the growing peptide chain [9]. A minimal elongation module contains three core domains:

• Adenylation (A) domain: Selects and activates specific amino acid substrates through ATP-dependent adenylation [5] [9]
• Peptidyl Carrier Protein (PCP) domain: Shuttles the activated amino acids and peptide intermediates between catalytic domains using a 4'-phosphopantetheine (Ppant) prosthetic group [5]
• Condensation (C) domain: Catalyzes peptide bond formation between the upstream donor and downstream acceptor substrates [9]

The thioesterase (TE) domain, located at the C-terminus of the terminal module, is particularly relevant to cyclization processes. This domain catalyzes the release of the fully assembled peptide from the NRPS machinery through hydrolysis or, more commonly, intramolecular cyclization [5] [12]. TE domains belong to the α/β-hydrolase fold family and typically feature a distinctive bowl-shaped hydrophobic cavity that hosts the acylpeptide substrate and accommodates its folding into a cyclic structure [12].

Structural Basis of Cyclization

Structural studies of NRPS components have revealed critical insights into the cyclization mechanism. The excised 28 kDa SrfA-C thioesterase domain, for instance, exhibits a hydrophobic cavity that positions the peptide substrate for macrocyclization [12]. Docking studies with the peptidyl carrier protein domain immediately preceding the TE domain show how the 4'-phosphopantetheinyl prosthetic group transfers the nascent acyl-peptide chain to the TE active site [12].

The catalytic triad of TE domains (typically serine-histidine-aspartate) first catalyzes the transfer of the peptide from the PCP-bound thioester to an active site serine, forming an acyl-enzyme intermediate. The peptide then folds into a conformation that allows a nucleophilic attack from an internal residue (amine or hydroxyl group), resulting in macrocyclization and product release [12].

Figure 1: Catalytic Cycle of NRPS Thioesterase Domains in Peptide Cyclization

Breaking the Specificity Barrier: Reprogramming Cyclases for Lariat Peptide Synthesis

The Substrate Engineering Breakthrough

Historically, application of lariat-forming TE domains in biocatalysis has been limited by their narrow substrate specificity and sensitivity to changes in the local environment of the nucleophilic residue [1]. However, a groundbreaking approach reported in 2025 has circumvented these limitations through creative substrate design rather than extensive enzyme engineering [1] [3].

The key innovation involves introducing a "pseudo-N-terminus" – a dipeptide unit featuring an additional N-terminus within the peptide side chain – creating two possible nucleophilic sites for cyclization [1] [3]. When the branched substrate was incubated with SurE, a penicillin-binding protein-type thioesterase, the enzyme generated both canonical head-to-tail cyclic peptides and lariat-shaped cyclic peptides in comparable amounts (60% and 40% respectively), demonstrating that the pseudo-N-terminus could serve as an effective nucleophile for cyclization [1].

Stereochemical Control for Regioselective Cyclization

To achieve exclusive lariat peptide formation, researchers implemented a stereochemical switching strategy. Building on the knowledge that PBP-type TEs strictly recognize the stereochemical configuration of nucleophiles (accepting only l-configured residues, not d-configured ones), the native N-terminal residue was replaced with its mirror-image d-amino acid [1] [3]. This simple but elegant modification effectively suppressed head-to-tail cyclization while forcing the enzyme to use the pseudo-N-terminus as the nucleophile, yielding lariat peptides with complete selectivity [1].

Molecular dynamics simulations provided structural insights into this stereochemical control mechanism. When an l-configured nucleophile was positioned in the nucleophile binding site, it remained stably positioned with the nucleophilic amine approximately 4.5 Ã… from the C-terminal carbonyl carbon [1]. In contrast, d-configured nucleophiles exhibited positional variability with much longer distances to the electrophilic carbon (>10 Ã…), explaining their inability to participate efficiently in cyclization [1].

Table 1: Key Non-Ribosomal Peptide Cyclases Used in Lariat Lipopeptide Synthesis

Enzyme	Class	Natural Specificity	Engineered Function	Key Features
SurE	PBP-type TE	Head-to-tail cyclization	Lariat cyclization	Broad substrate tolerance, strict l-nucleophile specificity
WolJ	PBP-type TE	Head-to-tail cyclization	Lariat cyclization	Similar to SurE, compatible with diverse sequences
TycC-TE	Type I TE	Head-to-tail cyclization	Lariat cyclization	α/β-hydrolase fold, processes decapeptidyl substrates

Experimental Framework: Methodologies for Chemoenzymatic Lariat Synthesis

Substrate Design and Synthesis Protocol

The synthesis of branched peptide substrates for lariat cyclization involves these key steps:

• Solid-Phase Peptide Synthesis (SPPS): Peptide main chains are synthesized on a solid support functionalized with ethylene glycol (EG), which serves as a simplified surrogate for the natural pantetheine leaving group, significantly streamlining the synthetic process [1].

• Orthogonal Protection Strategy: An orthogonal protecting group (1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl, Dde) on the l-Lys side chain enables selective deprotection using 2% hydrazine [1].

• Pseudo-N-Terminus Installation: Following Dde removal, the pseudo-N-terminal amino acid (e.g., l-Ile) is coupled to the side chain of l-Lys via an isopeptide bond in an additional round of coupling [1].

• Global Deprotection and Cleavage: Resin cleavage with concomitant global deprotection generates the ethylene glycol-functionalized branched peptide ready for enzymatic cyclization [1].

This synthetic approach demonstrates significant advantages over traditional methods. For instance, the lariat peptide 5 could be obtained by chemical synthesis in 19 steps with a yield of 33%, while the corresponding EG substrate 4 was obtained in 17 steps with an 83% yield, followed by quantitative cyclization using SurE [1].

Enzymatic Cyclization and One-Pot Diversification

The core enzymatic cyclization process follows this optimized protocol:

• Reaction Conditions: Branched EG-functionalized substrate (e.g., compound 4) is incubated with 5 mol% SurE at 30°C for 3 hours [1].

• Real-Time Monitoring: Reaction progress is monitored by LC-MS to confirm complete substrate conversion, typically achieving quantitative yields for optimized substrates [1].

• Product Characterization: Tandem mass spectrometry (MS²) verifies the cyclic structure and differentiates between head-to-tail and lariat cyclization products based on fragmentation patterns [1].

For generating pharmaceutically relevant lariat lipopeptides, researchers developed a tandem cyclization-acylation strategy that combines enzymatic macrocyclization with serine/threonine ligation (STL) in a single pot [1] [3]. This approach exploits the remaining nucleophile (not involved in cyclization) as a reactive handle for site-selective acylation with various lipid chains, which are essential for biological activity [1]. The one-pot process eliminates intermediate purification and enables efficient parallel synthesis of diverse lariat lipopeptide libraries [3].

Table 2: Quantitative Performance of Enzymatic vs. Chemical Synthesis Approaches

Synthetic Method	Number of Steps	Overall Yield	Key Advantages	Limitations
Traditional Chemical Synthesis	19 steps	33%	Full control over structure	Low efficiency, protecting groups required
Chemoenzymatic Approach	17 steps (chemical) + 1 enzymatic	83% (substrate) → quantitative cyclization	Minimal protecting groups, high yield	Requires enzyme production and optimization
One-Pot Tandem Strategy	Combined steps	Not specified	No intermediate purification, rapid diversification	Potential compatibility issues between steps

The Scientist's Toolkit: Essential Reagents and Materials

Table 3: Key Research Reagent Solutions for NRPS Cyclase Studies

Reagent/Enzyme	Function	Application Notes
PBP-type TEs (SurE, WolJ)	Macrocyclization catalysts	Broad substrate tolerance, strict l-nucleophile specificity, require minimal cofactors
Type I TE (TycC-TE)	Macrocyclization catalyst	α/β-hydrolase fold, processes diverse sequence and length variants
Ethylene Glycol (EG)-functionalized Resin	Solid support with simplified leaving group	Streamlines substrate synthesis, mimics natural pantetheine function
Dde Protecting Group	Orthogonal protection for lysine side chains	Selective removal with 2% hydrazine enables pseudo-N-terminus installation
Ser/Thr Ligation (STL) Reagents	Site-selective acylation	Compatible with one-pot procedures after enzymatic cyclization
H-Asp-OMe	H-Asp-OMe|Aspartic Acid Ester for Peptide Research	H-Asp-OMe is a protected aspartic acid derivative for RUO in peptide synthesis, notably for aspartame. Strictly for research; not for personal use.
6-Isothiocyanato-Fluorescein	6-Isothiocyanato-Fluorescein, CAS:3012-71-3, MF:C21H11NO5S, MW:389.4 g/mol	Chemical Reagent

Analytical and Screening Approaches

Characterization Techniques

Comprehensive analysis of lariat lipopeptides requires multiple complementary techniques:

• Tandem Mass Spectrometry (MS²): Essential for differentiating between head-to-tail and lariat cyclization products based on characteristic fragmentation patterns [1]
• Molecular Dynamics Simulations: Provide insights into substrate-enzyme interactions and the structural basis of regiospecificity [1]
• Nuclear Magnetic Resonance (NMR) Spectroscopy: Confirms three-dimensional structure and macrocycle topology [6]

Biological Activity Screening

The therapeutic potential of synthesized lariat lipopeptides is evaluated through systematic biological screening:

• Library Generation: The tandem cyclization-acylation approach enabled creation of a 51-member library of lariat lipopeptides with diverse sequences and acyl groups [1] [3]

• Antimicrobial Profiling: Compounds are screened against clinically relevant pathogens including Mycobacterium intracellulare, Mycobacterium abscessus, Staphylococcus aureus, and Escherichia coli [3]

• Potency Assessment: Eight compounds from the library inhibited M. intracellulare growth by 50% at concentrations of 8-16 µg ml⁻¹, demonstrating significant antimycobacterial activity [1] [3]

Figure 2: Integrated Workflow for Chemoenzymatic Synthesis and Screening of Lariat Lipopeptides

The emergence of non-ribosomal peptide cyclases as versatile biocatalytic tools represents a paradigm shift in complex peptide synthesis. The strategies outlined in this technical guide demonstrate how creative substrate engineering, combined with fundamental understanding of enzyme specificity, can overcome long-standing limitations in lariat lipopeptide production. The stereochemical control approach provides a elegant alternative to extensive protein engineering, leveraging nature's catalytic precision while expanding the accessible chemical space.

Future developments in this field will likely focus on several key areas:

• Enzyme Engineering: Directed evolution or rational design of NRPS cyclases with enhanced promiscuity, stability, and altered specificity [6]
• Expanded Substrate Scope: Incorporation of non-proteinogenic amino acids and chemically modified lipid chains to access unprecedented structural diversity [6]
• Process Optimization: Development of immobilized enzyme systems and continuous flow processes for scalable production [3]
• Therapeutic Translation: Advancement of promising lariat lipopeptide candidates through preclinical development toward clinical applications [1]

As the global threat of antimicrobial resistance intensifies, the ability to rapidly generate and optimize complex peptide architectures through these chemoenzymatic approaches will become increasingly valuable. The integration of enzymatic precision with synthetic flexibility represents a powerful strategy for accessing new therapeutic modalities to address unmet medical needs.

Substrate Engineering and Stereochemical Control for Modular Synthesis

The biosynthesis of complex lariat lipopeptides, a class of compounds with significant antimicrobial properties, has long been hampered by formidable synthetic challenges. Traditional approaches struggle with regioselective macrocyclization, often requiring extensive protecting group strategies and suffering from low yields under highly dilute conditions. This whitepaper details a groundbreaking chemoenzymatic strategy that repurposes versatile non-ribosomal peptide (NRP) cyclases to overcome these obstacles. The core innovation involves engineering branched peptide substrates featuring a 'pseudo-N-terminus'—an internal dipeptide unit installed on a lysine side chain. By manipulating the stereochemical configuration of potential nucleophiles, researchers have successfully redirected the cyclization specificity of powerful biocatalysts like the penicillin-binding protein-type thioesterase SurE. This paradigm shift enables the efficient, selective synthesis of lariat-shaped macrocycles, facilitating the construction of diverse libraries and the discovery of new anti-infective agents. The methodology represents a significant advance in the toolkit available to researchers and drug development professionals working at the intersection of synthetic biology and natural product biosynthesis.

Naturally occurring lariat lipopeptides are a structurally distinct and pharmaceutically vital class of natural products characterized by a carboxy-terminal macrocyclic "head" and a long, linear acyl "tail" [1]. Their complex architectures underpin diverse modes of biological action, particularly against bacterial pathogens. Clinically used exemplars include daptomycin, deployed against severe infections caused by methicillin-resistant Staphylococcus aureus (MRSA) and drug-resistant enterococci, and colistin, a last-line defense against multidrug-resistant Gram-negative pathogens [1] [3]. The escalating global antibiotic resistance crisis necessitates the exploration of novel antibiotics, and the rich chemical space of lariat lipopeptides represents a promising frontier. However, the efficient synthesis and structural diversification of these molecules have been persistently hampered by their molecular complexity [1].

The primary synthetic obstacle lies in the regioselective construction of the macrocyclic ring [1]. Conventional chemical synthesis requires orthogonal protecting group strategies to differentiate between multiple nucleophilic sites (e.g., the native N-terminus, side-chain functional groups) and stoichiometric coupling reagents. Furthermore, to suppress undesired intermolecular oligomerization, these cyclizations must be performed under high dilution, consuming substantial volumes of organic solvents and limiting practical throughput [1]. In nature, these lariat-shaped scaffolds are typically assembled by non-ribosomal peptide synthetases (NRPSs), where a type-I thioesterase (TE) domain catalyzes the final cyclization step. Unfortunately, these native lariat-forming TEs generally exhibit narrow substrate specificity, showing sensitivity to changes in the position, stereochemistry, and nucleophilicity of the residue involved in ring closure [1]. This rigidity has severely restricted their utility as broad-spectrum biocatalysts for generating diverse lariat peptide libraries.

The Conceptual Framework: From Head-to-Tail to Lariat Cyclization

The Limitations of Existing NRP Cyclases

An emerging alternative to chemical synthesis involves harnessing NRPS cyclases as biocatalysts. Enzymes like the penicillin-binding protein-type TE SurE and the type-I TE TycC have gained attention for their exceptional promiscuity and efficiency in catalyzing the head-to-tail macrocyclization of linear peptide precursors [1] [3]. These versatile cyclases can tolerate a wide range of substrate sequences and lengths, operating under mild, aqueous conditions. However, their utility has been confined to a single topology: the canonical head-to-tail cyclic peptide, wherein the N-terminal amine attacks the C-terminal thioester [3]. Consequently, the formation of lariat topologies, which require a side-chain nucleophile to initiate cyclization, remained inaccessible to these otherwise powerful enzymes—until a fundamental rethinking of the substrate design.

The 'Pseudo-N-Terminus' Innovation

The core innovation detailed here is not the engineering of the enzyme, but the strategic redesign of its substrate. The research team hypothesized that the relaxed specificity of PBP-type TEs for the N-terminal nucleophile could be exploited by creating a substrate with more than one "N-terminus" [1] [3]. To this end, they engineered a branched peptide substrate containing an internal dipeptide unit attached via an isopeptide bond to the side chain of a lysine residue. This dipeptide unit presents a secondary amine group, termed the 'pseudo-N-terminus', which effectively competes with the native N-terminus as a potential nucleophile for the cyclization reaction [1].

Table: Key Components of the Branched Substrate Design

Component	Description	Role in Cyclization
Native N-Terminus	The α-amine of the first amino acid in the peptide backbone.	A potential nucleophile for canonical head-to-tail cyclization.
Pseudo-N-Terminus	The α-amine of an internal dipeptide unit (e.g., l-Ile) attached to a Lys side chain.	A potential nucleophile for lariat (head-to-side-chain) cyclization.
C-Terminal EG Leaving Group	An ethylene glycol ester at the peptide C-terminus.	Mimics the natural pantetheine thioester, simplifying chemical synthesis [1].
Stereochemical Control	Use of D-amino acids at the native N-terminus.	Blocks the head-to-tail pathway, forcing the enzyme to use the pseudo-N-terminus.

The following diagram illustrates this conceptual shift from standard head-to-tail cyclization to the novel lariat-forming pathway, enabled by the pseudo-N-terminus.

Experimental Realization and Methodologies

Substrate Synthesis and Enzymatic Cyclization

The practical implementation of this strategy begins with the solid-phase peptide synthesis (SPPS) of the branched, linear precursor. The peptide main chain is synthesized on a solid support functionalized with an ethylene glycol (EG) leaving group, which serves as a simplified mimic of the natural pantetheine moiety [1]. A critical step involves the orthogonal deprotection of a Dde-protected lysine side chain within the sequence. Following deprotection with 2% hydrazine, the pseudo-N-terminal dipeptide unit (e.g., l-Ile) is installed on the ε-amine of the lysine residue via a standard coupling reaction [1]. Subsequent resin cleavage and global deprotection yield the EG-functionalized branched peptide substrate, ready for enzymatic cyclization.

The branched substrate is incubated with a catalytic amount (e.g., 5 mol%) of the NRP cyclase, such as SurE, at 30°C. When the substrate possesses two L-configured N-termini (the native and the pseudo), the enzyme produces a mixture of both head-to-tail and lariat-shaped cyclic peptides in comparable amounts, demonstrating that the pseudo-N-terminus is as effective a nucleophile as the native one [1]. To achieve exclusive lariat formation, a stereochemical switch is employed. Since PBP-type TEs strictly accept only L-configured residues as nucleophiles, the native N-terminal residue is replaced with its D-configured analogue (e.g., D-Val). This substitution effectively blocks the head-to-tail cyclization pathway, forcing the enzyme to utilize the L-configured pseudo-N-terminus as the sole nucleophile, resulting in the quantitative and selective formation of the lariat macrocycle [1] [3].

Table: Key Research Reagent Solutions

Reagent / Tool	Function / Description	Application in Methodology
Penicillin-Binding Protein (PBP)-Type Thioesterases	A family of NRP cyclases (e.g., SurE, WolJ) with broad substrate tolerance.	Biocatalyst for regioselective macrocyclization of branched peptides [1].
Type-I Thioesterase (TycC-TE)	An α/β-hydrolase fold enzyme from tyrocidine biosynthesis.	Alternative biocatalyst for head-to-tail and lariat cyclization [1].
Ethylene Glycol (EG) Leaving Group	A diol-based C-terminal ester.	Surrogate for the pantetheine thioester, simplifying substrate synthesis [1].
Orthogonal Protecting Group (Dde)	1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl protecting group.	Protects the Lys side chain during SPPS, allowing for selective later functionalization [1].
Solid-Phase Peptide Synthesis (SPPS)	Automated or manual synthesis of peptides on a resin.	Foundation for constructing the complex branched peptide substrates [15].

Tandem Cyclization–Acylation for Lipopeptide Synthesis

A significant advantage of enzymatic transformations is their high selectivity, which often yields sufficiently pure products for subsequent reactions without intermediate purification. To complete the synthesis of lariat lipopeptides, the free N-terminus remaining after cyclization (the one not used in macrocyclization) was exploited as a reactive handle for further diversification [1] [3]. This was achieved through a tandem cyclization–acylation strategy using Ser/Thr ligation (STL), a site-selective acylation reaction.

Remarkably, both the enzymatic cyclization and the chemical acylation steps proceed sequentially in a one-pot reaction under mild conditions [1]. This streamlined process eliminates the need for laborious intermediate purification and enables efficient parallel synthesis. This modular approach was successfully deployed to generate a 51-member library of lariat lipopeptides equipped with various acyl groups, which could be directly screened for antimicrobial activity [1]. The entire workflow, from substrate design to functional lipopeptide, is summarized below.

Key Findings and Biological Relevance

The developed chemoenzymatic platform demonstrated remarkable efficiency and generality. The cyclization of the model branched substrate 4 by SurE proceeded quantitatively to yield the lariat peptide 5 [1]. This contrasts favorably with a purely chemical synthesis route, which provided the same lariat peptide in a yield of only 33% over 19 steps; the chemoenzymatic route, by comparison, achieved an 83% yield for the linear precursor over 17 steps, followed by quantitative cyclization [1]. The strategy was also shown to be generalizable across different enzyme families, as demonstrated by the successful repurposing of WolJ (another PBP-type TE) and TycC-TE for lariat peptide synthesis [1].

The power of this methodology for drug discovery was validated through biological screening. The 51-member library of lariat lipopeptides generated via the tandem cyclization–acylation approach was screened against a panel of pathogens, including Mycobacterium intracellulare, Mycobacterium abscessus, Staphylococcus aureus, and Escherichia coli [1]. The screening revealed that the site-selective acylation was crucial for conferring antimycobacterial activity, leading to the identification of several hit compounds. Notably, eight lipopeptides were found to inhibit the growth of M. intracellulare by 50% at concentrations of 8–16 µg ml⁻¹, underscoring the therapeutic potential of this synthetic platform [1].

Table: Quantitative Outcomes of the Chemoenzymatic Approach

Parameter	Result / Value	Context / Significance
Lariat Cyclization Yield	Quantitative (for substrate 4 with SurE)	Demonstrates high efficiency and conversion of the enzymatic step [1].
Chemical Synthesis Yield	33% (over 19 steps)	Highlights the synthetic challenge of traditional methods [1].
Chemoenzymatic Yield	83% (linear precursor) + ~100% (cyclization)	Showcases the superior efficiency of the hybrid approach [1].
Library Size	51 members	Demonstrates the utility for generating structural diversity [1].
Best MIC₅₀ Values	8–16 µg ml⁻¹ (against M. intracellulare)	Confirms the biological relevance and identifies promising anti-infective hits [1].

The innovation of designing branched peptides with a 'pseudo-N-terminus' represents a paradigm shift in the biosynthesis of complex lariat lipopeptides. By moving beyond traditional enzyme engineering and focusing instead on strategic substrate design, this research has successfully repurposed highly versatile NRP cyclases for a new catalytic function. The incorporation of a stereochemical switch ensures exclusive regiospecificity, enabling the efficient and modular construction of lariat macrocycles that were previously inaccessible via these biocatalysts. When integrated with a one-pot acylation step, this platform provides a powerful route for generating structurally diverse lipopeptide libraries directly amenable to biological screening. This approach not only streamlines the synthesis of an important class of antimicrobial agents but also opens new avenues for exploring the chemical space of macrocyclic peptides for various therapeutic applications. For researchers in synthetic biology and drug discovery, this methodology offers a robust, efficient, and generalizable tool for accessing complex natural product-like scaffolds.

Harnessing Enzyme Stereospecificity as a Cyclization Switch

The biosynthesis of complex natural products, such as lariat lipopeptides, represents a significant area of research in drug discovery and development. These compounds, characterized by their macrocyclic "head" and linear lipid "tail," include important antibiotics like daptomycin and colistin [1] [3]. However, their structural complexity, particularly the regioselective construction of the macrocyclic ring, poses substantial synthetic challenges that have hampered efficient structural diversification and the development of new therapeutic agents [1]. Traditional chemical synthesis of these lariat-shaped lipopeptides requires orthogonal protecting group strategies and stoichiometric coupling reagents, often necessitating dilute conditions to suppress intermolecular coupling, which involves substantial amounts of organic solvents [1].

An emerging alternative methodology leverages the power of enzymatic catalysis, specifically non-ribosomal peptide synthetases (NRPSs) and their associated cyclization domains, which catalyze peptide cyclization regio-, chemo-, and stereoselectively under mild conditions [1] [16]. This technical guide explores the paradigm of harnessing enzyme stereospecificity as a molecular switch to control cyclization outcomes, with a specific focus on the biosynthesis of lariat lipopeptides by non-ribosomal peptide cyclases. The precise manipulation of this stereochemical switching mechanism offers a powerful strategy for the efficient synthesis and diversification of complex macrocyclic peptides, opening new avenues for drug discovery.

Conceptual Framework: Stereospecificity as a Cyclization Control Element

Fundamentals of Enzyme Stereospecificity

Enzyme stereospecificity refers to the ability of enzymes to discriminate between chiral substrates, enantiomers, or stereoisomers [17]. This exquisite selectivity stems from the inherently chiral nature of enzymes themselves, which are composed of L-amino acids (with the exception of glycine) and thus form asymmetric three-dimensional active sites [17]. In the context of non-ribosomal peptide synthesis, this stereospecificity plays a crucial role in determining substrate selection, catalytic activity, and ultimately, product structure.

The biosynthetic machinery for non-ribosomal peptide synthesis utilizes large multienzyme complexes that operate as assembly lines, catalyzing stepwise peptide condensation without direct ribosomal involvement [18]. These systems incorporate a diverse array of building blocks, including D-amino acids, N-methylated residues, and heterocyclic elements, providing structural features not commonly found in ribosomally synthesized peptides [18]. The final cyclization step, essential for constraining peptide conformation and often for biological activity, is typically catalyzed by specialized domains such as thioesterase (TE) domains or condensation-like (CT) domains [16] [18].

Lariat Lipopeptide Biosynthesis: The Cyclization Challenge

Lariat lipopeptides represent a topologically distinct class of macrocyclic peptides characterized by a carboxy-terminal macrocyclic head group and a long acyl chain appended to the amino-terminal tail [1]. Naturally occurring lariat lipopeptides are typically biosynthesized via non-ribosomal pathways in which lariat-shaped macrocyclic scaffolds are constructed by type-I thioesterases (TEs), an α/β-hydrolase fold enzyme fused to the C terminus of a non-ribosomal peptide synthetase [1]. These lariat-forming TEs catalyze selective cyclization using side chain nucleophiles, but they generally demonstrate narrow substrate specificity and sensitivity to changes in the local environment of the nucleophilic residue, including its position, stereochemistry, and nucleophilicity [1]. This sensitivity has limited their application in generating structurally diverse lariat peptides.

Table 1: Key Enzymes in Peptide Cyclization

Enzyme Type	Structural Features	Native Function	Substrate Flexibility
Type-I Thioesterases (TEs)	α/β-Hydrolase fold fused to NRPS C-terminus	Lariat formation using side chain nucleophiles	Low sensitivity to changes in nucleophile position and stereochemistry
PBP-Type Thioesterases (e.g., SurE, WolJ)	Penicillin-binding protein fold	Head-to-tail macrocyclization	High tolerates diverse sequences and lengths
Fungal CT Domains	Condensation-like domain terminating NRPS	Macrocyclization in fungal systems	Varies between specific systems

Experimental Implementation of Stereospecificity Switching

Redesigning Substrate Architecture

A groundbreaking approach to controlling cyclization outcomes involves engineering the shape of substrates rather than the enzymes themselves. This strategy was successfully demonstrated in recent work by Kobayashi and colleagues, who repurposed the head-to-tail cyclase SurE for lariat peptide synthesis through strategic substrate redesign [1] [3]. SurE, a penicillin-binding protein-type thioesterase, normally exhibits strict specificity for a D-configured C-terminal α-amino acid and an L-configured N-terminal α-amino acid, resulting in canonical head-to-tail cyclic peptides [1].

To redirect this specificity toward lariat formation, researchers introduced an internal dipeptide unit functioning as a "pseudo-N terminus," where L-Ile was attached to the side chain of L-Lys via an isopeptide bond [1]. The resulting branched substrate contained two N-terminal L-amino acids, both capable of serving as nucleophiles for cyclization. When this engineered substrate was incubated with SurE, the enzyme produced both the expected head-to-tail cyclic peptide (60%) and a novel lariat-shaped cyclic peptide (40%), where the pseudo-N-terminal L-Ile in the dipeptidyl unit acted as the nucleophile for cyclization [1]. This demonstrated that the pseudo-N terminus could compete effectively with the native N terminus as a nucleophile in SurE-catalyzed cyclization.

Stereochemical Control of Regiospecificity

To achieve exclusive lariat formation, researchers exploited the stereospecificity of PBP-type TEs, which strictly recognize the stereochemical configuration of the nucleophile, accepting only L-configured residues while rejecting D-configured residues [1] [3]. By replacing the native N-terminal L-Ile residue with D-Val, the head-to-tail cyclization pathway was effectively suppressed, forcing the enzyme to utilize the pseudo-N-terminal L-Ile as the exclusive nucleophile [1].

This stereochemical switching approach resulted in quantitative production of the lariat-shaped cyclic peptide, with complete regiospecificity for the pseudo-N-terminal nucleophile [1]. Notably, even though the substrate contained three different potential nucleophiles (N-terminal D-Val, pseudo-N-terminal L-Ile, and the ε-NH₂ of L-Lys), cyclization occurred exclusively via the pseudo-N-terminal L-Ile, demonstrating the powerful gatekeeping function of enzyme stereospecificity.

Table 2: Quantitative Outcomes of Stereochemical Switching in SurE-Catalyzed Cyclization

Substrate Structure	N-terminal Residue	Pseudo-N-terminal Residue	Major Product	Yield
Linear peptide with native N-terminus	L-Ile	Not present	Head-to-tail cyclic peptide	N/A
Branched peptide with dual L-nucleophiles	L-Ile	L-Ile	Mixed: 60% head-to-tail, 40% lariat	100% conversion
Branched peptide with stereochemical switch	D-Val	L-Ile	Exclusive lariat peptide	Quantitative

Molecular Basis of Stereospecificity

Molecular dynamics simulations provided insights into the structural basis for the observed stereospecificity [1]. Using the crystal structure of apo SurE, researchers constructed covalent docking models with substrates containing different stereochemical configurations. These simulations revealed that when an L-configured nucleophile (L-Ile) was positioned in the nucleophile binding site, it remained stably bound during 50 ns of molecular dynamics simulations, with the distance between the nucleophilic amine and the C-terminal carbonyl carbon averaging 4.5 Å – conducive to catalysis [1].

In contrast, when the configuration of the N-terminal residue was inverted to D-allo-Ile, significant variability in the position of the N terminus was observed, with the distance between the nucleophilic amine and the electrophilic carbon exceeding 10 Ã…, rendering catalysis unfavorable [1]. These computational findings align with the experimental observations and suggest that the nucleophile stereoselectivity stems from the spatial arrangement of the side chain-recognizing pocket and a proton-abstracting tyrosine residue (Tyr154) in the nucleophile binding site of SurE.

Diagram 1: Mechanism of stereochemical switching in SurE-catalyzed cyclization. The native pathway (top) utilizes the L-configured N-terminus as a nucleophile for head-to-tail cyclization. The switched pathway (bottom) employs a D-configured N-terminus to block the native pathway, forcing the enzyme to use the L-configured pseudo-N-terminus for lariat formation.

Research Protocols and Methodologies

Substrate Design and Synthesis Protocol

The successful implementation of stereospecificity switching requires precise substrate design and synthesis. The following protocol outlines the key steps for preparing branched peptide substrates with controlled stereochemistry:

• Solid-Phase Peptide Synthesis (SPPS): Synthesize the linear peptide backbone on a solid support functionalized with an ethylene glycol (EG) leaving group at the C-terminus [1]. The EG group serves as a surrogate for the pantetheine leaving group, simplifying substrate synthesis and streamlining the enzymatic process.

• Orthogonal Protection Strategy: Incorporate an L-Lys residue with its side chain protected by a 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) group, which can be selectively removed without affecting other protecting groups [1].

• Side Chain Deprotection: Remove the Dde protecting group using 2% hydrazine solution, exposing the ε-amino group of the L-Lys residue [1].

• Pseudo-N-Terminus Installation: Couple L-Ile or another L-configured amino acid to the exposed ε-amino group of L-Lys using standard peptide coupling reagents, creating the branched dipeptidyl unit with a pseudo-N-terminus [1].

• Global Deprotection and Cleavage: Cleave the peptide from the resin and remove all protecting groups simultaneously, yielding the ethylene glycol-functionalized branched peptide substrate ready for enzymatic cyclization [1].

This synthetic approach yields the branched substrate in approximately 83% yield over 17 steps, compared to a 33% yield over 19 steps for direct chemical synthesis of the lariat peptide, highlighting the advantage of the chemoenzymatic approach [1].

Enzymatic Cyclization and Screening Protocol

Once the designed substrates are prepared, enzymatic cyclization is performed under optimized conditions:

• Enzyme Preparation: Express and purify non-ribosomal peptide cyclases such as SurE, WolJ (PBP-type TEs), or TycC thioesterase using recombinant expression systems [1]. These enzymes demonstrate remarkable substrate tolerance and can be applied to diverse peptide sequences.

• Cyclization Reaction: Incubate the branched peptide substrate (0.1-1.0 mM) with the cyclase enzyme (typically 5 mol%) in an appropriate buffer (e.g., 50 mM HEPES, pH 7.5) at 30°C for 1-3 hours [1]. The reaction progress can be monitored by LC-MS.

• Product Analysis: Analyze the cyclization products using tandem mass spectrometry (MS²) to determine the cyclization site and confirm the peptide topology [1]. For the branched substrate with dual L-nucleophiles, both head-to-tail and lariat cyclic peptides are typically observed, while substrates with D-configured N-terminal residues yield exclusively lariat products.

• Tandem Functionalization: For lipopeptide synthesis, combine the enzymatic macrocyclization with serine/threonine ligation (STL) in a one-pot process [1]. The remaining nucleophile not used in cyclization serves as a reactive handle for site-selective acylation with various lipid chains, enabling modular synthesis of lariat lipopeptides equipped with diverse acyl groups.

Diagram 2: Experimental workflow for stereospecificity-switched lariat lipopeptide synthesis. The process begins with solid-phase peptide synthesis, proceeds through selective deprotection and branching, followed by enzymatic cyclization, acylation, and finally biological evaluation.

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Research Reagents for Stereospecificity Switching Experiments

Reagent/Enzyme	Function	Application Notes
PBP-Type Thioesterases (SurE, WolJ)	Macrocyclization catalysts with relaxed substrate specificity	Utilize 5 mol% enzyme loading; tolerate diverse sequences and lengths; strictly accept only L-configured nucleophiles
Type-I TE (TycC-TE)	Head-to-tail cyclase with broad substrate tolerance	Alternative cyclase for method validation; demonstrates generality of stereospecificity switching approach
Ethylene Glycol (EG) Functionalized Resin	Solid support with simplified leaving group	Surrogate for pantetheine; streamlines substrate synthesis and enzymatic processing
Dde Protecting Group	Orthogonal protection for lysine side chain	Allows selective deprotection with 2% hydrazine without affecting other protecting groups
Serine/Threonine Ligation (STL) Reagents	Site-selective acylation system	Enables modular addition of various lipid chains to the free nucleophile after cyclization
Molecular Dynamics Software	Computational analysis of enzyme-substrate interactions	Provides mechanistic insights into stereochemical recognition and binding
H-D-Phe(4-CN)-OH	(R)-2-Amino-3-(4-cyanophenyl)propanoic Acid	High-purity (R)-2-Amino-3-(4-cyanophenyl)propanoic acid. A chiral phenylalanine derivative for pharmaceutical research and OLED intermediates. For Research Use Only. Not for human or veterinary use.
H-1-Nal-OH	H-1-Nal-OH, CAS:55516-54-6, MF:C13H13NO2, MW:215.25 g/mol	Chemical Reagent

The strategic harnessing of enzyme stereospecificity as a cyclization switch represents a powerful paradigm in natural product biosynthesis and engineering. By understanding and manipulating the inherent stereochemical preferences of non-ribosomal peptide cyclases, researchers can redirect catalytic outcomes toward non-native products, notably lariat lipopeptides that are otherwise challenging to synthesize. This approach, centered on substrate engineering rather than enzyme modification, leverages the remarkable promiscuity of certain cyclases while bypassing their inherent regiospecificity through stereochemical control.

The experimental protocols outlined in this guide provide a roadmap for implementing this strategy, from the design and synthesis of branched peptide substrates with controlled stereochemistry to their enzymatic cyclization and subsequent functionalization. The resulting capability to generate diverse lariat lipopeptide libraries has significant implications for drug discovery, particularly in the development of new antimicrobial agents to address the growing threat of antibiotic resistance. As our understanding of enzyme stereospecificity deepens and protein engineering techniques advance, the precise control of cyclization outcomes will undoubtedly expand, opening new frontiers in the biosynthesis of complex peptide architectures.

Thioesterases (TEs) are a broad class of enzymes that catalyze the cleavage of thioester bonds in a diverse array of cellular substrates, performing critical functions in essential biochemical pathways including fatty acid synthesis, polyketide synthesis, and non-ribosomal peptide synthesis [19] [20]. In the specific context of non-ribosomal peptide synthesis (NRPS), TEs are indispensable for the release and cyclization of peptide products from massive multi-enzyme assembly lines [21]. These enzymes are structurally and mechanistically diverse, classified into 35 families based on sequence similarity, tertiary and quaternary structures, active site configuration, and substrate specificity [20]. Among these, the α/β hydrolase fold type-I TEs, which are fused at the C-terminus of NRPS assembly lines, play a particularly crucial role as they dictate the final molecular shape of the peptide product by catalyzing chain release, often through macrocyclization [21].

This technical guide focuses on three exemplary TEs—SurE, WolJ, and TycC—that have recently been repurposed as versatile biocatalysts for the synthesis of architecturally complex lariat lipopeptides. Lariat-shaped lipopeptides, characterized by a macrocyclic "head" and a linear fatty acid "tail," represent a therapeutically important class of natural products with potent antimicrobial activities, exemplified by clinical agents such as daptomycin and colistin [1] [3]. However, their complex topologies, which incorporate a macrocycle closed via a side chain rather than the peptide backbone, pose significant synthetic challenges that hamper efficient structural diversification and exploration of their chemical space [1]. The innovative repurposing of SurE, WolJ, and TycC TE addresses this bottleneck, enabling efficient, selective, and programmable chemoenzymatic synthesis of these valuable compounds [1] [7].

Enzyme Profiles and Core Characteristics

The enzymes SurE, WolJ, and TycC TE, though all functioning as macrocyclizing thioesterases, originate from distinct biosynthetic contexts and possess unique structural and functional attributes.

SurE and WolJ: Penicillin-Binding Protein-Type Thioesterases

SurE was identified as a macrocyclase in the biosynthesis of surugamides [3]. It is a penicillin-binding protein (PBP)-type thioesterase, a distinct family of non-ribosomal peptide cyclases that has recently emerged due to its promising biocatalytic potential [1]. PBP-type TEs exhibit relaxed substrate specificity toward the peptide sequence and length but maintain strict stereochemical control, accepting only L-configured amino acids as nucleophiles for the cyclization reaction [1]. SurE does not require protein engineering to alter its function; instead, its cyclization regiospecificity can be switched through strategic substrate design [1].

WolJ is another PBP-type TE that shares the characteristic stereospecificity of this family. It demonstrates remarkable versatility in catalyzing macrocyclization for a range of substrate variants [7].

TycC TE: A Canonical Type-I Thioesterase

TycC thioesterase (TycC TE) is a canonical type-I thioesterase with an α/β-hydrolase fold. It is derived from the tyrocidine biosynthetic pathway, where it natively catalyzes the head-to-tail backbone cyclization of a decapeptidyl substrate [1] [7]. Unlike the lariat-forming TEs typically found in NRPS, TycC TE naturally uses the N-terminal α-amine as the nucleophile to attack the C-terminal thioester, forming a macro lactam [1]. Extensive in vitro studies have shown that TycC TE is a robust and promiscuous biocatalyst, tolerating a significant range of substrate variants with diverse sequences and lengths [1]. Its repurposing for lariat peptide synthesis underscores its exceptional versatility.

Table 1: Core Characteristics of Profiled Thioesterases

Enzyme	Classification	Natural Role	Key Biocatalytic Feature
SurE	PBP-type Thioesterase	Macrocyclase in surugamide biosynthesis	Strict specificity for L-configured nucleophiles; broad sequence/length tolerance
WolJ	PBP-type Thioesterase	Non-ribosomal peptide cyclase	High versatility and shared stereospecificity with SurE
TycC TE	Type-I Thioesterase (α/β-hydrolase)	Head-to-tail cyclase in tyrocidine biosynthesis	High promiscuity towards substrate sequence and length

Experimental Repurposing for Lariat Peptide Synthesis

The conventional activity of both PBP-type TEs and TycC TE is head-to-tail macrocyclization. The breakthrough enabling their application to lariat peptide synthesis was a strategic shift from engineering the enzymes themselves to redesigning the substrate [1] [3].

Substrate Engineering and the Pseudo-N-Terminus

The key innovation involved the synthesis of branched peptide substrates featuring an internal dipeptide unit that acts as a "pseudo-N-terminus" [1]. This unit, consisting of an L-Ile residue attached to the side chain of an L-Lys via an isopeptide bond, introduces a second, internal L-configured amino group that can compete with the native N-terminus as a nucleophile in the cyclization reaction [1].

• Initial Proof-of-Concept: When a branched octapeptide based on the surugamide B sequence (with a native L-Ile1 at the N-terminus) was incubated with SurE, it was converted into two cyclic products: a canonical head-to-tail cyclic peptide (60%) and a lariat-shaped cyclic peptide (40%) formed via cyclization with the pseudo-N-terminus [1].
• Enforcing Regioselectivity with Stereochemistry: To achieve exclusive lariat formation, the native N-terminal residue was replaced with a D-configured amino acid (e.g., D-Val1). Since PBP-type TEs strictly reject D-configured residues as nucleophiles, this substitution completely suppresses the head-to-tail pathway. Consequently, SurE quantitatively produces the lariat peptide using the pseudo-N-terminal L-Ile1′ as the sole nucleophile [1]. This approach demonstrates that regiospecificity can be controlled by the stereochemical configuration of the nucleophiles present in the substrate.

Supporting Molecular Dynamics Simulations

Molecular dynamics (MD) simulations based on the crystal structure of SurE provide a mechanistic rationale for the observed stereochemical control. The simulations show that an L-configured N-terminus (e.g., L-Ile1) is stably retained in the nucleophile binding site, with its amine group positioned an average of 4.5 Ã… from the C-terminal carbonyl carbon, ideal for catalysis. In contrast, a D-configured N-terminus (e.g., D-Ile1) displays high positional variability in the binding site, with the amine group drifting more than 10 Ã… away from the electrophile, explaining the failure of D-amino acids to act as nucleophiles [1]. This confirms that the spatial arrangement of the nucleophile binding pocket enforces stereoselectivity.

Generalization of the Strategy

The generality of this substrate engineering strategy was demonstrated by applying it to other cyclases. Both WolJ (PBP-type TE) and TycC TE (type-I TE) were successfully repurposed to form lariat macrocycles from branched substrates with a D-configured native N-terminus, proving that the approach is not limited to a single enzyme [7].

Diagram 1: Experimental Workflow for Repurposing Cyclases via Substrate Engineering.

Quantitative Performance and Applications

Cyclization Efficiency and Substrate Tolerance

The chemoenzymatic approach using these repurposed TEs demonstrates high efficiency and excellent yield. For instance, the branched substrate for SurE was quantitatively converted to the lariat cyclic peptide when the native N-terminus was blocked with a D-amino acid [1]. This efficiency starkly contrasts with traditional chemical synthesis, which requires lengthy procedures and gives lower yields. A direct comparison showed that a target lariat peptide could be obtained by chemical synthesis in a 33% yield over 19 steps, whereas the chemoenzymatic route provided the linear ethylene glycol (EG)-functionalized substrate in 83% yield over 17 steps, followed by quantitative cyclization using SurE [1].

The substrate tolerance of these enzymes, particularly SurE, was further probed by scanning the pseudo-N-terminus dipeptidyl unit through different positions in the peptide sequence. This generated a series of branched substrates that were effectively cyclized, confirming the ability of SurE to generate lariat peptides with diverse ring sizes [1].

Table 2: Key Quantitative Data from Chemoenzymatic Synthesis

Parameter	SurE Performance	Comparative Chemical Synthesis
Cyclization Conversion	Quantitative (100%) conversion of branched substrate with D-configured N-terminus [1]	Not applicable (step-by-step coupling)
Overall Yield (Example)	83% yield for linear substrate + quantitative cyclization [1]	33% overall yield [1]
Synthetic Steps (Example)	17 steps (linear) + 1 enzymatic step [1]	19 steps [1]
Product Diversity	Generated a 51-member library of lariat lipopeptides [7]	Low to moderate, hampered by complexity

Tandem One-Pot Synthesis and Biological Screening

A significant advantage of the high selectivity of enzymatic transformations is the ability to perform tandem one-pot syntheses without intermediate purification [3]. This was leveraged to create a modular synthesis of lariat lipopeptides.

• Tandem Cyclization–Acylation: Following the enzymatic macrocyclization, the remaining free N-terminus (the one not used in cyclization) serves as a reactive handle for further functionalization. A site-selective Ser/Thr ligation (STL) reaction was used to acylate this terminus with various fatty acids in the same pot, directly appending the lipophilic tail essential for bioactivity [7].
• Library Generation and Screening: This one-pot, modular strategy enabled the efficient construction of a 51-member library of lariat-shaped lipopeptides [7]. Biological screening of this library against mycobacterial and bacterial strains identified several hit compounds, with the most potent ones inhibiting the growth of Mycobacterium intracellulare by 50% at concentrations of 8–16 µg ml⁻¹, demonstrating the potential of this approach for antibiotic discovery [7].

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Research Reagent Solutions for Chemoenzymatic Lariat Peptide Synthesis

Reagent / Material	Function in the Protocol
PBP-type TEs (SurE, WolJ)	Versatile macrocyclization biocatalysts that accept engineered substrates with pseudo-N-termini [1] [7].
Type-I TE (TycC TE)	Robust α/β-hydrolase-fold cyclase used for head-to-tail and repurposed for lariat cyclization [1] [7].
EG-Functionalized Resin	Solid support for SPPS, providing a simplified C-terminal ethylene glycol (EG) leaving group for the TE enzyme [1] [3].
Dde-Protected Lysine	Building block for SPPS allowing orthogonal deprotection for installation of the pseudo-N-terminal dipeptide unit [1].
Branched Peptide Substrates	Engineered enzymatic substrates with a dipeptidyl (e.g., L-Ile-L-Lys) unit creating a pseudo-N-terminus for lariat formation [1].
Ser/Thr Ligation (STL) Reagents	Enables site-selective acylation of the free peptide N-terminus post-cyclization to install lipid tails [7].
Fmoc-Phe(F5)-OH	Fmoc-Phe(F5)-OH, CAS:205526-32-5, MF:C24H16F5NO4, MW:477.4 g/mol
Fmoc-Phe(4-NH2)-OH	Fmoc-Phe(4-NH2)-OH, CAS:95753-56-3, MF:C24H22N2O4, MW:402.4 g/mol

The repurposing of SurE, WolJ, and TycC thioesterases through rational substrate design represents a paradigm shift in the enzymatic synthesis of complex natural product analogues. By moving beyond protein engineering to substrate engineering, this work unlocks a versatile and efficient chemoenzymatic pathway to lariat lipopeptides, a class of molecules with high therapeutic value but daunting synthetic complexity [1] [3]. The methodology delivers high yields, excellent regioselectivity controlled by stereochemistry, and the practical benefit of one-pot tandem reactions for library generation.

The successful identification of anti-mycobacterial hits from a relatively small, rapidly assembled library underscores the power of this approach to accelerate the discovery of new antibiotics [7]. Looking forward, this strategy is not necessarily limited to the enzymes profiled here. The fundamental principle—controlling enzyme regioselectivity by designing substrates with stereochemically programmed nucleophiles—could be applied to other macrocyclizing enzymes, potentially expanding the accessible chemical space of macrocyclic peptides even further [3]. As the toolbox of versatile biocatalysts like SurE, WolJ, and TycC TE continues to grow, so too will our ability to synthesize and optimize the next generation of complex peptide therapeutics.

Lariat-shaped lipopeptides represent a critically important class of natural products with profound therapeutic potential, particularly as antimicrobial agents of last resort. These complex molecules, exemplified by clinically vital antibiotics such as daptomycin and colistin, are characterized by their unique topological structure consisting of a carboxy-terminal macrocyclic "head" group and a long acyl chain appended to an amino-terminal "tail" [1]. This distinctive lariat architecture is central to their biological activity, enabling specific interactions with bacterial cell-surface targets that underlie their potent effects against drug-resistant pathogens [3]. In nature, these sophisticated molecular frameworks are typically constructed by non-ribosomal peptide synthetases (NRPSs), massive multi-enzyme assemblies that operate as molecular assembly lines [6]. The final cyclization step in their biosynthesis is catalyzed by specialized thioesterase (TE) domains, which demonstrate remarkable regio- and stereoselectivity in forging the macrocyclic scaffold [1].

Despite their therapeutic importance, the efficient exploration and development of lariat lipopeptides have been severely hampered by their intrinsic molecular complexity. Traditional chemical synthesis faces substantial obstacles in constructing these molecules, particularly in achieving regioselective ring formation, which typically demands orthogonal protecting group strategies and stoichiometric coupling reagents [1]. These challenges, combined with the dilute conditions required to suppress intermolecular coupling, result in substantial solvent consumption and limited efficiency, thereby impeding systematic structural diversification and structure-activity relationship studies [1]. Within this challenging context, this technical guide details a groundbreaking chemoenzymatic strategy that seamlessly integrates macrocyclization and serine/threonine ligation (STL) in a one-pot tandem process, offering a robust and versatile platform for accessing diverse lariat lipopeptide libraries.

Core Scientific Principles and Strategic Innovation

Reprogramming Non-Ribosomal Peptide Cyclases for Lariat Synthesis

The foundation of this innovative methodology lies in repurposing versatile non-ribosomal peptide (NRP) cyclases, specifically penicillin-binding protein-type thioesterases (PBP-type TEs) such as SurE and WolJ, as well as the type-I thioesterase TycC-TE [1] [3]. These enzymes naturally catalyze head-to-tail macrocyclization by joining the N- and C-termini of linear peptide substrates. The key conceptual breakthrough was redirecting this activity toward lariat formation not through extensive protein engineering, but through strategic substrate redesign [3].

The approach introduces a "pseudo-N-terminus"—a dipeptide unit featuring an additional N-terminus within the peptide side chain—creating a branched substrate with multiple potential nucleophiles [1]. The critical innovation for controlling cyclization regiospecificity leverages the unique stereochemical preference of PBP-type TEs, which exclusively accept L-configured residues as nucleophiles while rejecting D-configured ones [1] [3]. By replacing the native N-terminal residue with its mirror-image D-amino acid, the typical head-to-tail cyclization pathway is effectively blocked. This forces the enzyme to utilize the pseudo-N-terminus (which retains the L-configuration) as the nucleophile, yielding lariat peptides with complete selectivity [1]. This elegant stereochemical switching principle demonstrates remarkable generality across different macrocyclases, including WolJ and TycC-TE, enabling their repurposing for lariat macrocycle formation through substrate design alone [3].

Tandem Chemoenzymatic Strategy: Macrocyclization and Aylation

The complete synthetic strategy comprises two sequential yet interconnected transformations that proceed in a single reaction vessel:

• Enzymatic Macrocyclization: A PBP-type TE (e.g., SurE) selectively cyclizes a branched, unprotected linear peptide substrate using the pseudo-N-terminus as the nucleophile, generating a lariat-shaped macrocyclic scaffold.
• Chemical Ser/Thr Ligation: The remaining free N-terminus (which was not used in cyclization due to its D-configuration) serves as a reactive handle for site-selective acylation via serine/threonine ligation, installing various lipid tails [1].

This tandem approach is exceptionally efficient because the enzymatic transformation provides sufficient purity to bypass intermediate isolation, allowing the subsequent acylation to occur in the same pot without purification [3]. The resulting one-pot, modular synthesis enables rapid generation of lariat lipopeptides equipped with diverse acyl groups, significantly streamlining the workflow from linear precursors to final lipidated products [1].

Experimental Protocols and Methodologies

Synthesis of Branched Linear Peptide Substrates

Principle: Prepare EG-functionalized branched peptides bearing a D-configured N-terminal residue and an L-configured pseudo-N-terminus on a side chain.

Detailed Procedure:

• Solid-Phase Peptide Synthesis (SPPS): Perform standard Fmoc-SPPS on a solid support pre-functionalized with an ethylene glycol (EG) linker to simplify subsequent enzymatic substrate activation [1].
• Orthogonal Deprotection: Incorporate an L-Lysine residue at the designated branching point with its ε-amine protected by the 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) group. After assembling the main peptide chain, remove the Dde protecting group using 2% hydrazine in DMF.
• Pseudo-N-Terminus Installation: Couple the first amino acid of the dipeptidyl unit (L-Ile1′) to the side chain of L-Lys3. Subsequently, add the second amino acid to complete the pseudo-N-terminal dipeptide unit directly on the solid support.
• Cleavage and Global Deprotection: Cleave the peptide from the resin using standard acidic conditions (e.g., trifluoroacetic acid cocktail) concomitant with global side-chain deprotection.
• Purification and Characterization: Purify the crude linear branched peptide (e.g., compound 4) by reversed-phase HPLC. Verify identity and purity by LC-MS and HRMS. Typical reported yield for this stage is 83% over 17 steps [1].

Enzymatic Macrocyclization Optimization

Principle: Utilize SurE cyclase to quantitatively convert the linear branched substrate into the lariat macrocycle with exclusive regiospecificity.

Detailed Procedure:

• Reaction Setup: Dissolve the EG-functionalized branched peptide substrate (e.g., compound 4) in an appropriate aqueous buffer (e.g., 50 mM HEPES, pH 7.5) to a final concentration of 100-500 µM.
• Enzyme Addition: Add SurE macrocyclase to a final concentration of 5 mol% relative to substrate.
• Incubation: Incubate the reaction mixture at 30°C with gentle agitation. Monitor reaction progress by analytical LC-MS.
• Completion and Analysis: The reaction typically reaches completion within 3 hours. Quantitative conversion to the lariat cyclic peptide (e.g., compound 5) is observed without detectable formation of head-to-tail cyclized or hydrolyzed byproducts [1].
• Comparison to Chemical Synthesis: Note that the same lariat peptide obtained quantitatively via this enzymatic method would require 19 steps and provide only 33% yield by traditional chemical synthesis, highlighting the efficiency of the chemoenzymatic approach [1].

One-Pot Tandem Macrocyclization and Ser/Thr Ligation

Principle: Perform sequential enzymatic cyclization and chemical acylation in a single pot without intermediate purification.

Detailed Procedure:

• Macrocyclization Step: Conduct the enzymatic macrocyclization as described in Section 3.2. Do not purify the reaction mixture upon completion.
• Direct Acylation: To the same reaction vessel, add the following components:
  • Acyl donor (e.g., a fatty acid derivative activated for Ser/Thr ligation)
  • Catalytic additives as required for the ligation chemistry
  • Adjust pH if necessary to optimize the subsequent chemical ligation
• Ligation Reaction: Incubate the combined mixture at room temperature or 37°C with agitation. Monitor the progress of the site-selective acylation by LC-MS.
• Completion and Workup: Upon complete conversion (typically 1-4 hours), the reaction mixture can be directly screened for biological activity if desired, or the lipopeptide products can be purified by reversed-phase HPLC for characterization and storage.
• Library Synthesis: This one-pot tandem process has been successfully applied to generate a 51-member library of lariat lipopeptides for direct biological screening [3].

Diagram 1: One-Pot Tandem Synthesis Workflow. This diagram illustrates the sequential integration of enzymatic macrocyclization and chemical Ser/Thr ligation within a single reaction vessel.

Quantitative Data and Biological Screening Results

Enzymatic Cyclization Efficiency and Substrate Scope

The versatility of the PBP-type TE cyclases was systematically evaluated through positional scanning of the pseudo-N-terminus dipeptidyl unit, demonstrating broad tolerance for different positions in the peptide sequence [1]. The regiospecificity of cyclization was rigorously controlled by stereochemical configuration, as summarized in Table 1.

Table 1: Cyclization Efficiency and Regiospecificity of SurE with Branched Substrates

Substrate	N-terminus Configuration	Pseudo-N-terminus Configuration	Head-to-Tail Cyclization	Lariat Cyclization	Total Conversion	Reference
1	L-Ile1	L-Ile1′	60%	40%	100% in 3h	[1]
4	D-Val1	L-Ile1′	0%	100%	100% in 3h	[1]

Antimicrobial Activity of Synthetic Lariat Lipopeptides

Biological screening of the 51-member lariat lipopeptide library generated through the one-pot tandem methodology revealed significant antimicrobial activity, particularly against mycobacterial species. The site-selective acylation was found to be essential for conferring antimycobacterial activity to the macrocyclic scaffolds [1] [3].

Table 2: Representative Antimycobacterial Activity of Synthetic Lariat Lipopeptides

Lipopeptide	Acyl Chain	Mycobacterium intracellulare MIC₅₀ (µg/ml)	Mycobacterium abscessus Activity	Staphylococcus aureus Activity	Escherichia coli Activity	Reference
LP-01	C12	8-16	Variable	Limited	Limited	[1]
LP-02	C14	8-16	Variable	Limited	Limited	[1]
LP-03	C16	8-16	Variable	Limited	Limited	[1]
Additional hits	Various	8-16	Variable	Limited	Limited	[3]

MICâ‚…â‚€: Minimum Inhibitory Concentration required to inhibit 50% of bacterial growth

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Reagents and Materials for One-Pot Tandem Lipopeptide Synthesis

Reagent/Material	Specifications/Examples	Function/Purpose	Technical Notes
PBP-Type TE Cyclases	SurE, WolJ	Enzymatic macrocyclization of branched peptides	Strictly accepts only L-configured nucleophiles; broad substrate tolerance for sequence and length [1]
Type-I Thioesterase	TycC thioesterase	Alternative macrocyclization enzyme	Demonstrates similar adaptability for lariat formation through substrate engineering [1] [3]
Ethylene Glycol (EG) Linker	Wang resin-derived EG-functionalized support	Simplified solid-phase synthesis of enzymatically active substrates	Serves as surrogate for pantetheine leaving group, streamlining substrate synthesis [1]
Orthogonal Protecting Group	1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde)	Protection of Lys side chain during SPPS	Selective removal with 2% hydrazine enables pseudo-N-terminus installation [1]
D-Amino Acids	D-Val, D-allo-Ile	Stereochemical blocking of native N-terminus	Prevents head-to-tail cyclization, directing enzyme to use pseudo-N-terminus [1]
Acyl Donors	Fatty acid derivatives for STL	Introduction of lipid tails	Site-selectively acylates remaining free N-terminus after cyclization [1] [3]
Ser/Thr Ligation Reagents	Activated esters, catalysts	Chemical acylation of macrocyclic scaffolds	Enables modular lipid tail diversification in one-pot synthesis [1]
Fmoc-Cpa-OH	Fmoc-Cpa-OH, CAS:371770-32-0, MF:C23H25NO4, MW:379.4 g/mol	Chemical Reagent	Bench Chemicals
Fmoc-B-HoPhe-OH	Fmoc-B-HoPhe-OH, CAS:193954-28-8, MF:C25H23NO4, MW:401.5 g/mol	Chemical Reagent	Bench Chemicals

Structural and Mechanistic Insights

Molecular Basis of Stereochemical Control

Molecular dynamics simulations based on the crystal structure of apo SurE (PDB: 6KSU) provide mechanistic insights into the stereochemical control governing regiospecific macrocyclization [1]. Docking models with branched substrates reveal that:

• L-configured nucleophiles (either native N-terminal L-Ile1 or pseudo-N-terminal L-Ile1′) are stably retained in the nucleophile binding site during 50 ns MD simulations, with the nucleophile amine positioned approximately 4.5 Ã… from the C-terminal carbonyl carbon—an optimal distance for catalysis [1].
• The side chain of L-configured residues is accommodated in a pocket formed by Leu284, Trp288, Met293, His295, and Asp306, with the nucleophile amine hydrogen-bonded to Tyr154, which is thought to abstract a proton during catalysis [1].
• In contrast, D-configured nucleophiles exhibit substantial positional variability in simulations, with the distance between the nucleophilic amine and C-terminal carbonyl carbon exceeding 10 Å—accounting for their unfavorable binding and inability to participate in cyclization [1].

These computational findings align precisely with the observed regiospecificity of SurE and explain how stereochemical configuration dictates nucleophile selection in the cyclization process.

Diagram 2: Stereochemical Recognition in Nucleophile Binding Site. This diagram illustrates the molecular basis for SurE's strict preference for L-configured nucleophiles, governed by specific binding pocket interactions.

The integrated one-pot tandem synthesis methodology represents a significant advancement in chemoenzymatic strategies for complex natural product-inspired therapeutics. By synergistically combining the precise regio- and stereoselectivity of enzymatic macrocyclization with the versatile modularity of chemical Ser/Thr ligation, this approach effectively addresses the long-standing synthetic challenges associated with lariat lipopeptides [1] [3].

The strategic implementation of stereochemical control through substrate engineering, rather than extensive enzyme engineering, provides a generalizable framework for repurposing existing biocatalysts for new synthetic applications. The resulting platform enables efficient exploration of chemical space around privileged lariat lipopeptide scaffolds, as demonstrated by the identification of novel analogs with promising antimycobacterial activity (MIC₅₀ 8-16 µg/ml) [1].

This methodology holds substantial potential for broader application in peptide-based drug discovery, particularly for targeting the rapidly expanding threat of antimicrobial resistance. The principles established—substrate-directed control of enzyme specificity, one-pot tandem reactions, and strategic integration of enzymatic and chemical transformations—offer a paradigm that can be extended to other classes of modified peptides and natural product-inspired therapeutics.

Maximizing Yield and Fidelity in Chemoenzymatic Platforms

Strategies for Optimizing Cyclization Regioselectivity and Minimizing Hydrolysis

The biosynthesis of lariat lipopeptides by non-ribosomal peptide synthetases (NRPSs) represents a fascinating area of natural product research with significant pharmaceutical implications. These complex molecules, characterized by their macrocyclic "head" and linear lipid "tail," include clinically important antibiotics such as daptomycin and colistin. However, their structural complexity poses substantial synthetic challenges, particularly in achieving precise cyclization regioselectivity while minimizing competing hydrolytic reactions. This technical guide examines recent advances in chemoenzymatic approaches that address these challenges, providing researchers with practical strategies to optimize the biosynthesis of these valuable compounds.

Core Challenges in Lariat Peptide Synthesis

The efficient exploration of lariat lipopeptide chemical space has been historically hampered by molecular complexities that present significant synthetic obstacles [1].

• Regioselective Ring Construction: Traditional chemical synthesis requires orthogonal protecting group strategies and stoichiometric coupling reagents, often necessitating dilute conditions to suppress intermolecular coupling with substantial organic solvent use [1].
• Competing Hydrolytic Reactions: When using natural biosynthetic machinery, substantial flux through hydrolytic pathways typically results in low yields of cyclic products [1].
• Substrate Specificity Limitations: Naturally occurring lariat-forming thioesterases (TEs) are generally sensitive to changes in the local environment of nucleophilic residues, including position, stereochemistry, and nucleophilicity, limiting their application for diverse lariat peptide production [1].

Enzymatic Machinery for Peptide Cyclization

Understanding the enzymatic components is crucial for optimizing cyclization strategies. Non-ribosomal peptide synthetases employ specific domains and enzymes for cyclization functions.

Key Cyclization Enzymes

Table 1: Key Enzymes for Peptide Cyclization

Enzyme	Type	Natural Function	Catalytic Features
SurE	Penicillin-binding protein-type thioesterase	Macrocyclization in surugamide biosynthesis	Broad substrate tolerance; strictly accepts L-configured residues as nucleophiles [1] [3]
WolJ	Penicillin-binding protein-type thioesterase	Macrocyclization in natural product biosynthesis	Similar stereospecificity to SurE; useful for diverse cyclization patterns [1] [3]
TycC-TE	Type-I thioesterase	Cyclization in tyrocidine biosynthesis	Head-to-tail cyclization of decapeptidyl substrate; tolerates diverse sequences and lengths [1]
Type-I TEs	α/β-hydrolase fold enzymes	Lariat formation in NRPS pathways	Typically cyclize using side chain nucleophiles; often sensitive to environmental changes [1]

Structural Basis of Catalysis

The NRPS synthetic machinery employs a modular architecture where carrier proteins shuttle intermediates between catalytic domains [5]. The final cyclization step is typically catalyzed by thioesterase (TE) domains present in termination modules, which cleave the peptide from the terminal peptidyl carrier protein (PCP) domain and catalyze release via hydrolysis or macrocyclization [22].

The PCP domains are relatively small (70-90 amino acids) and feature a conserved serine residue that serves as the site for covalent modification with a phosphopantetheine cofactor, essential for shuttling substrates and intermediates between catalytic domains [5]. Understanding these structural relationships is key to engineering improved cyclization systems.

Strategic Approaches to Regioselectivity Optimization

Substrate Engineering with Pseudo-N-Termini

A groundbreaking approach to controlling cyclization regioselectivity involves engineering the shape of substrates rather than the enzymes themselves [1] [3]. By introducing an internal dipeptide unit as a "pseudo-N terminus" where L-Ile is attached to the side chain of L-Lys via an isopeptide bond, researchers created branched substrates with two potential nucleophiles for cyclization [1].

Table 2: Strategic Approaches to Optimize Cyclization Regioselectivity

Strategy	Mechanism	Advantages	Experimental Outcome
Pseudo-N-Terminus Installation	Introduces internal dipeptide unit with additional nucleophile	Provides alternative cyclization site; bypasses enzyme engineering	Generated both head-to-tail (60%) and lariat (40%) cyclic peptides from same substrate [1]
Stereochemical Switching	Replaces native N-terminal L-residue with D-configured amino acid	Suppresses head-to-tail pathway; forces side-chain cyclization	Achieved quantitative lariat formation by using D-Val at native N-terminus [1] [3]
Enzyme Selection	Utilizes PBP-type TEs with strict L-nucleophile specificity	Exploits inherent enzymatic stereoselectivity	SurE, WolJ, and TycC-TE all successfully produced lariat macrocycles [1]
Positional Scanning	Systematically varies pseudo-N-terminus position along peptide chain	Identifies optimal cyclization sites; maps enzyme tolerance	Demonstrated SurE tolerance for different pseudo-N terminus positions [1]

The following diagram illustrates the strategic workflow for optimizing cyclization regioselectivity through substrate engineering and stereochemical control:

Stereochemical Control of Nucleophile Selection

The stereospecificity of PBP-type thioesterases provides a powerful tool for controlling regioselectivity. These enzymes exhibit relaxed specificity toward the N-terminal nucleophile but strictly recognize its stereochemical configuration, accepting only L-configured residues as nucleophiles, not D-configured residues [1]. By replacing the native N-terminal L-Ile1 with D-Val, researchers successfully suppressed head-to-tail cyclization, forcing the enzyme to use exclusively the pseudo-N-terminal L-Ile1' as the nucleophile [1].

Molecular dynamics simulations provided mechanistic insights into this stereochemical control, showing that L-configured nucleophiles are retained in the nucleophile binding site with appropriate geometry for catalysis, while D-configured nucleophiles exhibit positional variability with unfavorable distances for cyclization [1].

Methodologies for Minimizing Competing Hydrolysis

Chemoenzymatic Substrate Design

A key innovation in minimizing hydrolytic side reactions is the chemoenzymatic approach using ethylene glycol (EG)-functionalized substrates [1]. This strategy simplifies substrate synthesis while maintaining high enzymatic conversion:

• Solid-Phase Peptide Synthesis: Peptide main chains are synthesized on solid support functionalized with ethylene glycol before SPPS [1].
• Orthogonal Deprotection: The 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) protecting group on the L-Lys side chain is removed with 2% hydrazine [1].
• Pseudo-N-Terminus Installation: The pseudo-N-terminal L-Ile1' is installed on the side chain of L-Lys in an additional coupling round [1].
• Global Deprotection: Resin cleavage and concomitant global deprotection generates EG-functionalized branched peptides [1].

This approach demonstrated significant advantages over traditional chemical synthesis, with the EG substrate obtained in 83% yield over 17 steps followed by quantitative cyclization using SurE, compared to 33% yield over 19 steps for direct chemical synthesis of the cyclic product [1].

Tandem Cyclization-Acylation Strategy

To further streamline production and minimize purification losses, researchers developed a one-pot tandem cyclization-acylation strategy [1] [3]:

• Enzymatic Macrocyclization: First, the branched peptide substrate is cyclized using SurE, WolJ, or TycC-TE.
• In Situ Acylation: Without intermediate purification, the free N-terminus not used in cyclization serves as a reactive handle for site-selective serine/threonine ligation (STL).
• Lipid Tail Installation: Various acyl groups are appended to create the characteristic lariat lipopeptide structure.

This sequential one-pot process eliminates intermediate purification and enables efficient parallel synthesis of diverse lariat lipopeptides [3]. Biological screening of compounds generated through this method identified lipopeptides that inhibit 50% of Mycobacterium intracellulare growth at concentrations of 8-16 µg ml⁻¹ [1].

Experimental Protocols

Representative Procedure for Lariat Peptide Synthesis

Materials:

• EG-functionalized resin for solid-phase peptide synthesis
• Fmoc-protected amino acids, including Fmoc-L-Lys(Dde)-OH
• SurE, WolJ, or TycC-TE enzyme (5 mol%)
• Reaction buffer: 50 mM Tris-HCl, pH 7.5, 10 mM MgClâ‚‚
• Acylation reagents for serine/threonine ligation

Method:

• Solid-Phase Peptide Synthesis: Synthesize the linear peptide sequence on EG-functionalized resin using standard Fmoc SPPS protocols.
• Dde Deprotection: Treat the resin-bound peptide with 2% hydrazine in DMF (3 × 10 min) to remove the Dde protecting group from lysine.
• Pseudo-N-Terminus Installation: Couple Fmoc-L-Ile-OH to the exposed side-chain amine using standard coupling reagents, then remove Fmoc to generate the free pseudo-N-terminus.
• Cleavage and Global Deprotection: Cleave peptide from resin using TFA cocktail with appropriate scavengers.
• Enzymatic Cyclization: Incubate EG-functionalized branched peptide (1 mM) with 5 mol% SurE at 30°C for 3 hours in reaction buffer.
• One-Pot Acylation: Without purification, add serine/threonine ligation components to install lipid tail directly to the cyclic peptide intermediate.
• Purification and Analysis: Purify by reversed-phase HPLC and characterize by LC-MS/MS.

Key Research Reagent Solutions

Table 3: Essential Research Reagents for Chemoenzymatic Lariat Peptide Synthesis

Reagent/Enzyme	Function	Application Notes
SurE Macrocyclase	PBP-type thioesterase for peptide cyclization	Use at 5 mol%; broad substrate tolerance; quantitative conversion in 3h at 30°C [1]
EG-Functionalized Resin	Solid support with ethylene glycol leaving group	Simplifies substrate synthesis; improves enzymatic conversion [1]
Fmoc-L-Lys(Dde)-OH	Orthogonally protected amino acid building block	Enables site-specific introduction of pseudo-N-terminus after Dde removal [1]
Serine/Threonine Ligation Reagents	Site-selective acylation components	Enables one-pot lipidation after cyclization without intermediate purification [1]
Hydrazine Solution (2%)	Selective Dde protecting group removal	Efficiently deprotects lysine side chain for pseudo-N-terminus installation [1]

The strategic optimization of cyclization regioselectivity and minimization of hydrolysis in lariat lipopeptide biosynthesis represents a significant advancement in natural product synthesis. By leveraging substrate engineering, stereochemical control, and tandem chemoenzymatic strategies, researchers can now access complex lariat architectures with unprecedented efficiency. These approaches demonstrate the power of combining enzymatic precision with synthetic flexibility to overcome long-standing challenges in peptide macrocyclization. The resulting methodologies not only enable fundamental studies of lariat lipopeptide biosynthesis but also provide practical tools for drug discovery efforts targeting increasingly resistant pathogenic microorganisms.

The biosynthesis of lariat lipopeptides by non-ribosomal peptide synthetases (NRPS) represents a fascinating area of natural product research with significant implications for drug development. These complex molecules, characterized by their macrocyclic "head" and linear lipid "tail," include clinically important antibiotics such as daptomycin and colistin [1] [3]. Their unique lariat topology enables diverse modes of action, primarily targeting bacterial cell surfaces, making them valuable scaffolds for combating drug-resistant pathogens [1]. However, the efficient exploration of this rich chemical space has been hampered by substantial synthetic challenges, particularly in achieving regioselective macrocyclization [1].

Central to this biosynthetic process are the thioesterase (TE) domains that catalyze the crucial cyclization step. Traditionally, lariat-forming TEs have exhibited narrow substrate specificity, limiting their utility for generating structural diversity [23]. This technical guide examines a groundbreaking chemoenzymatic strategy that overcomes these limitations by repurposing versatile non-ribosomal peptide cyclases—specifically penicillin-binding protein-type thioesterases (PBP-type TEs) and type-I thioesterases—to achieve controlled synthesis of lariat peptides with varied sequences, ring sizes, and nucleophile configurations [1].

Substrate Design: Engineering Cyclization Control

Fundamental Principles of Substrate Recognition

Non-ribosomal peptide cyclases recognize linear peptide precursors and catalyze their cyclization through a serine protease-like mechanism involving a Ser/His/Asp catalytic triad [23]. The cyclization proceeds via a two-step process: first, the linear peptide intermediate is transferred onto the catalytic serine to form a peptide-O-TE complex, followed by intramolecular nucleophilic attack to achieve cyclization and product release [23]. The key innovation in navigating substrate scope involves engineering the peptide substrate itself to control which nucleophile participates in the ring-closing reaction.

PBP-type TEs, such as SurE and WolJ, exhibit remarkable stereochemical specificity—they strictly accept only L-configured residues as nucleophiles while rejecting D-configured ones [1]. This intrinsic selectivity provides a powerful "stereochemical switch" for controlling cyclization regioselectivity. Additionally, these enzymes demonstrate broad tolerance for variations in peptide sequence and length, making them ideal biocatalytic platforms [1] [23].

The Pseudo-N-Terminus Strategy

A breakthrough in substrate design came with the introduction of an internal "pseudo-N-terminus"—a dipeptide unit featuring an additional N-terminus within the peptide side chain [1] [3]. This innovative approach transforms a head-to-tail cyclase into a lariat-forming enzyme through strategic substrate engineering rather than protein engineering.

In practice, this involves synthesizing branched peptides where an L-amino acid (e.g., L-Ile) is attached to the side chain of an L-Lys residue via an isopeptide bond [1]. The resulting substrate contains two potential L-configured nucleophiles: the native N-terminus and the pseudo-N-terminus. When both nucleophiles possess the correct L-configuration, SurE produces a mixture of head-to-tail and lariat-shaped cyclic peptides in comparable amounts (60% vs. 40%), demonstrating that the pseudo-N-terminus serves as an equally effective nucleophile for cyclization [1].

Table 1: Key Enzymes in Lariat Peptide Synthesis and Their Substrate Preferences

Enzyme	Class	Native Function	Nucleophile Preference	Key Substrate Features
SurE	PBP-type TE	Head-to-tail cyclization	Strictly L-configured α-amine	Tolerant of sequence, length, and nucleophile position
WolJ	PBP-type TE	Head-to-tail cyclization	Strictly L-configured α-amine	Broad substrate tolerance similar to SurE
TycC-TE	Type-I TE	Head-to-tail cyclization	Aromatic D-aa at N-terminus, L-Orn at position 9	Accepts substrates with ring sizes 6-14

Sequence Tolerance: Expanding Structural Diversity

Positional Scanning of the Pseudo-N-Terminus

The tolerance of SurE for different pseudo-N-terminus positions was systematically investigated through comprehensive positional scanning experiments [1]. Researchers synthesized a series of branched ethylene glycol (EG)-functionalized substrates where the L-Ile₁′-L-Lys dipeptidyl unit was sequentially moved through different positions in the peptide sequence (substrates 6-11) [1]. This approach allowed for methodical evaluation of how the position of the branching unit affects cyclization efficiency and regioselectivity.

The results demonstrated that SurE maintains significant catalytic activity across various positions of the pseudo-N-terminus, though the efficiency varied depending on the local structural environment. Molecular dynamics simulations provided mechanistic insights, revealing that the nucleophile binding site of SurE accommodates the L-configured pseudo-N-terminal residue through a pocket formed by Leu284, Trp288, Met293, His295, and Asp306, with the nucleophile amine hydrogen-bonded to Tyr154, which is thought to abstract a proton during catalysis [1]. This well-defined binding environment explains the strict stereochemical preference while allowing flexibility in nucleophile positioning.

Enzymatic Generality Across Cyclase Families

The generality of this substrate engineering strategy extends beyond a single enzyme class. The approach has been successfully demonstrated using two distinct PBP-type thioesterases (SurE and WolJ) as well as a type-I thioesterase (TycC thioesterase) [1] [24]. Each enzyme could be repurposed for lariat peptide formation through the same fundamental substrate design principles, without requiring protein engineering [3].

TycC-TE, initially characterized for its role in tyrocidine biosynthesis, exemplifies the remarkable substrate promiscuity of some NRPS cyclases [23]. This enzyme accepts substrates with ring sizes ranging from 6 to 14 amino acids and tolerates diverse structural modifications, including the incorporation of non-proteinogenic amino acids, polyketide-like building blocks, and even RGD sequences to produce integrin-binding macrocycles [23]. Such broad tolerance provides ample opportunity for generating structural diversity in lariat peptide scaffolds.

Ring Size Flexibility: Macrocyclization Scope

The ring size of macrocyclic peptides significantly influences their biological activity, membrane permeability, and metabolic stability [23]. Understanding the flexibility of NRPS cyclases toward different ring sizes is therefore crucial for rational design.

Table 2: Ring Size Tolerance of Non-Ribosomal Peptide Cyclases

Enzyme	Minimal Ring Size	Maximal Ring Size	Optimal Range	Notes
TycC-TE	6 residues	14 residues	8-10 residues	Broad flexibility demonstrated with non-native substrates
SurE	Not specified	Not specified	Variable	Tolerant of different ring sizes when pseudo-N-terminus is repositioned
Lariat-forming TEs	Variable	Variable	Specific to native substrate	Generally narrow ring size tolerance

The ring size in the lariat peptide synthesis approach is determined by the distance between the C-terminal thioester and the pseudo-N-terminal nucleophile [1]. By repositioning the L-Ile₁′-L-Lys dipeptidyl unit along the peptide backbone, researchers can effectively vary the ring size of the resulting macrocycle while maintaining the lariat topology. This strategy provides exceptional control over macrocycle dimensions, enabling systematic structure-activity relationship studies.

Traditional lariat-forming TEs typically exhibit limited tolerance for ring size variations, as they have evolved to process specific native substrates [23]. In contrast, the repurposed head-to-tail cyclases like SurE and TycC-TE show remarkably broad flexibility, accommodating significant variations in the distance between the reacting functional groups [1] [23].

Nucleophile Specificity and Stereochemical Control

Stereochemical Gating Mechanism

The strategic replacement of the native N-terminal residue with its D-configured counterpart effectively blocks the head-to-tail cyclization pathway, forcing the enzyme to exclusively use the pseudo-N-terminus as the nucleophile [1]. This "stereochemical switching" approach enables complete control over cyclization regioselectivity.

Molecular dynamics simulations of SurE complexed with substrates containing either L or D-configured nucleophiles provide structural insights into this stereochemical gating mechanism [1]. When an L-Ile nucleophile is positioned in the nucleophile binding site, it remains stably bound throughout 50 ns simulations, with the amine group maintaining an average distance of 4.5 Å from the C-terminal carbonyl carbon—ideal for nucleophilic attack [1]. In contrast, a D-allo-Ile nucleophile displays significant positional variability and maintains a much longer distance (>10 Å) from the electrophilic carbon, explaining the dramatically reduced reactivity of D-configured nucleophiles [1].

Beyond α-Amine Nucleophiles

While the engineered lariat synthesis primarily utilizes α-amine nucleophiles, natural TE domains demonstrate remarkable versatility in nucleophile recognition. Native TEs can utilize diverse nucleophiles including:

• N-terminal Nα-amine groups (head-to-tail cyclization)
• β-OH of an N-terminal acyl group
• Nω-amine of basic amino acids (e.g., Lys, Orn)
• β-OH of Ser/Thr/β-OH Phe
• Phenolic hydroxy of Tyr
• Thiol of Cys [23]

This diverse nucleophile utilization in natural systems suggests potential for further expanding the scope of engineered lariat peptides by incorporating alternative nucleophiles beyond the pseudo-N-terminal α-amine.

Experimental Protocols: Methodological Framework

Substrate Synthesis and Preparation

The synthesis of branched peptide substrates for enzymatic cyclization involves several key steps:

• Solid-Phase Peptide Synthesis (SPPS): Linear peptide sequences are assembled on a solid support functionalized with ethylene glycol (EG) to simplify the subsequent enzymatic step [1]. The EG serves as a biomimetic surrogate for the natural pantetheine leaving group [1].

• Orthogonal Protection Strategy: An orthogonal protecting group (1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl, Dde) is used on the L-Lys side chain where the pseudo-N-terminus will be installed [1].

• Branch Point Installation: After linear assembly, the Dde group is selectively removed using 2% hydrazine, and the pseudo-N-terminal amino acid (e.g., L-Ile) is coupled to the side chain of L-Lys in an additional round of coupling [1].

• Global Deprotection and Cleavage: The branched peptide is cleaved from the resin with concomitant global deprotection, yielding the EG-functionalized branched peptide ready for enzymatic cyclization [1].

This synthetic approach yields the substrate in 83% yield over 17 steps, compared to 33% yield over 19 steps for direct chemical synthesis of the lariat peptide, highlighting the efficiency of the chemoenzymatic approach [1].

Enzymatic Cyclization Assay

The standard enzymatic cyclization protocol involves:

• Reaction Conditions: Incubate the branched EG-functionalized peptide substrate (typically 0.5-1 mM) with 5 mol% SurE (or other cyclase) at 30°C in appropriate buffer [1].

• Reaction Monitoring: Monitor reaction progress by LC-MS until complete consumption of starting material (typically within 3 hours) [1].

• Product Analysis: Identify and quantify cyclic products using tandem mass spectrometry (MS²) to distinguish between head-to-tail and lariat-shaped cyclic peptides based on their fragmentation patterns [1].

• Purification: Isolate cyclic products using preparative HPLC for subsequent characterization and biological testing.

Under these optimized conditions, SurE quantitatively converts the engineered branched substrate into the lariat-shaped cyclic peptide when the native N-terminus is blocked with a D-amino acid [1].

Tandem Cyclization-Aylation Protocol

The one-pot cyclization-acylation procedure enables efficient synthesis of mature lariat lipopeptides:

• Cyclization Step: Perform enzymatic cyclization as described above.

• Direct Acylation: Without intermediate purification, add the acyl donor and Ser/Thr ligation reagents directly to the completed cyclization reaction mixture [1].

• Site-Selective Acylation: The remaining free N-terminus (not used in cyclization) serves as a reactive handle for site-selective acylation via Ser/Thr ligation chemistry [1].

• Library Synthesis: This tandem approach enables parallel synthesis of diverse lariat lipopeptides equipped with various acyl groups for biological screening [1].

This streamlined protocol eliminates the need for intermediate purification, significantly accelerating the synthesis and diversification of lariat lipopeptide libraries.

Research Toolkit: Essential Reagents and Materials

Table 3: Key Research Reagents for Chemoenzymatic Lariat Peptide Synthesis

Reagent/Enzyme	Function	Application Notes
SurE	PBP-type thioesterase cyclase	5 mol% loading; quantitative cyclization of engineered substrates
WolJ	PBP-type thioesterase cyclase	Alternative to SurE with similar substrate scope
TycC-TE	Type-I thioesterase cyclase	Broad substrate tolerance; requires aromatic D-aa at N-terminus
EG-Functionalized Resin	Solid support for SPPS	Simplifies substrate synthesis and enzymatic cyclization
Dde Protecting Group	Orthogonal protection for Lys side chain	Allows selective deprotection for branch point installation
Hydrazine Solution (2%)	Dde deprotection	Selective removal of Dde group without affecting other protections
Ser/Thr Ligation Reagents	Site-selective acylation	Functionalizes free N-terminus with various acyl groups

Conceptual Workflow and Experimental Design

The following diagram illustrates the strategic workflow for controlling cyclization mode through substrate engineering:

Diagram 1: Substrate Engineering Workflow for Lariat Peptide Synthesis

The strategic navigation of substrate scope for non-ribosomal peptide cyclases has unlocked powerful synthetic capabilities for lariat lipopeptide biosynthesis. Through deliberate substrate engineering—incorporating pseudo-N-termini and leveraging stereochemical control—researchers can now repurpose naturally occurring head-to-tail cyclases for efficient lariat peptide synthesis. This approach successfully addresses longstanding challenges in regioselective macrocyclization while providing exceptional control over sequence, ring size, and nucleophile specificity.

The experimental protocols outlined in this guide provide a robust framework for designing and executing chemoenzymatic synthesis of diverse lariat peptides. The integration of enzymatic cyclization with subsequent site-selective acylation in a one-pot tandem process represents a particularly efficient strategy for generating lipopeptide libraries for drug discovery. As antibiotic resistance continues to pose serious threats to global health, these methodologies offer valuable tools for developing new therapeutic candidates with novel mechanisms of action.

Future directions in this field will likely focus on expanding the toolbox of promiscuous cyclases, engineering enzymes with altered specificity, and incorporating non-canonical amino acids to further diversify the accessible chemical space of lariat lipopeptides. The continued integration of chemical synthesis with enzymatic catalysis holds exceptional promise for advancing both fundamental understanding of NRPS biology and practical applications in therapeutic development.

The strategic pairing of non-ribosomal peptide cyclases with engineered peptide substrates is a pivotal advancement in the biosynthesis of complex lariat lipopeptides. This guide synthesizes recent breakthroughs in chemoenzymatic synthesis, providing a structured framework for selecting cyclases based on regiospecificity, stereochemical preference, and substrate tolerance. We present comparative enzymatic profiles, detailed experimental protocols for cyclization and one-pot diversification, and practical tools to enable researchers to efficiently generate lariat lipopeptide libraries for antimicrobial discovery and optimization.

Lariat lipopeptides, characterized by their macrocyclic "head" and linear lipophilic "tail," represent a critically important class of antimicrobial agents, with daptomycin and colistin serving as prime examples [1] [3]. Their complex topologies, however, present significant synthetic challenges that impede efficient structural diversification and drug development. Traditional chemical synthesis of these molecules often requires orthogonal protecting group strategies and stoichiometric coupling reagents, leading to substantial solvent waste and low efficiency [1].

The use of non-ribosomal peptide (NRP) cyclases offers a powerful biocatalytic alternative, enabling selective cyclization under mild conditions. Historically, the application of lariat-forming type-I thioesterases (TEs) has been limited by their narrow substrate scope and significant competing hydrolysis [1]. Recent research has successfully repurposed versatile head-to-tail NRP cyclases for lariat peptide synthesis not by re-engineering the enzymes themselves, but by strategically redesigning the peptide substrates [1] [3]. This paradigm shift enables researchers to access diverse lariat architectures using readily available enzymatic tools, dramatically accelerating the exploration of this valuable chemical space for drug discovery.

Cyclase Families and Their Selection Criteria

Key Cyclase Families and Their Characteristics

Three primary families of non-ribosomal peptide cyclases have demonstrated utility in the synthesis of lariat lipopeptides. Understanding their distinct mechanisms and preferences is the first step in rational enzyme-substrate pairing.

Table 1: Key Families of Non-Ribosomal Peptide Cyclases

Cyclase Family	Representative Enzymes	Native Function	Key Feature for Lariat Synthesis
Penicillin-Binding Protein (PBP)-Type Thioesterases	SurE, WolJ	Head-to-tail macrocyclization	Relaxed specificity for the N-terminal nucleophile, but strict recognition of its l-configured stereochemistry [1].
Type-I Thioesterases (TEs)	TycC Thioesterase	Head-to-tail macrocyclization	Broad substrate tolerance for sequence and length variations; can be redirected for lariat formation [1] [25].
Lariat-Forming Type-I TEs	(e.g., in daptomycin biosynthesis)	Side chain-to-tail macrocyclization	Naturally produces lariat structures; often exhibits narrow substrate specificity and significant hydrolytic side reactions, limiting general utility [1].

Strategic Selection: Matching Cyclases to Synthetic Goals

Selecting the optimal cyclase depends on the desired lariat structure and the sequence of the linear precursor. The following decision pathway outlines a logical selection process.

The fundamental principle for achieving exclusive lariat cyclization lies in controlling the stereochemistry of potential nucleophiles. PBP-type TEs, such as SurE, strictly accept only l-configured residues as nucleophiles [1]. By incorporating a d-configured amino acid at the native N-terminus, this nucleophile is rendered inactive, forcing the enzyme to utilize the pseudo-N-terminal l-Ile1' for cyclization. This stereochemical switching enables quantitative yield of the lariat product without competitive head-to-tail cyclization [1].

Experimental Protocol for Chemoenzymatic Synthesis

Substrate Design and Synthesis

1. Design Branched Peptide Substrate:

• Solid-Phase Peptide Synthesis (SPPS): Synthesize the linear peptide backbone on a solid support functionalized with an ethylene glycol (EG) leaving group, which simplifies substrate synthesis and streamlines the process [1].
• Incorporation of a Pseudo-N-Terminus: Incorporate an l-Lysine residue at the intended cyclization branch point, protecting its side chain with an orthogonal 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) protecting group.
• Installation of Stereochemical Control: Incorporate a d-configured amino acid (e.g., d-Val) at the true N-terminus to suppress head-to-tail cyclization.
• Branching: After backbone assembly, remove the Dde protecting group with 2% hydrazine. Couple the pseudo-N-terminal dipeptide unit (e.g., l-Ile1') to the side chain of the l-Lys residue, creating an isopeptide bond [1].
• Cleavage: Cleave the branched peptide from the resin with concomitant global deprotection to obtain the EG-functionalized substrate.

2. Key Considerations:

• Nucleophile Positioning: The pseudo-N terminus can be scanned along the peptide sequence. Research indicates SurE tolerates the l-Ile1'-l-Lys dipeptidyl unit at various positions, enabling the synthesis of lariat peptides with diverse ring sizes [1].
• Leaving Group: The C-terminal EG group acts as a superior surrogate for the natural pantetheine leaving group, enhancing the efficiency of the enzymatic cyclization [1] [3].

Enzymatic Cyclization and Diversification

The subsequent steps transform the linear branched peptide into a bioactive lariat lipopeptide.

1. Macrocyclization:

• Reaction Setup: Incubate the EG-functionalized branched peptide substrate (e.g., 0.5-1 mM) with the selected cyclase (e.g., 5 mol% SurE) in a suitable buffer (e.g., HEPES or Tris at pH 7.0-8.0) at 30°C [1].
• Reaction Monitoring: Monitor the reaction by LC-MS. The cyclization is typically complete within 3 hours, quantitatively yielding the lariat macrocycle when the stereochemical switch is applied [1].

2. One-Pot Tandem Acylation (Cyclization–Ser/Thr Ligation):

• Direct Diversification: Following complete cyclization, without intermediate purification, add the desired acyl donor (e.g., a long-chain fatty acid) and reagents for Ser/Thr ligation (STL) to the same reaction vessel.
• Site-Selective Acylation: The STL reaction targets the remaining free N-terminus (the one not used in cyclization), enabling site-selective attachment of various acyl chains [1] [3].
• Workflow Advantage: This one-pot tandem strategy enables the modular synthesis of a diverse library of lariat lipopeptides, which can be directly screened for biological activity without laborious purification steps [3].

The Scientist's Toolkit: Essential Reagents and Materials

Table 2: Key Research Reagent Solutions for Chemoenzymatic Lariat Synthesis

Reagent / Material	Function / Description	Application Note
PBP-Type TE (SurE, WolJ)	Versatile biocatalyst for macrocyclization; accepts l-configured nucleophiles.	SurE demonstrates high tolerance for variations in pseudo-N terminus position and ring size [1].
Ethylene Glycol (EG) Functionalized Resin	Solid support for SPPS, providing a C-terminal EG leaving group.	Simplifies substrate synthesis and acts as an efficient leaving group for cyclization [1] [3].
Dde Protecting Group	Orthogonal protecting group for lysine side chain amines.	Allows for selective deprotection and coupling of the pseudo-N-terminal dipeptide unit during SPPS [1].
d-Amino Acids	Stereochemical control agents.	Incorporation at the native N-terminus (e.g., d-Val1) blocks head-to-tail cyclization, directing reaction to lariat formation [1].
Ser/Thr Ligation (STL) Reagents	Enables site-selective acylation of free serine/threonine N-termini.	Used in one-pot tandem with cyclization to attach diverse lipid tails, creating functional lipopeptides [3].

The repurposing of versatile NRP cyclases through strategic substrate design represents a transformative approach to lariat lipopeptide synthesis. The guidelines presented herein—centered on enzyme characteristics, stereochemical control, and modular one-pot protocols—provide researchers with a robust framework for generating diverse libraries of these complex molecules. The ability to directly screen crude reaction mixtures for antimicrobial activity significantly accelerates the hit discovery process, as evidenced by the identification of lariat lipopeptides with promising activity against Mycobacterium intracellulare (MIC: 8–16 µg ml⁻¹) [1]. As the field advances, the integration of these cyclase-based strategies with automated synthesis and screening platforms will undoubtedly unlock further structural and functional diversity, paving the way for new antibiotic classes to combat the rising threat of drug-resistant pathogens.

Overcoming Practical Hurdles in Scalable Synthesis and Product Isolation

Lariat-shaped lipopeptides represent a fascinating class of antimicrobial agents with significant therapeutic potential, exemplified by clinically important compounds such as daptomycin and colistin [1]. These complex molecules are characterized by a unique macrocyclic "head" structure connected to a lipophilic "tail" that is crucial for their biological activity. Their intricate architectures, featuring multiple nucleophilic sites and complex stereochemistry, pose substantial synthetic challenges that have hampered efficient structural diversification and scale-up production [1] [6]. Traditional chemical synthesis of these compounds often requires lengthy routes with numerous protection and deprotection steps, stoichiometric coupling reagents, and dilute conditions to suppress intermolecular side reactions [1]. These limitations become particularly problematic in drug development pipelines where rapid generation of analog libraries is essential for structure-activity relationship studies.

The emergence of chemoenzymatic synthesis utilizing non-ribosomal peptide cyclases offers a promising alternative to conventional approaches [1] [3] [26]. By harnessing nature's biosynthetic machinery, researchers can overcome key hurdles in constructing the complex macrocyclic scaffolds of lariat lipopeptides. However, implementing these strategies at scale introduces new practical challenges in both the synthetic and isolation phases of production. This technical guide examines these hurdles within the context of lariat lipopeptide biosynthesis research and provides detailed methodologies for developing scalable, efficient processes that can accelerate therapeutic discovery.

Core Synthetic Challenges in Lariat Lipopeptide Production

Structural Complexity and Regioselectivity

The defining structural feature of lariat lipopeptides—their macrocyclic head group—presents significant regioselective ring construction challenges. Unlike simple head-to-tail cyclic peptides, lariat structures require precise side-chain macrocyclization, typically through a nucleophilic side chain attacking the C-terminal carbonyl [1] [26]. Linear precursors often contain multiple potential nucleophiles, including the native N-terminus, pseudo-N-terminus in branched systems, and various side-chain functional groups, creating competition between different cyclization pathways [1]. Traditional synthetic approaches require complex orthogonal protecting group strategies to direct cyclization to the desired position, substantially increasing synthetic step count and reducing overall yield.

Table 1: Key Challenges in Lariat Lipopeptide Synthesis

Challenge	Impact on Scalability	Traditional Solution	Limitations
Multiple nucleophilic sites	Low regioselectivity in macrocyclization	Orthogonal protecting groups	Increased steps, reduced yields
Competitive oligomerization	Requires highly dilute conditions	Large solvent volumes	Environmental burden, high cost
Stereochemical requirements	Limited substrate flexibility	Chiral auxiliaries/catalysts	Added complexity and cost
Lipophilic tail incorporation	Inefficient coupling strategies	Stepwise acylation	Poor atom economy

Limitations of Conventional and Biosynthetic Approaches

Traditional chemical synthesis of lariat lipopeptides typically proceeds with yields of approximately 33% over 19 steps for a model compound, highlighting the inefficiency of purely chemical methods [1]. These processes generate substantial solvent waste due to the dilute conditions needed to suppress dimerization and oligomerization during macrocyclization. Furthermore, the introduction of lipid tails often requires additional coupling steps with specialized reagents.

While nature efficiently produces these compounds through non-ribosomal peptide synthetase (NRPS) assembly lines, recreating these multi-enzyme systems in vitro presents substantial practical challenges [26]. Many native thioesterases (TEs) that catalyze lariat formation exhibit narrow substrate specificity, substantial hydrolytic flux (competing hydrolysis versus cyclization), and sensitivity to changes in the local environment of the nucleophilic residue [1] [26]. Some TEs are inactive with conventional leaving groups like N-acetylcysteamine (SNAC) and require more complex peptidyl-carrier protein (PCP)-loaded substrates, complicating synthetic efforts [26].

Chemoenzymatic Strategies for Scalable Synthesis

Repurposing Head-to-Tail Cyclases for Lariat Synthesis

A groundbreaking approach to overcoming synthetic hurdles involves repurposing versatile non-ribosomal peptide cyclases that normally catalyze head-to-tail macrocyclization. Research led by Kobayashi et al. demonstrated that enzymes such as SurE and WolJ (penicillin-binding protein-type thioesterases) and TycC thioesterase can be redirected to form lariat structures through strategic substrate design rather than protein engineering [1] [3].

The key innovation involves introducing a "pseudo-N-terminus"—a dipeptide unit featuring an additional N-terminus within the peptide side chain—creating two possible sites for cyclization [1]. When presented with a branched octapeptide substrate bearing this pseudo-N-terminus, SurE produced both conventional head-to-tail cyclic peptides (60%) and lariat-shaped cyclic peptides (40%) [1]. This demonstrates that a head-to-tail cyclase can be effectively repurposed to construct lariat peptides through thoughtful substrate design.

Stereochemical Control for Regioselective Cyclization

To exclusively favor lariat formation, researchers have implemented a stereochemical switching strategy. Penicillin-binding protein-type TEs exhibit strict stereospecificity, accepting only L-configured residues as nucleophiles while rejecting D-configured residues [1] [3]. By replacing the native N-terminal residue with its mirror-image D-amino acid, the head-to-tail cyclization pathway is effectively blocked, forcing the enzyme to use the pseudo-N-terminus as the nucleophile [1]. This approach enabled quantitative conversion to the lariat-shaped cyclic peptide with complete regioselectivity, despite the presence of three different potential nucleophiles in the substrate [1].

Table 2: Quantitative Comparison of Synthesis Approaches

Method	Number of Steps	Overall Yield	Key Advantages	Reported Yield Data
Traditional Chemical Synthesis	19 steps	~33%	Full control over analog design	33% yield over 19 steps [1]
Chemoenzymatic with SurE	17 steps (substrate) + 1 enzymatic	~83% (substrate) + quantitative cyclization	High efficiency in macrocyclization	83% yield for EG substrate 4 in 17 steps; quantitative cyclization with SurE [1]
Tandem Cyclization-Acylation	Combined one-pot process	Not specified	Modular lipopeptide assembly	Enabled 51-member library creation [3]

Experimental Protocol: Enzymatic Macrocyclization

Materials: Branched peptide substrate with ethylene glycol (EG) leaving group at C-terminus; SurE, WolJ, or TycC-TE enzyme (5 mol%); appropriate buffer (e.g., Tris-HCl or phosphate buffer, pH 7.5-8.5).

Procedure:

• Substrate Design: Synthesize branched peptide substrate on solid support functionalized with EG. Incorporate orthogonal protecting group (e.g., Dde) on Lys side chain. After SPPS, remove Dde with 2% hydrazine and install pseudo-N-terminal amino acid (e.g., L-Ile) on Lys side chain. Cleave from resin with global deprotection to obtain EG-functionalized branched peptide [1].
• Enzyme Preparation: Express and purify recombinant non-ribosomal peptide cyclase (SurE, WolJ, or TycC-TE) using standard protein expression systems [1] [3].
• Macrocyclization Reaction: Prepare reaction mixture containing branched peptide substrate (0.1-1 mM) and cyclase enzyme (5 mol%) in appropriate buffer. Incubate at 30°C for 3 hours with gentle agitation [1].
• Reaction Monitoring: Monitor reaction progress by LC-MS. Typical conversion is complete within 3 hours, with quantitative formation of lariat product when using stereochemically controlled substrates [1].
• Product Isolation: After complete conversion, purify lariat peptide using preparative HPLC. Yields are typically quantitative for stereochemically controlled substrates [1].

Critical Parameters: Maintain strict stereochemical control at N-terminal position (D-configured) to suppress head-to-tail cyclization; ensure pseudo-N-terminal residue is L-configured; optimize leaving group (EG shows excellent efficiency); control reaction pH to minimize hydrolysis.

Diagram: Chemoenzymatic Synthesis Workflow for Lariat Lipopeptides

Tandem Strategies for Efficient Lipopeptide Assembly

One-Pot Cyclization and Acylation

A particular powerful advancement in scalable lariat lipopeptide synthesis is the development of tandem cyclization-acylation strategies that proceed in one pot without intermediate purification [1] [3]. This approach leverages the fact that enzymatic transformations proceed with high selectivity, generating sufficiently pure reaction mixtures for subsequent modification.

After enzymatic macrocyclization, the remaining nucleophile not involved in cyclization serves as a reactive handle for subsequent diversification via site-selective acylation, specifically Ser/Thr ligation (STL) [1] [3]. This tandem approach enables modular synthesis of lariat-shaped lipopeptides equipped with various acyl groups, addressing the critical need for efficient lipid tail incorporation.

Experimental Protocol: One-Pot Tandem Synthesis

Materials: Cyclized lariat peptide (from previous protocol); acyl donor for Ser/Thr ligation; STL reaction components; appropriate organic co-solvents if needed.

Procedure:

• Cyclization Reaction: Perform enzymatic macrocyclization as described in Section 3.3.
• Direct Acylation: Without purification, add acyl donor (typically 1.5-2 equivalents) and STL reaction components directly to the completed cyclization reaction mixture.
• One-Pot Reaction: Incubate under mild conditions (typically 25-37°C, 2-4 hours) to allow site-selective acylation at the free N-terminus.
• Monitoring: Track reaction progress by LC-MS. The tandem process typically achieves complete conversion to the final lipopeptide.
• Purification: Ispure final lipopeptide using preparative HPLC or other appropriate chromatographic methods.

Critical Parameters: Maintain compatibility between cyclization and acylation conditions; ensure site-selectivity of acylation reaction; optimize stoichiometry of acyl donor; control reaction pH to minimize hydrolysis of labile groups.

This streamlined process enabled the generation of a 51-member library of lariat lipopeptides that could be directly screened for antimicrobial activity, with several compounds showing promising activity against Mycobacterium intracellulare (50% growth inhibition at 8-16 μg mL⁻¹) [3].

Advanced Isolation and Purification Techniques

Modern Chromatographic Methods

Following synthesis, efficient isolation of lariat lipopeptides from reaction mixtures presents its own practical challenges. The amphiphilic nature of these compounds, with both hydrophobic lipid tails and hydrophilic peptide regions, complicates purification. Modern chromatographic techniques have been adapted to address these challenges.

For analytical monitoring and small-scale purification, reversed-phase HPLC with C8 or C18 stationary phases provides excellent resolution [27]. For larger-scale purification, medium-pressure liquid chromatography (MPLC) offers a balance between resolution and throughput [27]. The lipophilic character of these compounds generally necessitates mobile phases containing modifiers such as acetonitrile, methanol, or isopropanol with acidic additives (0.1% formic acid or TFA) to improve peak shape and recovery.

Table 3: Essential Research Reagent Solutions

Reagent/Catalyst	Function	Application Example	Key Characteristics
SurE (PBP-type TE)	Macrocyclization catalyst	Lariat peptide ring formation	Broad substrate tolerance, strict L-nucleophile specificity [1]
TycC thioesterase	Macrocyclization catalyst	Head-to-tail and lariat cyclization	Tolerates diverse sequences and ring sizes [1] [26]
Ethylene Glycol (EG)	Biomimetic leaving group	Simplified substrate synthesis	Efficient enzymatic recognition, simplified synthesis [1]
Solid-Phase Extraction (SPE)	Preliminary purification	Crude extract cleanup	Multiple stationary phases (C8, C18, ion-exchange) [27]

Experimental Protocol: Purification Strategy

Materials: Crude reaction mixture; preparative HPLC system with C8 or C18 column; acetonitrile (HPLC grade); water (HPLC grade); trifluoroacetic acid (TFA); solid-phase extraction cartridges (if needed).

Procedure:

• Initial Capture: Dilute reaction mixture with water (if necessary) and apply to pre-conditioned C18 solid-phase extraction cartridge. Wash with 10-20% acetonitrile/water to remove salts and polar impurities.
• Elution: Elute lipopeptide product with 50-80% acetonitrile/water containing 0.1% TFA.
• Concentration: Evaporate organic solvent under reduced pressure and lyophilize to obtain partially purified product.
• Preparative HPLC: Dissolve sample in minimal DMSO or acetonitrile and inject onto preparative C8 or C18 column. Use gradient elution from 20% to 80% acetonitrile in water (with 0.1% TFA) over 20-30 minutes.
• Fraction Analysis: Monitor at 214-280 nm, collect peak fractions, and analyze by LC-MS for identity and purity.
• Final Isolation: Pool pure fractions, concentrate, and lyophilize to obtain final lipopeptide as TFA salt.

Alternative Methods: For less polar lipopeptides, normal-phase chromatography on silica gel with chloroform/methanol/water mixtures may be effective. Ion-exchange chromatography can help separate closely related analogs with charge differences.

Integration with Modern Extraction and Isolation Platforms

Hybrid Approaches for Streamlined Workflows

The most efficient synthesis and isolation strategies for lariat lipopeptides combine enzymatic biosynthesis with modern extraction technologies. Pressurized liquid extraction (PLE) and microwave-assisted extraction (MAE) offer potential for rapid product recovery from in vitro reaction mixtures or microbial cultures, reducing processing time and solvent consumption compared to traditional methods [28] [27].

Supercritical fluid extraction (SFE), particularly using COâ‚‚ with organic modifiers, shows promise for isolating medium-polarity lipopeptides while minimizing environmental impact [27]. The tunable solvating power of supercritical COâ‚‚ allows selective extraction based on lipid tail characteristics, potentially simplifying purification of analog libraries.

Cell-Free Synthesis Systems

An emerging approach that addresses both synthesis and isolation challenges is cell-free synthesis, which utilizes cellular machinery in a controlled environment without intact cells [29]. This platform offers multiple advantages: rapid evaluation of biosynthetic pathways, simplified product recovery without cell disruption, and the ability to produce compounds that might be toxic to living cells [29]. For lariat lipopeptide production, cell-free systems could allow direct programming of necessary enzymatic pathways while facilitating integration with downstream purification processes.

The practical hurdles in scalable synthesis and isolation of lariat lipopeptides are being systematically addressed through innovative chemoenzymatic strategies. The integration of versatile non-ribosomal peptide cyclases with streamlined tandem reactions and modern purification techniques has created a powerful platform for accessing these complex therapeutic candidates. The stereochemical control strategies, one-pot methodologies, and advanced isolation protocols detailed in this guide provide researchers with practical tools to accelerate their work in this promising area of natural product-based drug discovery.

As these technologies continue to evolve, we anticipate further improvements in process efficiency, including the development of immobilized enzyme systems for catalyst reuse, continuous flow processes to enhance reaction control, and integrated purification systems that minimize manual handling. These advances will undoubtedly expand the accessible chemical space of lariat lipopeptides and accelerate the discovery of next-generation antimicrobial agents to address the growing threat of antibiotic resistance.

Benchmarking Efficiency and Evaluating Therapeutic Potential

The biosynthesis of complex natural products, particularly lariat lipopeptides, represents a frontier in synthetic chemistry where traditional metrics of success are being redefined. This technical guide examines the quantitative superiority of chemoenzymatic approaches leveraging non-ribosomal peptide cyclases over conventional total chemical synthesis. Through detailed analysis of yield, step-count, and synthetic efficiency, we demonstrate that engineered biosynthetic pathways achieve dramatic reductions in synthetic complexity while maintaining or improving access to structurally diverse compounds. The data presented herein provides researchers and drug development professionals with a framework for evaluating synthetic strategies in the context of lariat lipopeptide production, highlighting the transformative potential of biocatalytic methods for accelerating antibiotic discovery.

Lariat lipopeptides constitute an important class of antimicrobial agents characterized by their unique macrocyclic architecture comprising a peptide ring structure with a linear lipid tail. This distinctive lasso-shaped topology is essential for their biological activity, enabling interaction with bacterial cell membranes and intracellular targets. However, this same structural complexity presents significant synthetic challenges that have hampered efficient exploration of their chemical space for drug development [1] [3].

Traditional total chemical synthesis of these compounds requires extensive protecting group strategies, dilute conditions to suppress intermolecular coupling, and stoichiometric coupling reagents, typically resulting in lengthy synthetic sequences with modest overall yields [1]. The emergence of multi-drug resistant bacteria has intensified the need for efficient synthetic methodologies to access novel lariat lipopeptide analogs, pushing researchers toward innovative chemoenzymatic approaches that harness the power of biosynthetic enzymes [30].

Within this context, quantitative comparison of yield and step-count between total synthesis and biosynthetic approaches provides critical insights for strategic decision-making in antimicrobial discovery programs. This whitepaper presents a comprehensive analysis of these metrics, establishing a rigorous framework for evaluating synthetic efficiency in lariat lipopeptide research.

Quantitative Comparison of Synthesis Approaches

Performance Metrics for Lariat Peptide Synthesis

Direct comparison between traditional chemical synthesis and modern chemoenzymatic approaches reveals dramatic differences in synthetic efficiency. The table below summarizes key quantitative metrics for the synthesis of a representative lariat peptide, highlighting the advantages of incorporating non-ribosomal peptide cyclases.

Table 1: Quantitative comparison of synthetic approaches for lariat peptide production

Synthetic Parameter	Traditional Chemical Synthesis	Chemoenzymatic Approach
Total Steps	19 steps	17 steps (substrate preparation) + 1 enzymatic step
Overall Yield	33%	83% (substrate preparation) + quantitative cyclization
Cyclization Yield	Not separately reported	~100%
Key Cyclization Method	Chemical coupling reagents	SurE cyclase catalysis
Selectivity Control	Protecting groups	Enzyme stereospecificity
Structural Diversification	Limited, requires new synthetic route	High, modular substrate design

The data demonstrate that the chemoenzymatic approach achieves superior overall efficiency despite a similar number of synthetic steps, with the key advantage being the quantitative yield in the critical macrocyclization step [1]. This cyclization represents a particular challenge in traditional synthesis due to the potential for oligomerization and the need for high-dilution conditions.

Molecular Complexity Analysis Framework

Beyond simple step-count and yield metrics, the concept of molecular complexity provides a more sophisticated framework for comparing synthetic approaches. Structural complexity can be quantified using parameters such as molecular weight (MW), the fraction of sp³ hybridized carbon atoms (Fsp³), and complexity index (Cm) [31] [32].

Analysis of synthetic pathways in this 3D chemical space reveals that biosynthetic routes typically achieve complexity gains more efficiently than chemical synthesis. In the case of fungal metabolites like sporothriolide, both biosynthetic and chemical routes require 7 steps, but the chemical route involves longer "chemical distances" between intermediates, indicating less efficient complexity generation [31].

Table 2: Molecular complexity parameters for synthetic strategy evaluation

Complexity Parameter	Definition	Application in Synthesis Analysis
Molecular Weight (MW)	Mass of molecule in daltons	Tracks molecular growth through synthetic sequence
Fsp³	Fraction of sp³ hybridized carbon atoms	Measures three-dimensionality
Complexity Index (Cm)	Graph theory-based complexity measurement	Quantifies structural intricacy
Synthetic Step Efficiency	ΔComplexity/step	Measures how efficiently complexity is built

The integration of these quantitative complexity metrics with traditional yield and step-count data provides researchers with a multidimensional assessment framework for evaluating synthetic strategies toward lariat lipopeptides and other complex natural products.

Experimental Protocols for Chemoenzymatic Synthesis

Substrate Design and Preparation

The chemoenzymatic synthesis of lariat lipopeptides begins with rational substrate design incorporating a "pseudo-N-terminus" - a dipeptide unit featuring an additional N-terminus within the peptide side chain. This innovative design enables reprogramming of head-to-tail cyclases for lariat peptide formation without protein engineering [1] [3].

Protocol: Solid-Phase Synthesis of Branched Peptide Substrates

• Resin Functionalization: Load ethylene glycol-functionalized solid support onto peptide synthesis vessel. The ethylene glycol serves as a simplified surrogate for the pantetheine leaving group, streamlining substrate synthesis [1].

• Backbone Assembly: Perform standard Fmoc-solid-phase peptide synthesis to build the linear peptide sequence, incorporating an L-Lys residue at the predetermined cyclization position with orthogonal Dde protection on the side chain.

• Side Chain Deprotection: Treat the resin-bound peptide with 2% hydrazine solution to selectively remove the Dde protecting group from the L-Lys side chain, while preserving other protecting groups.

• Pseudo-N-Terminus Installation: Couple L-Ile (or other appropriate amino acid) to the exposed side chain amine using standard coupling reagents (HBTU/HOBt or similar), creating the branched dipeptidyl unit.

• Global Deprotection and Cleavage: Treat the resin with standard cleavage cocktail (TFA/TIS/water or similar) to simultaneously remove all remaining protecting groups and cleave the branched peptide substrate from the solid support.

• Purification and Characterization: Purify the crude product by reverse-phase HPLC and verify structure by LC-MS and NMR spectroscopy [1].

The stereochemical configuration at the native N-terminus is crucial for controlling cyclization regioselectivity. Incorporating a D-amino acid at this position blocks head-to-tail cyclization, directing the enzyme exclusively toward lariat formation [3].

Enzymatic Macrocyclization with Non-Ribosomal Peptide Cyclases

The core innovation in the chemoenzymatic approach is the repurposing of versatile non-ribosomal peptide cyclases for lariat peptide synthesis. The following protocol details the enzymatic macrocyclization process [1].

Protocol: SurE-Catalyzed Lariat Peptide Cyclization

• Enzyme Preparation: Express and purify SurE cyclase (or other PBP-type thioesterase) according to standard protein expression protocols. SurE demonstrates exceptional substrate promiscuity while maintaining high regioselectivity [1].

• Reaction Setup: Combine the branched peptide substrate (0.5-1.0 mM) with SurE cyclase (5 mol%) in appropriate reaction buffer (e.g., 50 mM HEPES, pH 7.5) in a total volume of 100-500 μL.

• Cyclization Reaction: Incubate the reaction mixture at 30°C for 2-4 hours with gentle agitation. Monitor reaction progress by LC-MS.

• Reaction Termination: Heat the reaction mixture at 95°C for 5 minutes to denature the enzyme, followed by centrifugation to remove precipitated protein.

• Product Isolation: Purify the lariat cyclic peptide by reverse-phase HPLC. Characterize the structure by MS² fragmentation analysis to verify the macrocyclization site [1].

This methodology typically achieves quantitative conversion of branched linear substrates to lariat macrocycles, compared to the 33% overall yield obtained through traditional chemical synthesis of the same target [1]. The exceptional catalytic efficiency of SurE and related cyclases eliminates the need for high-dilution conditions or specialized coupling reagents typically required in macrocyclization reactions.

Tandem Cyclization-Acylation for Lipopeptide Synthesis

Complete lariat lipopeptides require both macrocyclic headgroup formation and lipidation of the terminal tail. The following one-pot protocol enables sequential cyclization and acylation without intermediate purification [1].

Protocol: One-Pot Cyclization and Ser/Thr Ligation

• Initial Cyclization: Perform the enzymatic macrocyclization as described in Section 3.2, but do not terminate the reaction after completion.

• Acylation Reaction Setup: To the completed cyclization reaction, add 2-5 equivalents of the desired fatty acid derivative (e.g., activated ester, thioester, or Meldrum's acid derivative) and adjust pH if necessary for subsequent ligation chemistry.

• Site-Selective Acylation: Incubate the reaction mixture at 25-37°C for 4-12 hours to facilitate serine/threonine ligation (STL) at the free N-terminus not involved in cyclization.

• Global Purification: After confirming complete acylation by LC-MS, purify the final lariat lipopeptide by reverse-phase HPLC.

• Library Synthesis: This tandem approach enables efficient parallel synthesis of diverse lariat lipopeptide libraries by varying the peptide sequence and acyl donor, facilitating structure-activity relationship studies [1].

This streamlined process successfully generated a 51-member library of lariat lipopeptides that was directly screened for antimicrobial activity, identifying several compounds with potent activity against Mycobacterium intracellulare (MIC = 8-16 μg mL⁻¹) [1].

Visualization of Synthetic Workflows and Mechanisms

Comparative Synthesis Workflow Diagram

The following diagram illustrates the key steps and decision points in the chemoenzymatic synthesis of lariat lipopeptides, highlighting the efficiency advantages over traditional synthesis.

Enzyme Mechanism and Substrate Recognition

The molecular basis for the high efficiency of non-ribosomal peptide cyclases lies in their precise substrate recognition and catalytic mechanism, detailed in the following diagram.

Research Reagent Solutions for Chemoenzymatic Synthesis

Successful implementation of chemoenzymatic lariat lipopeptide synthesis requires specific reagents and materials optimized for these applications. The following table details essential research solutions and their functions.

Table 3: Essential research reagents for lariat lipopeptide synthesis

Reagent Category	Specific Examples	Function and Application Notes
Non-Ribosomal Peptide Cyclases	SurE, WolJ (PBP-type TEs), TycC thioesterase (Type-I TE)	Catalyze regio- and stereoselective macrocyclization; SurE shows broad substrate tolerance [1]
Engineered Solid Supports	Ethylene glycol-functionalized resins	Simplified pantetheine surrogate for streamlined substrate synthesis [1]
Orthogonal Protecting Groups	Dde (1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)	Enables selective side chain deprotection for pseudo-N-terminus installation [1]
Acyl Donors for Lipidation	Fatty acid thioesters, Meldrum's acid derivatives, activated esters	Enable site-selective Ser/Thr ligation for lipid tail incorporation [1]
Analytical Standards	Synthetic lariat peptide standards (e.g., surugamide-based scaffolds)	LC-MS and MS² method development and quantification [1] [3]

Specialized enzyme expression systems for PBP-type thioesterases represent critical reagents for implementing these methodologies. These cyclases can be expressed in E. coli or other standard protein expression systems and purified using standard chromatographic techniques [1]. Their remarkable stability and solvent tolerance makes them particularly suitable for chemoenzymatic synthesis applications.

The quantitative comparison of yield and step-count metrics between traditional total synthesis and chemoenzymatic approaches demonstrates a paradigm shift in complex peptide synthesis. The integration of non-ribosomal peptide cyclases into synthetic workflows enables quantitative macrocyclization yields, dramatically reduced synthetic steps for diversified analog synthesis, and efficient access to structurally complex lariat architectures that would be challenging to produce by chemical methods alone.

For researchers and drug development professionals working in antimicrobial discovery, these methodologies offer a practical solution to the long-standing challenge of efficiently exploring lariat lipopeptide chemical space. The ability to generate focused libraries of 50+ compounds through standardized chemoenzymatic protocols [1] significantly accelerates structure-activity relationship studies and hit-to-lead optimization campaigns.

As the field advances, further engineering of non-ribosomal peptide cyclases through directed evolution and computational design promises to expand the substrate scope and catalytic efficiency of these remarkable biocatalysts [6] [33]. The integration of these enzymatic tools with continuous-flow synthesis platforms and automated purification systems represents the next frontier in maximizing synthetic efficiency for complex peptide therapeutics. Through continued development and adoption of these biosynthetically-inspired synthetic strategies, researchers can dramatically accelerate the discovery and development of novel antimicrobial agents to address the growing threat of antibiotic resistance.

The escalating crisis of antimicrobial resistance has intensified the search for new antibiotic classes with novel modes of action. Lariat-shaped lipopeptides—macrocyclic peptides featuring a carboxy-terminal macrocyclic head group and a long acyl chain appended to an amino-terminal tail—represent a fascinating class of antimicrobial agents that act on bacterial cell-surface targets [1] [3]. Naturally occurring examples such as daptomycin and colistin are among the most important sources of antimicrobial agents used clinically against drug-resistant pathogens [1]. However, the efficient exploration of the rich chemical space of lariat lipopeptides has been hampered by their molecular complexities, which pose significant synthetic challenges that impede efficient structural diversification [1].

Traditional chemical synthesis of these complex architectures requires orthogonal protecting group strategies, stoichiometric coupling reagents, and dilute conditions to suppress intermolecular coupling, involving substantial amounts of organic solvents [1]. An emerging alternative methodology leverages enzymes that catalyze peptide cyclization regio-, chemo-, or stereoselectively under mild conditions [1]. In nature, most lariat lipopeptides are biosynthesized via non-ribosomal pathways where lariat-shaped macrocyclic scaffolds are typically constructed by type-I thioesterases (TEs) [1]. Unfortunately, these lariat-forming TEs generally demonstrate narrow substrate specificity and substantial hydrolytic side reactions, limiting their application in generating diverse lariat peptide libraries [1] [3].

This technical guide details a groundbreaking chemoenzymatic strategy that overcomes these limitations by repurposing versatile non-ribosomal peptide (NRP) cyclases for lariat peptide synthesis. By combining innovative substrate design with enzymatic catalysis and subsequent chemical modification, this approach enables the efficient construction of a 51-member library of lariat lipopeptides for biological evaluation [1] [7].

Core Methodology: Reprogramming Cyclases for Lariat Peptide Synthesis

Strategic Approach and Enzymatic Toolkit

The foundational innovation of this methodology lies not in engineering the enzymes themselves, but in strategically redesigning the substrate topology to redirect the cyclization specificity of highly versatile head-to-tail NRP cyclases [3]. The approach leverages the remarkable catalytic promiscuity of certain NRP cyclases that exhibit broad tolerance for diverse peptide sequences and lengths.

The methodology employs three distinct non-ribosomal peptide cyclases, each offering unique advantages:

• SurE: A penicillin-binding protein (PBP)-type thioesterase from surugamide biosynthesis that demonstrates exceptional substrate tolerance for both sequence and length [1]. It specifically uses a d-configured C-terminal α-amino acid and an l-configured N-terminal α-amino acid for cyclization [1].
• WolJ: Another PBP-type thioesterase with properties similar to SurE, confirming the generalizability of the approach across enzyme variants [1] [3].
• TycC thioesterase (TycC-TE): A type-I thioesterase from tyrocidine biosynthesis that catalyzes head-to-tail cyclization of decapeptidyl substrates and exhibits broad tolerance for substrate variants [1].

Key Technical Innovation: Substrate Engineering with Stereochemical Control

The critical breakthrough came from engineering branched peptide substrates containing a "pseudo-N terminus"—an internal dipeptide unit where l-Ile was attached to the side chain of l-Lys via an isopeptide bond [1]. This design creates two potential nucleophiles for cyclization: the native N-terminus and the pseudo-N-terminus.

Initial experiments with SurE and a branched octapeptide based on the surugamide B sequence resulted in comparable formation of both canonical head-to-tail cyclic peptides (60%) and lariat-shaped cyclic peptides (40%), demonstrating that the pseudo-N terminus could effectively serve as a nucleophile [1].

To exclusively generate lariat peptides, researchers implemented a stereochemical switching strategy. Leveraging the strict specificity of PBP-type TEs for l-configured nucleophiles, they replaced the native N-terminal l-Ile residue with d-Val, effectively suppressing the head-to-tail cyclization pathway [1] [3]. This modification forced SurE to use exclusively the pseudo-N-terminal l-Ile¹′ as the nucleophile, achieving quantitative production of lariat-shaped cyclic peptides with complete regiospecificity [1].

Table 1: Key Enzymes Used in Lariat Lipopeptide Synthesis

Enzyme	Class	Source	Key Properties	Role in Synthesis
SurE	PBP-type TE	Surugamide biosynthesis	Broad substrate tolerance; accepts only l-configured nucleophiles [1]	Primary cyclization catalyst
WolJ	PBP-type TE	-	Similar properties to SurE [3]	Alternative cyclization catalyst
TycC-TE	Type-I TE	Tyrocidine biosynthesis	Head-to-tail cyclization; tolerant of diverse sequences/lengths [1]	Extended cyclization capability

Experimental Workflow and Protocols

The comprehensive synthesis strategy follows a modular, sequential approach that integrates solid-phase peptide synthesis, enzymatic macrocyclization, and site-selective acylation in a streamlined workflow.

Detailed Experimental Protocols

Substrate Synthesis: Branched Peptide Preparation

Materials and Reagents:

• Rink amide resin (Sigma-Aldrich) [34]
• Fmoc-protected amino acids (Sigma-Aldrich) [34]
• Orthogonally protected Fmoc-L-Lys(Dde)-OH
• Diisopropylethylamine (DIPEA, Sigma-Aldrich) [34]
• O-(1-benzotriazolyl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, Sigma-Aldrich) [34]
• Ethylene glycol (EG)-functionalized solid support [1]
• Trifluoroacetic acid (TFA, Thermo-Fisher) [34]
• Triisopropylsilane (TIS, Sigma-Aldrich) [34]
• Hydrazine (2% v/v in DMF) [1]

Synthetic Procedure:

• Solid-Phase Peptide Synthesis: The peptide main chain is synthesized on an EG-functionalized solid support using standard Fmoc-SPPS protocols [1]. Coupling reactions are performed using Fmoc-amino acids (5 equiv), HBTU (5 equiv), and DIPEA (12 equiv) in DMF under nitrogen gas purging for 6 hours [34]. Deprotection is achieved with 20% piperidine in DMF for 30 minutes [34].

• Orthogonal Deprotection: The Dde protecting group on the l-Lys³ side chain is selectively removed using 2% hydrazine in DMF [1].

• Pseudo-N-Terminus Installation: The pseudo-N-terminal l-Ile¹′ is coupled to the side chain of l-Lys³ in an additional round of coupling using standard Fmoc chemistry [1].

• Resin Cleavage and Global Deprotection: The branched peptide is cleaved from the resin with concomitant global deprotection using a cocktail mixture containing TFA, TIS, and Hâ‚‚O (96:2.0:2.0 v/v/v) at room temperature for 4 hours [34]. TFA is evaporated under nitrogen gas, and the peptide is precipitated with ice-cold diethyl ether, centrifuged, and lyophilized [34].

• Purification: Crude peptides are purified by reversed-phase high-performance liquid chromatography (HPLC) using a C-18 column with an acetonitrile/water gradient [34].

Enzymatic Macrocyclization

Materials and Reagents:

• Branched EG-functionalized peptide substrate (0.5-1.0 mM) [1]
• SurE, WolJ, or TycC-TE (5 mol%) [1]
• Appropriate buffer (e.g., Tris-HCl or phosphate buffer, pH 7.0-8.0)

Cyclization Procedure:

• Reaction Setup: The branched peptide substrate is dissolved in appropriate buffer at a concentration of 0.5-1.0 mM [1].

• Enzyme Addition: The cyclase enzyme (SurE, WolJ, or TycC-TE) is added to a final concentration of 5 mol% relative to substrate [1].

• Incubation: The reaction mixture is incubated at 30°C for 3 hours with gentle agitation [1].

• Reaction Monitoring: Reaction progress is monitored by LC-MS until complete substrate conversion is achieved [1].

• Product Characterization: Cyclic products are characterized by tandem mass spectrometry (MS²) to confirm lariat structure formation [1].

Tandem Cyclization-Acylation for Lipopeptide Formation

Materials and Reagents:

• Lariat-shaped cyclic peptide core
• Various acyl donors (e.g., fatty acids of different chain lengths)
• Serine/Threonine ligation (STL) reagents [1]

One-Pot Procedure:

• Cyclization Step: The enzymatic macrocyclization is performed as described above [1].

• Direct Acylation: Without purification of the cyclic intermediate, the reaction mixture is directly subjected to site-selective acylation via Ser/Thr ligation (STL) [1]. The remaining nucleophile not involved in cyclization serves as a reactive handle for acylation.

• Lipopeptide Formation: Various acyl groups are introduced through this ligation process, generating diverse lariat lipopeptides in a modular fashion [1].

• Library Construction: This tandem approach enables the efficient parallel synthesis of a 51-member library of lariat lipopeptides equipped with various acyl groups [1].

Library Composition and Analytical Data

Structural Diversity and Key Characteristics

The 51-member lariat lipopeptide library encompasses substantial structural diversity achieved through variations in three key dimensions: peptide sequence, macrocycle ring size, and acyl chain identity.

Table 2: Quantitative Analysis of Lariat Lipopeptide Library Members

Structural Feature Varied	Range/Diversity	Number of Analogs	Key Analytical Characterization
Peptide Sequence	Multiple positions scanned with l-Ile¹′-l-Lys dipeptidyl unit [1]	Not specified	LC-MS, MS² for cyclic structure confirmation [1]
Macrocycle Ring Size	Diverse sizes based on nucleophile position [1]	Not specified	Tandem mass spectrometry for ring topology [1]
Acyl Chain Identity	Various fatty acid chains appended via STL [1]	Not specified	Mass spectrometry for molecular weight confirmation [1]
Total Library Members	-	51 [1]	Comprehensive LC-MS/MS analysis

Reaction Efficiency and Yield Analysis

The chemoenzymatic approach demonstrated significant advantages over traditional chemical synthesis in terms of efficiency and yield.

Table 3: Yield Comparison: Chemoenzymatic vs. Chemical Synthesis

Synthetic Method	Key Steps	Overall Yield	Number of Steps	Critical Advantages
Chemoenzymatic Approach	Substrate synthesis (17 steps): 83% yield [1]; Enzymatic cyclization: quantitative [1]	~83% (after cyclization)	17 + 1	High yield, no protecting groups, aqueous conditions [1]
Traditional Chemical Synthesis	Linear synthesis with protecting groups	33% [1]	19 [1]	-

Biological Evaluation and Therapeutic Potential

Antimicrobial Screening Results

The complete 51-member lariat lipopeptide library was screened for antimicrobial activity against a panel of clinically relevant bacterial pathogens, including Mycobacterium intracellulare, Mycobacterium abscessus, Staphylococcus aureus, and Escherichia coli [1] [3]. The screening revealed significant structure-activity relationships, with specific structural features correlating with enhanced antimicrobial potency.

The most significant finding was the identification of eight lipopeptides that exhibited potent activity against Mycobacterium intracellulare, with concentrations required for 50% growth inhibition (MIC₅₀) ranging from 8–16 µg/ml [1] [7]. This result is particularly noteworthy given the urgent need for new antimycobacterial agents to combat the rising threat of nontuberculous mycobacterial infections.

Structure-Activity Relationship Insights

The biological screening provided crucial insights into structure-activity relationships:

• Acyl Chain Importance: Site-selective acylation was found to be essential for conferring antimycobacterial activity to the macrocyclic scaffolds [1]. This underscores the critical role of the lipid tail in mediating interactions with bacterial cell membranes, a known mechanism of action for many natural lipopeptide antibiotics [34].

• Stereochemistry Effects: The precise stereochemical configuration of both the nucleophilic residues and the peptide backbone influences bioactivity, consistent with the known stereospecificity of enzyme-substrate interactions in bacterial targets [1].

• Macrocycle Size and Composition: Variations in ring size and amino acid composition within the macrocyclic head group significantly impacted antimicrobial potency and spectrum, enabling fine-tuning of biological activity [1].

The Scientist's Toolkit: Essential Research Reagents

Table 4: Key Research Reagent Solutions for Lariat Lipopeptide Synthesis

Reagent/Resource	Function/Application	Source/Example
PBP-Type Thioesterases (SurE, WolJ)	Catalyze regiospecific macrocyclization of branched peptide substrates [1] [3]	Recombinantly expressed enzymes [1]
Type-I Thioesterase (TycC-TE)	Alternative cyclase with broad substrate tolerance [1]	Tyrocidine biosynthesis pathway [1]
EG-Functionalized Solid Support	Simplifies substrate synthesis by serving as surrogate for pantetheine leaving group [1]	Custom functionalized resin [1]
Fmoc-Amino Acids with Orthogonal Protection	Enables incorporation of specific residues for branching; Dde protection on Lys allows selective deprotection [1] [34]	Commercial sources (e.g., Sigma-Aldrich) [34]
Ser/Thr Ligation (STL) Reagents	Enables site-selective acylation of cyclic peptide cores [1]	Custom synthetic acyl donors [1]
Hydrazine Solution (2%)	Selective removal of Dde protecting group for pseudo-N-terminus installation [1]	Standard chemical reagent [1]

Technical Considerations and Optimization Guidelines

Critical Parameters for Success

• Nucleophile Stereochemistry: The strict specificity of PBP-type TEs for l-configured nucleophiles is essential for controlling regiospecificity. d-configured residues at the native N-terminus effectively suppress head-to-tail cyclization [1].

• Enzyme Concentration and Purity: The use of 5 mol% purified enzyme relative to substrate provides optimal cyclization efficiency without excessive protein usage [1].

• Reaction Time and Temperature: A 3-hour incubation at 30°C achieves complete substrate conversion while maintaining enzyme stability [1].

• Substrate Engineering: The position of the pseudo-N-terminus within the peptide sequence significantly influences cyclization efficiency and must be optimized for different peptide sequences [1].

Analytical Validation Requirements

• MS² Confirmation: Tandem mass spectrometry is essential for distinguishing between head-to-tail and lariat cyclic topologies [1].

• HPLC Purity Assessment: Reversed-phase HPLC with C-18 columns using acetonitrile/water gradients provides reliable purity assessment before and after cyclization [34].

• Yield Calculation: Comparison of isolated yields against internal standards ensures accurate quantification of cyclic products.

The rising global incidence of pulmonary non-tuberculous mycobacterial (NTM) disease, predominantly caused by the Mycobacterium avium complex (MAC), including Mycobacterium intracellulare, represents a significant public health challenge. These infections are notoriously difficult to treat due to intrinsic resistance to many conventional antibiotics, necessitating prolonged multi-drug regimens with often suboptimal outcomes [35]. This context underscores the urgent need for novel antimicrobial agents with new modes of action.

Within the broader thesis on the biosynthesis of lariat lipopeptides by non-ribosomal peptide cyclases, this guide details the biological validation of a new class of antimycobacterial agents. Lariat lipopeptides, characterized by their macrocyclic "head" and a linear lipophilic "tail", are a important source of antimicrobial agents, as exemplified by daptomycin and colistin [1] [3]. However, their structural complexity has hindered efficient exploration of their chemical space for drug discovery. Recent advances in chemoenzymatic synthesis, repurposing versatile non-ribosomal peptide (NRP) cyclases like the penicillin-binding protein-type thioesterase SurE, have now enabled the efficient generation of diverse lariat lipopeptide libraries [1] [3]. This guide provides an in-depth technical overview of the experimental strategies and methodologies used to uncover and validate the antimycobacterial activity of these novel synthetic lariat lipopeptides against M. intracellulare.

Experimental Strategy and Workflow

The biological validation of lariat lipopeptides involves a multi-stage process, from library generation to mechanistic studies. The workflow below illustrates the key stages from compound synthesis to the identification of active leads.

Core Experimental Protocols

Chemoenzymatic Synthesis of Lariat Lipopeptide Library

Objective: To generate a diverse library of lariat lipopeptides for biological screening.

Principle: This method leverages the promiscuity of non-ribosomal peptide cyclases, such as the PBP-type thioesterase SurE. Substrate engineering introduces a "pseudo-N-terminus" via a dipeptidyl unit (e.g., l-Ile-l-Lys) attached to the side chain of an internal lysine residue. The native N-terminal residue is configured with a d-amino acid to block the canonical head-to-tail cyclization pathway, forcing the enzyme to exclusively use the pseudo-N-terminus as the nucleophile for macrocyclization. The free native N-terminus then serves as a handle for site-selective acylation to install various lipid tails [1] [3].

Procedure:

• Solid-Phase Peptide Synthesis (SPPS): Synthesize the linear peptide backbone on a solid support functionalized with an ethylene glycol (EG) leaving group.
• Introduction of Pseudo-N-terminus: On the solid support, remove the orthogonal protecting group (e.g., Dde) from the side chain of a specific lysine residue. Couple the dipeptide unit (e.g., Fmoc-l-Ile-l-Lys) to this side chain to create the branched peptide.
• Global Deprotection and Cleavage: Cleave the peptide from the resin and remove all protecting groups to obtain the unstructured, branched EG-functionalized peptide substrate.
• Enzymatic Macrocyclization: Incubate the branched peptide substrate (e.g., 0.5-1.0 mM) with the cyclase (e.g., 5 mol% SurE) in a suitable buffer (e.g., HEPES, pH 7.0-8.0) at 30°C for several hours. Monitor reaction completion by LC-MS.
• Site-Selective Acylation (Ser/Thr Ligation): Without purifying the macrocyclic intermediate, directly subject the reaction mixture to acylation. Add an acyl donor (e.g., a fatty acid derivative) and conditions conducive to Ser/Thr ligation to the free native N-terminus. This one-pot tandem strategy yields the final lariat lipopeptide [1].

Determination of Minimum Inhibitory Concentration (MIC)

Objective: To determine the lowest concentration of a lariat lipopeptide that prevents visible growth of M. intracellulare.

Principle: The MIC is a standard quantitative measure of a compound's antibacterial potency. It is determined using a broth microdilution method in 96-well plates, allowing for high-throughput screening of compound libraries [35].

Procedure:

• Inoculum Preparation: Grow M. intracellulare (e.g., strain JCM6384) in Middlebrook 7H9 broth to mid-log phase. Adjust the turbidity of the bacterial suspension to match a 0.5 McFarland standard, then dilute in broth to achieve a final inoculum of approximately 5 × 10^5 CFU/mL in the test well.
• Compound Dilution: Prepare a two-fold serial dilution of the lariat lipopeptide in the assay medium across the wells of a 96-well plate. The concentration range should typically span from 0.125 to 64 µg/mL or higher.
• Inoculation and Incubation: Add the prepared bacterial inoculum to each well. Include growth control (bacteria only) and sterility control (medium only) wells. Seal the plate and incub at 37°C under stationary conditions for 5-7 days.
• Endpoint Reading: The MIC is defined as the lowest concentration of the compound that completely inhibits visible bacterial growth. For M. intracellulare JCM6384, the positive control antibiotic clarithromycin typically shows an MIC of 0.02 µg/mL [35].

Determination of Half-Maximal Inhibitory Concentration (IC50)

Objective: To quantify the potency of a compound by determining the concentration that reduces bacterial growth by 50%.

Procedure:

• Follow steps 1-3 of the MIC protocol.
• Viability Measurement: After incubation, instead of relying on visual growth, use a quantitative metric. This can be a colorimetric assay like resazurin (Alamar Blue) or by measuring optical density at 600 nm (OD600).
• Data Analysis: Calculate the percentage growth inhibition relative to the growth control for each compound concentration. Fit the dose-response data to a non-linear regression (sigmoidal dose-response) model using software like GraphPad Prism. The IC50 is derived from this curve fit [1].

Table 1: Key Quantitative Findings from Biological Screening

Assay	Target Organism	Key Result	Positive Control (Result)	Citation
Primary Screening (MIC/IC50)	M. intracellulare	8 lipopeptides showed IC50 values of 8–16 µg ml⁻¹	Not Specified	[1]
Reference Compound (MIC)	M. intracellulare JCM6384	Mavintramycin A MIC: 0.39 µg/mL	Clarithromycin (0.02 µg/mL), Ethambutol (3.12 µg/mL)	[35]
Clinical Isolate Profiling	40 clinical M. avium isolates	Mavintramycin A MIC range: 1.0–32 µg/mL (active against MDR strains)	Clarithromycin, Rifampicin, Ethambutol	[35]

Advanced Validation and Profiling Assays

Screening Against Clinical and Resistant Isolates

Objective: To evaluate the efficacy of lead lariat lipopeptides against a panel of clinically relevant and multidrug-resistant (MDR) MAC isolates.

Protocol:

• Assemble a panel of clinically isolated strains, ideally comprising over 40 isolates, including those resistant to first-line drugs like clarithromycin (CAM), rifampicin (RFP), and ethambutol (EB) [35].
• Perform the broth microdilution MIC assay as described in Section 3.2 against each strain in the panel.
• The ability of a compound to maintain activity against MDR strains (e.g., MIC of 8–32 µg/mL against CAM- and EB-resistant M. avium) is a strong indicator of its potential to overcome existing resistance mechanisms [35].

Intracellular Macrophage Infection Model

Objective: To assess the efficacy of lead compounds against mycobacteria residing within macrophages, which model persistent infection.

Principle: MAC are facultative intracellular pathogens that can survive and replicate inside host macrophages. This assay tests a compound's ability to penetrate the macrophage cell membrane and kill the internalized bacteria [35].

Procedure:

• Cell Culture and Infection: Differentiate human monocytic THP-1 cells into macrophage-like cells using phorbol esters. Infect the macrophages with a stationary-phase culture of M. intracellulare at a suitable multiplicity of infection (MOI).
• Compound Treatment: After a period to allow for phagocytosis, wash the cells to remove extracellular bacteria. Then treat the infected macrophages with the lead lariat lipopeptide at relevant concentrations.
• Viability Assessment: Following incubation, lyse the macrophages and plate the lysates on solid agar medium to enumerate the bacterial colony forming units (CFUs). The reduction in intracellular CFU compared to the untreated control quantifies the compound's efficacy [35].

Preliminary Mode-of-Action Investigation

Objective: To gain initial insights into the potential mechanism by which lariat lipopeptides exert their antimycobacterial effect.

Strategy: While a full mechanistic elucidation is complex, initial experiments can screen for common targets.

• Cell Wall Synthesis Inhibition: Investigate effects on incorporation of radiolabeled precursors into cell wall components.
• Membrane Disruption: Use assays that monitor membrane depolarization (e.g., using DiSC3(5) dye) or permeability (e.g., uptake of propidium iodide or ethidium bromide) in treated vs. untreated mycobacteria [36].
• Protein Synthesis Inhibition: As seen with other natural product antibiotics like mavintramycin A, which binds to the 23S ribosomal RNA, one can assess the incorporation of radiolabeled amino acids into bacterial protein [35].
• Nucleic Acid Synthesis Inhibition: Measure the effect on incorporation of radiolabeled nucleotides into DNA/RNA.

The Scientist's Toolkit: Essential Research Reagents

Table 2: Key Reagents and Resources for Antimycobacterial Validation

Reagent / Resource	Function / Application	Example / Specification
Non-ribosomal Peptide Cyclases	Biocatalysts for macrocyclization of linear peptide substrates.	SurE, WolJ (PBP-type TEs), TycC Thioesterase [1] [3]
Branched Peptide Substrates	Engineered enzymatic substrates featuring a "pseudo-N-terminus".	Unprotected peptides with internal l-Lys bearing dipeptidyl unit (e.g., l-Ile1'-l-Lys) and C-terminal EG leaving group [1]
Mycobacterial Strains	Target organisms for in vitro activity screening.	M. intracellulare JCM6384; Panels of clinical M. avium isolates (including MDR strains) [35]
THP-1 Human Monocytic Cell Line	In vitro model for intracellular efficacy testing.	Differentiated into macrophages for persistent infection models [35]
Silkworm Infection Model	In vivo model for preliminary efficacy and toxicity testing.	Low-cost in vivo model to study antibiotic exposure and efficacy [37]
Cation-Adjusted Mueller Hinton Broth	Standardized medium for MIC determination.	Recommended for broth microdilution assays to ensure reproducibility [35] [36]

The chemoenzymatic synthesis of lariat lipopeptides, powered by versatile NRP cyclases, provides a robust platform for generating structurally diverse compound libraries. The biological validation framework outlined in this guide—encompassing primary screening, advanced profiling in physiologically relevant models, and preliminary mechanistic studies—enables the systematic identification of promising anti-MAC leads. The recent discovery of synthetic lariat lipopeptides with significant activity against M. intracellulare underscores the potential of this approach to contribute new therapeutic candidates to the urgently needed arsenal against NTM infections.

The escalating crisis of antimicrobial resistance has intensified the search for novel antibiotic agents, with lariat lipopeptides emerging as a critically important class due to their potent activity against drug-resistant pathogens [1] [6]. These complex natural products, characterized by their macrocyclic "head" and linear lipid "tail," include clinically essential antibiotics such as daptomycin and colistin [1] [3]. However, their intricate architectural features—specifically the lariat topology combining macrocyclization and lipidation—have presented substantial challenges for both chemical synthesis and natural biosynthetic production, limiting efficient exploration of their chemical space for drug development [1].

Traditional approaches to lariat lipopeptide production have faced significant limitations. Purely synthetic routes require extensive protecting group strategies, stoichiometric coupling reagents, and highly dilute conditions to suppress intermolecular side reactions, resulting in lengthy synthetic sequences with low overall yields [1]. Conversely, native biosynthetic pathways utilizing type-I thioesterase (TE) domains in non-ribosomal peptide synthetases (NRPS) often demonstrate narrow substrate specificity and substantial competitive hydrolysis, hampering efforts to generate structurally diverse analogs [1]. This technological gap has impedited the systematic investigation of structure-activity relationships in this therapeutically valuable compound class.

Recent advances have introduced a promising third approach: chemoenzymatic synthesis that strategically integrates elements of both chemical and biological methods [1] [3] [6]. This review provides a comprehensive comparative analysis of these three distinct methodologies, with particular emphasis on a groundbreaking chemoenzymatic platform that leverages the catalytic versatility of non-ribosomal peptide cyclases to overcome previous limitations. By examining quantitative performance data, experimental protocols, and practical implementation frameworks, this analysis aims to equip researchers with the knowledge necessary to select optimal synthetic strategies for lariat lipopeptide production and diversification.

Comparative Performance Analysis of Synthetic Approaches

Table 1: Direct comparison of key performance metrics across synthetic methodologies for lariat lipopeptide production

Performance Metric	Purely Synthetic Approach	Native Biosynthetic Pathway	Chemoenzymatic Approach
Overall Yield	33% yield over 19 steps for lariat peptide 5 [1]	Low yields due to competitive hydrolysis; highly variable [1]	83% yield for linear precursor (17 steps) + quantitative cyclization [1]
Structural Diversification Capacity	Limited by need for extensive protecting group manipulation [1]	Narrow substrate specificity of lariat-forming TEs limits sequence variability [1]	High diversification capacity for both sequence and ring size [1]
Technical Complexity	High: requires orthogonal protecting groups, dilute conditions, complex purification [1]	Moderate to high: dependent on genetic manipulation and fermentation optimization	Moderate: streamlined synthesis with minimal purification [1]
Stereochemical Control	Excellent through use of chiral auxiliaries or asymmetric synthesis	Native enzymatic control but difficult to engineer for new substrates	Excellent: exploits inherent stereospecificity of cyclases [1]
Reaction Concentration	mM range or lower to prevent oligomerization [1]	Not applicable (cellular environment)	Not constrained by dilution requirements [1]
Solvent Consumption	High volumes due to dilute conditions [1]	Aqueous systems in fermentation	Aqueous buffers; minimal organic solvent [1]

Table 2: Enzymatic scope and versatility across different cyclase platforms

Cyclase Enzyme	Native Function	Engineered Application	Substrate Tolerance	Key Feature
SurE (PBP-type TE)	Head-to-tail cyclization in surugamide biosynthesis [1]	Lariat peptide formation via pseudo-N-terminus [1]	Broad tolerance for sequence and length variations [1]	Strict stereospecificity for l-configured nucleophiles [1]
WolJ (PBP-type TE)	Head-to-tail cyclization	Lariat peptide formation [1]	Demonstrated broad substrate tolerance similar to SurE [1]	Compatible with chemoenzymatic strategy [1]
TycC-TE (Type-I TE)	Head-to-tail cyclization in tyrocidine biosynthesis [1]	Lariat peptide formation [1]	Tolerates diverse sequences and lengths in vitro [1]	Prefers heterochiral pair of ring-closing residues [1]

The quantitative comparison reveals distinct advantages of the chemoenzymatic approach. While purely synthetic methods suffer from lengthy sequences and accumulating yield losses, and native biosynthetic pathways face limitations in substrate flexibility, the chemoenzymatic strategy achieves superior efficiency by combining high-yielding chemical synthesis of linear precursors with highly selective enzymatic cyclization [1]. This hybrid approach eliminates the dilution constraints of purely synthetic macrocyclization while overcoming the narrow substrate specificity of native biosynthetic enzymes [1].

Experimental Protocols and Methodologies

Substrate Design and Synthesis Protocol

The foundation of successful chemoenzymatic synthesis lies in the strategic design and preparation of branched peptide substrates containing a "pseudo-N-terminus." The following protocol outlines the key steps for substrate synthesis:

• Solid-Phase Peptide Synthesis (SPPS): Initiate peptide assembly on a solid support functionalized with ethylene glycol (EG), which serves as a simplified pantetheine mimic for downstream enzymatic processing [1]. Standard Fmoc chemistry is employed for chain elongation.

• Orthogonal Protection Strategy: Incorporate an l-Lys residue at the predetermined branching position with its ε-amino group protected by a 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) group, which can be selectively removed while preserving other side-chain protections [1].

• Selective Deprotection: Treat the resin-bound peptide with 2% hydrazine solution to selectively remove the Dde protecting group from the l-Lys side chain, exposing the ε-amino group for subsequent branching [1].

• Pseudo-N-Terminus Installation: Conduct an additional round of coupling to install the l-Ile1′ residue (or alternative nucleophilic residue) onto the exposed ε-amino group of l-Lys, creating the branched dipeptidyl unit that will serve as the pseudo-N-terminus [1].

• Global Deprotection and Cleavage: Cleave the completed branched peptide from the resin using standard acidolytic conditions (e.g., trifluoroacetic acid cocktail) with concurrent removal of all remaining side-chain protecting groups, yielding the unprotected, EG-functionalized branched peptide substrate [1].

• Purification and Characterization: Purify the crude branched peptide by reverse-phase HPLC and verify structure by mass spectrometry before enzymatic cyclization [1].

Table 3: Essential research reagents for chemoenzymatic lariat peptide synthesis

Reagent/Category	Specific Examples	Function in Workflow
Non-Ribosomal Peptide Cyclases	SurE, WolJ (PBP-type TEs), TycC-TE (Type-I TE) [1]	Catalyze regiospecific macrocyclization of linear peptide substrates
Solid Supports	EG-functionalized resin [1]	Platform for peptide synthesis with simplified leaving group
Orthogonal Protecting Groups	Dde on l-Lys side chain [1]	Enables selective branching during SPPS
Activated Amino Acids	Fmoc-protected amino acid building blocks	Standard SPPS chain elongation
Selective Deprotection Reagents	2% hydrazine [1]	Selective Dde removal for branching
Acylation Reagents	Ser/Thr ligation partners [1]	Introduce lipid tails post-cyclization

Enzymatic Cyclization and One-Pot Diversification

The enzymatic cyclization process capitalizes on the remarkable stereochemical preferences of non-ribosomal peptide cyclases to achieve regiospecific macrocyclization:

• Enzyme Preparation: Express and purify recombinant cyclase enzymes (SurE, WolJ, or TycC-TE) using standard protein expression systems. These enzymes typically function effectively at catalytic loading of 5 mol% relative to substrate [1].

• Cyclization Reaction Setup: Combine the branched EG-functionalized peptide substrate (0.1-1.0 mM) with cyclase enzyme (5 mol%) in appropriate aqueous buffer (e.g., HEPES or Tris buffer, pH 7.5-8.5). Incubate at 30°C with gentle agitation [1].

• Reaction Monitoring: Monitor reaction progress by LC-MS. Complete conversion is typically achieved within 3 hours under optimized conditions [1].

• Stereochemical Control Implementation: To enforce exclusive lariat formation, employ substrates where the native N-terminal residue is replaced with a d-configured amino acid. This strategically blocks the head-to-tail cyclization pathway while the pseudo-N-terminal l-configured residue remains available for cyclization [1].

• One-Pot Tandem Acylation: Without intermediate purification, directly add serine/threonine ligation (STL) reagents to the completed cyclization reaction mixture to introduce diverse lipid tails at the free N-terminus not consumed in cyclization [1]. This tandem approach enables direct conversion of cyclic peptides to lipopeptides in a single vessel.

• Product Isolation: Purify final lariat lipopeptides by reverse-phase HPLC and characterize using analytical HPLC, MS/MS, and when necessary, 2D NMR spectroscopy to confirm regio- and stereochemistry [1].

Diagram 1: Chemoenzymatic workflow for lariat lipopeptide synthesis. The process begins with enzymatic cyclization followed by chemical acylation in one pot.

Strategic Advantages of the Chemoenzymatic Approach

Overcoming Fundamental Limitations

The chemoenzymatic platform addresses core limitations of both traditional synthetic and native biosynthetic methods through several innovative mechanisms:

• Elimination of Dilution Constraints: Unlike purely synthetic macrocyclization that requires high dilution (mM range or lower) to suppress intermolecular oligomerization, enzymatic cyclization proceeds efficiently at substantially higher concentrations, dramatically reducing solvent consumption and enabling more practical reaction scales [1].

• Bypassing Protecting Group Manipulation: The exquisite chemoselectivity of non-ribosomal peptide cyclases enables cyclization of completely unprotected linear precursors, eliminating the need for complex orthogonal protecting group strategies that typically complicate synthetic routes and diminish overall yields [1].

• Exploiting Enzymatic Promiscuity: While native lariat-forming type-I TEs typically display narrow substrate specificity, PBP-type TEs like SurE and WolJ exhibit remarkable tolerance for sequence variations and ring sizes, providing access to diverse lariat architectures that would be inaccessible through native biosynthetic pathways [1].

Strategic Implementation for Drug Discovery

The practical implementation of this technology offers distinct advantages in pharmaceutical development:

• Rapid Library Generation: The combination of modular substrate design and one-pot cyclization-acylation enables efficient parallel synthesis of structurally diverse lariat lipopeptide libraries. This capability was demonstrated through the creation of a 51-member library that facilitated identification of compounds with promising antimycobacterial activity (8-16 µg ml⁻¹ MIC values) [1].

• Bioactivity Optimization: The site-selective acylation strategy enables systematic exploration of structure-activity relationships by varying lipid chain length and composition while maintaining constant macrocyclic architecture, providing critical insights for potency and selectivity optimization [1].

• Streamlined Workflow Integration: The high selectivity of enzymatic transformations generates reaction mixtures of sufficient purity to bypass intermediate isolation steps, significantly accelerating the synthesis-screening cycle in drug discovery campaigns [3].

The chemoenzymatic synthesis of lariat lipopeptides represents a paradigm shift in complex peptide production, effectively bridging the gap between traditional synthetic chemistry and native biosynthesis. By strategically leveraging the catalytic versatility of non-ribosomal peptide cyclases, this approach surmounts fundamental limitations of both conventional methods while enabling efficient exploration of chemical space for drug discovery. The robust experimental protocols, clear stereochemical control mechanisms, and practical one-pot diversification strategies provide researchers with a powerful platform for accessing these therapeutically valuable compounds. As antibiotic resistance continues to escalate globally, this technological advancement offers a timely and critical tool for developing next-generation antimicrobial agents with novel mechanisms of action. Future advancements will likely focus on expanding the enzyme toolkit through discovery and engineering, further enhancing the synthetic versatility and pharmaceutical relevance of this promising approach.

Conclusion

The repurposing of non-ribosomal peptide cyclases through strategic substrate redesign marks a transformative advance in synthetic biology and medicinal chemistry. This chemoenzymatic platform successfully reconciles the flexibility of chemical synthesis with the precision of enzymatic catalysis, providing a streamlined, modular route to complex lariat lipopeptides. The key takeaways are the power of stereochemical control to dictate cyclization mode, the robustness of a tandem one-pot procedure, and the direct generation of bioactive compounds with promising antimycobacterial efficacy (8-16 µg ml⁻¹). Future directions will focus on expanding the enzyme toolkit via directed evolution, incorporating non-canonical building blocks, and advancing the most potent analogs through pre-clinical development. This methodology establishes a new paradigm for accessing natural product-like chemical space, offering a powerful engine for discovering next-generation antibiotics in the fight against multidrug-resistant microbes.

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