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The glycopeptide antibiotics (GPAs) are a clinically approved class of antimicrobial agents that classically function via inhibition of bacterial cell wall biosynthesis through sequestration of the precursor lipid II. The oxidative crosslinking of the core peptide by cytochrome P450 (Oxy) enzymes during GPA biosynthesis is both essential to their function and the source of their synthetic challenge. Thus, understanding the activity and selectivity of these Oxy enzymes is of key importance for the future engineering of this important compound class. Recent reports of GPAs that display an alternate mode of action and a wider range of core peptide structures compared to classic lipid II binding GPAs raises the question of the tolerance of Oxy enzymes for larger changes in their peptide substrates. In this work, we explore the ability of Oxy enzymes from the biosynthesis pathways of lipid II binding GPAs to accept altered peptide substrates based on a vancomycin template. Our results show that Oxy enzymes are more tolerant of changes at the N-terminus of their substrates, whilst C-terminal extension of their peptide substrates is deleterious to the activity of all Oxy enzymes. Thus, future studies should prioritise the study of Oxy enzymes from atypical GPA biosynthesis pathways bearing C-terminal peptide extension to increase the substrate scope of these important cyclisation enzymes.

Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains., Non-ribosomal peptide synthetases (NRPSs) are multi-modular enzymes assembling complex natural products. Here, the structures of a Thermobifida fusca NRPS condensation domain bound to the substrate-bearing peptidyl carrier protein (PCP) domain provide insight into the mechanisms of substrate selectivity and engagement within the catalytic pocket.

The glycopeptide antibiotics (GPAs) are a fascinating example of complex natural product biosynthesis, with the nonribosomal synthesis of the peptide core coupled to a cytochrome P450-mediated cyclisation cascade that crosslinks aromatic side chains within this peptide. Given that the challenges associated with the synthesis of GPAs stems from their highly crosslinked structure, there is great interest in understanding how biosynthesis accomplishes this challenging set of transformations. In this regard, the use of in vitro experiments has delivered important insights into this process, including the identification of the unique role of the X-domain as a platform for P450 recruitment. In this minireview, we present an analysis of the results of in vitro studies into the GPA cyclisation cascade that have demonstrated both the tolerances and limitations of this process for modified substrates, and in turn developed rules for the future reengineering of this important antibiotic class.

We report our investigation of the utility of peptide crosslinking cytochrome P450 enzymes from biarylitide biosynthesis to generate a range of cyclic tripeptides from simple synthons. The crosslinked tripeptides produced by this P450 include both tyrosine-histidine (A-N-B) and tyrosine-tryptophan (A-O-B) crosslinked tripeptides, the latter a rare example of a phenolic crosslink to an indole moiety. Tripeptides are easily isolated following proteolytic removal of the leader peptide and can incorporate a wide range of amino acids in the residue inside the crosslinked tripeptide. Given the utility of peptide crosslinks in important natural products and the synthetic challenge that these can represent, P450 enzymes have the potential to play roles as important tools in the generation of high-value cyclic tripeptides for incorporation in synthesis, which can be yet further diversified using selective chemical techniques through specific handles contained within these tripeptides.

Non-ribosomal peptide synthesis is an important biosynthesis pathway in secondary metabolism. In this study we have investigated modularisation and redesign strategies for the glycopeptide antibiotic teicoplanin. Using the relocation or exchange of domains within the NRPS modules, we have identified how to initiate peptide biosynthesis and explored the requirements for the functional reengineering of both the condensation/adenylation domain and epimerisation/condensation domain interfaces. We have also demonstrated strategies that ensure communication between isolated NRPS modules, leading to new peptide assembly pathways. This provides important insights into NRPS reengineering of glycopeptide antibiotic biosynthesis and has broad implications for the redesign of other NRPS systems., Redesign of the non-ribosomal peptide synthetase (NRPS) from teicoplanin biosynthesis has been extensively investigated via domain exchange, interface reengineering and through engineering communication between isolated NRPS modules.

Since the discovery of vancomycin in the 1950s, the glycopeptide antibiotics (GPAs) have been of great interest to the scientific community. These nonribosomally biosynthesized peptides are highly cross-linked, often glycosylated, and inhibit bacterial cell wall assembly by interfering with peptidoglycan synthesis. Interest in glycopeptide antibiotics covers many scientific disciplines, due to their challenging total syntheses, complex biosynthesis pathways, mechanism of action, and high potency. After intense efforts, early enthusiasm has given way to a recognition of the challenges in chemically synthesizing GPAs and of the effort needed to study and modify GPA-producing strains to prepare new GPAs to address the increasing threat of microbial antibiotic resistance. Although the preparation of GPAs, either by modifying the pendant groups such as saccharides or by functionalizing the N- or C-terminal moieties, is readily achievable, the peptide core of these molecules—the GPA aglycone—remains highly challenging to modify. This review aims to present a summary of the results of GPA modification obtained with the three major approaches developed to date: in vivo strain manipulation, total chemical synthesis, and chemoenzymatic synthesis methods.

Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report the first structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this previously uncharacterized complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains.

[Image: see text] The receptor for the neuropeptide relaxin 3, relaxin family peptide 3 (RXFP3) receptor, is an attractive pharmacological target for the control of eating, addictive, and psychiatric behaviors. Several structure–activity relationship studies on both human relaxin 3 (containing 3 disulfide bonds) and its analogue A2 (two disulfide bonds) suggest that the C-terminal carboxylic acid of the tryptophan residue in the B-chain is important for RXFP3 activity. In this study, we have added amide, alcohol, carbamate, and ester functionalities to the C-terminus of A2 and compared their structures and functions. As expected, the C-terminal amide form of A2 showed lower binding affinity for RXFP3 while ester and alcohol substitutions also demonstrated lower affinity. However, while these analogues showed slightly lower binding affinity, there was no significant difference in activation of RXFP3 compared to A2 bearing a C-terminal carboxylic acid, suggesting the binding pocket is able to accommodate additional atoms.

The pathogen Staphylococcus aureus can readily develop antibiotic resistance and evade the human immune system, which is associated with reduced levels of neutrophil recruitment. Here, we present a class of antibacterial peptides with potential to act both as antibiotics and as neutrophil chemoattractants. The compounds, which we term ‘antibiotic-chemoattractants’, consist of a formylated peptide (known to act as chemoattractant for neutrophil recruitment) that is covalently linked to the antibiotic vancomycin (known to bind to the bacterial cell wall). We use a combination of in vitro assays, cellular assays, infection-on-a-chip and in vivo mouse models to show that the compounds improve the recruitment, engulfment and killing of S. aureus by neutrophils. Furthermore, optimizing the formyl peptide sequence can enhance neutrophil activity through differential activation of formyl peptide receptors. Thus, we propose antibiotic-chemoattractants as an alternate approach for antibiotic development., Antibiotic resistance in Staphylococcus aureus is associated with reduced neutrophil recruitment. Here, Payne et al. link formylated peptides, which act as chemoattractants for neutrophils, with the antibiotic vancomycin and show that these hybrid compounds improve clearance of S. aureus by neutrophils.

The protein Ebony from Drosophila melanogaster plays a central role in the regulation of histamine and dopamine in various tissues through condensation of these amines with β-alanine. Ebony is a rare example of a nonribosomal peptide synthetase (NRPS) from a higher eukaryote and contains a C-terminal sequence that does not correspond to any previously characterized NRPS domain. We have structurally characterized this C-terminal domain and have discovered that it adopts the aryl-alkylamine- N -acetyl transferase (AANAT) fold, which is unprecedented in NRPS biology. Through analysis of ligand-bound structures, activity assays, and binding measurements, we have determined how this atypical condensation domain is able to provide selectivity for both the carrier protein-bound amino acid and the amine substrates, a situation that remains unclear for standard condensation domains identified to date from NRPS assembly lines. These results demonstrate that the C terminus of Ebony encodes a eukaryotic example of an alternative type of NRPS condensation domain; they also illustrate how the catalytic components of such assembly lines are significantly more diverse than a minimal set of conserved functional domains.

Non-ribosomal peptide biosynthesis produces highly diverse natural products through a complex cascade of enzymatic reactions that together function with high selectivity to produce bioactive peptides. The modification of non-ribosomal peptide synthetase (NRPS)-bound amino acids can introduce significant structural diversity into these peptides and has exciting potential for biosynthetic redesign. However, the control mechanisms ensuring selective modification of specific residues during NRPS biosynthesis have previously been unclear. Here, we have characterised the incorporation of the non-proteinogenic amino acid 3-chloro-β-hydroxytyrosine during glycopeptide antibiotic (GPA) biosynthesis. Our results demonstrate that the modification of this residue by trans-acting enzymes is controlled by the selectivity of the upstream condensation domain responsible for peptide synthesis. A proofreading thioesterase works together with this process to ensure that effective peptide biosynthesis proceeds even when the selectivity of key amino acid activation domains within the NRPS is low. Furthermore, the exchange of condensation domains with altered amino acid specificities allows the modification of such residues within NRPS biosynthesis to be controlled, which will doubtless prove important for reengineering of these assembly lines. Taken together, our results indicate the importance of the complex interplay of NRPS domains and trans-acting enzymes to ensure effective GPA biosynthesis, and in doing so reveals a process that is mechanistically comparable to the hydrolytic proofreading function of tRNA synthetases in ribosomal protein synthesis.

The glycopeptide antibiotics (GPAs) serve as an important example of the interplay of two powerful enzymatic classes in secondary metabolism: the coupling of nonribosomal peptide synthesis with oxidative aromatic cross-linking performed by cytochrome P450 enzymes. This interplay is responsible for the generation of the highly cross-linked peptide aglycone at the core of this compound class that is required for antibiotic activity and, as such, serves as an important point for the exploration of chemoenzymatic routes to understand the selectivity and mechanism of this complex cascade. Here, we demonstrate the effective reconstitution of enzymatic tetracyclization of synthetic teicoplanin-derived heptapeptides and furthermore discern the importance of the OxyE enzyme in maintaining effective cyclization of such peptides bearing 3,5-dihydroxyphenylglycine residues at position 3 in their structures. These results demonstrate the value of chemically synthesized probes for the elucidation of the enzyme mechanism underpinning the complex process of GPA cyclization and furthermore show the utility of the technique for probing the cyclization of non-natural GPA peptides by these powerful biosynthetic enzymes.

Natural products are the greatest source of antimicrobial agents, although their structural complexity often renders synthetic production and diversification of key classes impractical. One pertinent example is the glycopeptide antibiotics (GPAs), which are highly challenging to synthesize due to their heavily cross-linked structures. Here, we report an optimized method that generates >75% tricyclic peptides from synthetic precursors in order to explore the acceptance of novel GPA precursor peptides by these key existent biosynthetic enzymes.

Kistamicin is a divergent member of the glycopeptide antibiotics, a structurally complex class of important, clinically relevant antibiotics often used as the last resort against resistant bacteria. The extensively crosslinked structure of these antibiotics that is essential for their activity makes their chemical synthesis highly challenging and limits their production to bacterial fermentation. Kistamicin contains three crosslinks, including an unusual 15-membered A-O-B ring, despite the presence of only two Cytochrome P450 Oxy enzymes thought to catalyse formation of such crosslinks within the biosynthetic gene cluster. In this study, we characterise the kistamicin cyclisation pathway, showing that the two Oxy enzymes are responsible for these crosslinks within kistamicin and that they function through interactions with the X-domain, unique to glycopeptide antibiotic biosynthesis. We also show that the kistamicin OxyC enzyme is a promiscuous biocatalyst, able to install multiple crosslinks into peptides containing phenolic amino acids., Kistamicin is a structurally divergent glycopeptide antibiotic (GPA) that contains a unique 15-membered A-O-B ring. Here, the authors obtained a crystal structure of the kistamicin OxyA/X-domain complex and analysed the cyclisation cascade leading to the formation of the A-O-B ring.

Structure-activity relationship studies are a highly time-consuming aspect of peptide-based drug development, particularly in the assembly of disulfide-rich peptides, which often requires multiple synthetic steps and purifications. Therefore, it is vital to develop rapid and efficient chemical methods to readily access the desired peptides. We have developed a photolysis-mediated "one-pot" strategy for regioselective disulfide bond formation. The new pairing system utilises two ortho-nitroveratryl protected cysteines to generate two disulfide bridges in less than one hour in good yield. This strategy was applied to the synthesis of complex disulfide-rich peptides such as Rho-conotoxin ρ-TIA and native human insulin.

Halogenase enzymes involved in glycopeptide antibiotic biosynthesis accept aminoacyl-carrier protein substrates., Halogenation plays a significant role in the activity of the glycopeptide antibiotics (GPAs), although up until now the timing and therefore exact substrate involved was unclear. Here, we present results combined from in vivo and in vitro studies that reveal the substrates for the halogenase enzymes from GPA biosynthesis as amino acid residues bound to peptidyl carrier protein (PCP)-domains from the non-ribosomal peptide synthetase machinery: no activity was detected upon either free amino acids or PCP-bound peptides. Furthermore, we show that the selectivity of GPA halogenase enzymes depends upon both the structure of the bound amino acid and the PCP domain, rather than being driven solely via the PCP domain. These studies provide the first detailed understanding of how halogenation is performed during GPA biosynthesis and highlight the importance and versatility of trans-acting enzymes that operate during peptide assembly by non-ribosomal peptide synthetases.

Despite recent advances in the treatment of diabetes mellitus, storage of insulin formulations at 4 °C is still necessary to minimize chemical degradation. This is problematic in tropical regions where reliable refrigeration is not ubiquitous. Some degradation byproducts are caused by disulfide shuffling of cystine that leads to covalently bonded oligomers. Consequently we examined the utility of the non-reducible cystine isostere, cystathionine, within the A-chain. Reported herein is an efficient method for forming this mimic using simple monomeric building blocks. The intra-A-chain cystathionine insulin analogue was obtained in good overall yield, chemically characterized and demonstrated to possess native binding affinity for the insulin receptor isoform B. It was also shown to possess significantly enhanced thermal stability indicating potential application to next-generation insulin analogues.

Natural products such as the glycopeptide antibiotics (GPAs, including vancomycin and teicoplanin) are of great pharmaceutical importance due to their use against Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus. GPAs are assembled in a complex process based on nonribosomal peptide synthesis and late-stage, multistep cross-linking of the linear heptapeptide performed by cytochrome P450 monooxygenases. These P450 enzymes demonstrate varying degrees of substrate selectivity toward the linear peptide precursor, with limited information available about their tolerance regarding modifications to amino acid residues within the essential antibiotic core of the GPA. In order to test the acceptance of altered residues by the P450-catalyzed cyclization cascade, we have explored the use of β-amino acids in both variable and highly conserved positions within GPA peptides. Our results indicate that the incorporation of β-amino acids at the C-terminus of the peptide leads to a dramatic reduction in the efficiency of peptide cyclization by the P450s during GPA biosynthesis, whereas replacement of residue 3 is well tolerated by the same enzymes. These results show that maintaining the C-terminal 3,5-dihydroxyphenylglycine residue is of key importance to maintain the efficiency of this complex and essential enzymatic cross-linking process.

Non-ribosomal peptides contain an array of amino acid building blocks that can present challenges for the synthesis of important intermediates. Here, we report the synthesis of glycopeptide antibiotic (GPA) thioester peptides that retains the crucial stereochemical purity of the terminal phenylglycine residue, which we show is essential for the enzymatic GPA cyclisation cascade.

The mechanism of membrane disruption by melittin (MLT) of giant unilamellar vesicles (GUVs) and live cells was studied using fluorescence microscopy and two fluorescent synthetic analogues of MLT. The N-terminus of one of these was acylated with thiopropionic acid to enable labeling with maleimido-AlexaFluor 430 to study the interaction of MLT with live cells. It was compared with a second analogue labeled at P14C. The results indicated that the fluorescent peptides adhered to the membrane bilayer of phosphatidylcholine GUVs and inserted into the plasma membrane of HeLa cells. Fluorescence and light microscopy revealed changes in cell morphology after exposure to MLT peptides and showed bleb formation in the plasma membrane of HeLa cells. However, the membrane disruptive effect was dependent upon the location of the fluorescent label on the peptide and was greater when MLT was labeled at the N-terminus. Proline at position 14 appeared to be important for antimicrobial activity, hemolysis and cytotoxicity, but not essential for cell membrane disruption.

SummaryA3-APO, a de novo designed branched dimeric proline-rich antimicrobial peptide (PrAMP), is highly effective against a variety of in vivo bacterial infections. We undertook a selective examination of the mechanism for the Gram-negative Escherichia coli bacterial membrane interaction of the monomer (Chex-Arg20), dimer (A3-APO), and tetramer (A3-APO disulfide-linked dimer). All three synthetic peptides were effective at killing E. coli. However, the tetramer was 30-fold more membrane disruptive than the dimer while the monomer showed no membrane activity. Using flow cytometry and high-resolution fluorescent microscopy, it was observed that dimerization and tetramerization of the Chex-Arg20 monomer led to an alteration in the mechanism of action from non-lytic/membrane hyperpolarization to membrane disruption/depolarization. Our findings show that the membrane interaction and permeability of Chex-Arg20 was altered by multimerization.

Intramolecular acyl transfer equilibrium in peptides and proteins has stimulated the development of new methodologies for ligation, aggregation suppression or difficult peptide synthesis. Native chemical ligation or aggregation suppression methodologies are based on an X-to-N acyl transfer of a peptide chain (X = S, O). The reverse reaction from N-to-X has led to exciting developments in solving key synthetic problems such as peptide thioester preparation using Fmoc/tBu strategy. Depending on the target peptide or protein, variations of these methods, which are also based on acyl transfer equilibriums, are now available. In this review, we provide a detailed overview of development and utility of methodologies in peptide chemistry that are based on the control of intramolecular equilibrium. To this end, we outline the scaffolds that are favorable for acyl transfer, the conditions for controlling both sides of the acyl transfer equilibrium and their applications to peptide and protein chemistry. Additional new methodologies have been developed for the synthesis of difficult peptides such as peptide alcohols or head-to-tail cyclic peptides. Promising new applications of intramolecular acyl transfer reactions are also highlighted. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

Bioactive peptides play important roles in metabolic regulation and modulation and many are used as therapeutics. These peptides often possess disulfide bonds, which are important for their structure, function and stability. A systematic network of enzymes--a disulfide bond generating enzyme, a disulfide bond donor enzyme and a redox cofactor--that function inside the cell dictates the formation and maintenance of disulfide bonds. The main pathways that catalyze disulfide bond formation in peptides and proteins in prokaryotes and eukaryotes are remarkably similar and share several mechanistic features. This review summarizes the formation of disulfide bonds in peptides and proteins by cellular and recombinant machinery.

Two series of branched tetramers of the proline-rich antimicrobial peptide (PrAMP), Chex1-Arg20, were prepared to improve antibacterial selectivity and potency against a panel of Gram-negative nosocomial pathogens including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. First, tetramerization was achieved by dithiomaleimide (DTM) conjugation of two C-terminal-cysteine bearing dimers that also incorporated C-terminal peptide chemical modification. DTM-linked tetrameric peptides containing a C-terminal hydrazide moiety on each dimer exhibited highly potent activities in the minimum inhibitory concentration (MIC) range of 0.49-2.33 μm. A second series of tetrameric analogues with C-terminal hydrazide modification was prepared by using alternative conjugation linkers including trans-1,4-dibromo-2-butene, α,α'-dibromo-p-xylene, or 6-bismaleimidohexane to determine the effect of length on activity. Each displayed potent and broadened activity against Gram-negative nosocomial pathogens, particularly the butene-linked tetrameric hydrazide. Remarkably, the greatest MIC activity is against P. aeruginosa (0.77 μm/8 μg mL-1 ) where the monomer is inactive. None of these peptides showed any cytotoxicity to mammalian cells up to 25 times the MIC. A diffusion NMR study of the tetrameric hydrazides showed that the more active antibacterial analogues were those with a more compact structure having smaller hydrodynamic radii. The results show that C-terminal PrAMP hydrazidation together with its rational tetramerization is an effective means for increasing both diversity and potency of PrAMP action.

Structure-activity studies of the insulin superfamily member, relaxin-3, have shown that its G protein-coupled receptor (RXFP3) binding site is contained within its central B-chain α-helix and this helical structure is essential for receptor activation. We sought to develop a single B-chain mimetic that retained agonist activity. This was achieved by use of solid phase peptide synthesis together with on-resin ruthenium-catalyzed ring closure metathesis of a pair of judiciously placed i,i+4 α-methyl, α-alkenyl amino acids. The resulting hydrocarbon stapled peptide was shown by solution NMR spectroscopy to mimic the native helical conformation of relaxin-3 and to possess potent RXFP3 receptor binding and activation. Alternative stapling procedures were unsuccessful, highlighting the critical need to carefully consider both the peptide sequence and stapling methodology for optimal outcomes. Our result is the first successful minimization of an insulin-like peptide to a single-chain α-helical peptide agonist which will facilitate study of the function of relaxin-3.

Heterodimeric peptides linked by disulfide bonds are attractive drug targets. However, their chemical assembly can be tedious, time-consuming, and low yielding. Inspired by the cellular synthesis of pro-insulin in which the two constituent peptide chains are expressed as a single-chain precursor separated by a connecting C-peptide, we have developed a novel chemically cleavable bis-linker tether which allows the convenient assembly of two peptide chains as a single "pro"-peptide on the same solid support. Following the peptide cleavage and post-synthetic modifications, this bis-linker tether can be removed in one-step by chemical means. This method was used to synthesize a drug delivery-cargo conjugate, TAT-PKCi peptide, and a two-disulfide bridged heterodimeric peptide, thionin (7-19)-(24-32R), a thionin analogue. To our knowledge, this is the first report of a one-pot chemically cleavable bis-linker strategy for the facile synthesis of cross-bridged two-chain peptides.

A new strategy for the synthesis of polyamides from polyesters of hydroxyl-containing amino acids using a multi O-N acyl transfer reaction was developed. This original approach allowed the synthesis of three generations of polymers from the same starting monomer. The polymerization of N-benzyloxycarbonyl-serine and its γ-homologated derivative provided the Z-protected polyesters; then the water-soluble polycationic polyesters were obtained by removal of the Z-protecting group; and finally the polyamides were obtained by a base-induced multi O-N acyl transfer, both in aqueous or organic medium. The key step transfer reaction was monitored by the disappearance and appearance of characteristic NMR proton signals and IR bands of polyesters and polyamides.

Novel 5-Z-amino-δ-valerolactone (5-NHZ-VL) was synthesized with an aim to prepare degradable polyesters and copolyesters having amino pendant groups. Following a straightforward and efficient synthetic pathway, 5-NHZ-VL was obtained in only two steps and up to 50% yield. The monomer was fully characterized by 1H NMR, 13C NMR, ESI mass spectrometry, and HPLC. Various conventional conditions were tested for this lactone ring-opening polymerization and led to the novel corresponding poly(5-NHZ-VL) (Mn = 7000 g/mol; PD = 1.2). Following this homopolymerization, 5-NHZ-VL was copolymerized with e-caprolactone to generate a family of copolyesters with an amino-group content ranging from 10 to 80%. Finally, the polyelectrolyte poly(5-NH3+-VL) was recovered by removal of the protecting group under acidic conditions, and integrity of the polyester backbone was confirmed by 1H NMR. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010

Insulin-like peptide 5 (INSL5) has recently been discovered as only the second orexigenic gut hormone after ghrelin. As we have previously reported, INSL5 is extremely difficult to assemble and oxidize into its two-chain three-disulfide structure. The focus of this study was to generate structure-activity relationships (SARs) of INSL5 and use it to develop a potent and simpler INSL5 mimetic with RXFP4 agonist activity. A series of human and mouse INSL5 (hINSL5/mINSL5) analogues were designed and chemically synthesized, resulting in a chimeric INSL5 analogue exhibiting more than 10-fold higher potency (0.35 nM) at human RXFP4 compared with native hINSL5 (4.57 nM). The SAR study also identified a key residue (K(A15)) in the A-chain of mINSL5 that contributes to improved RXFP4 affinity and potency of mINSL5 compared with hINSL5. This knowledge ultimately led us to engineer a minimized hINSL5 mimetic agonist that retains native hINSL5-like RXFP4 affinity and potency at human RXFP4. This minimized analogue was synthesized in 17.5-fold higher yield and in less time compared with hINSL5.

Insulin-like peptide 5 (INSL5) is an orexigenic peptide hormone belonging to the relaxin family of peptides. It is expressed primarily in the L-cells of the colon and has a postulated key role in regulating food intake. Its G protein-coupled receptor, RXFP4, is a potential drug target for treating obesity and anorexia. We studied the effect of modification of the C-terminus of the A and B-chains of human INSL5 on RXFP4 binding and activation. Three variants of human INSL5 were prepared using solid phase peptide synthesis and subsequent sequential regioselective disulfide bond formation. The peptides were synthesized as C-terminal acids (both A- and B-chains with free C-termini, i.e., the native form), amides (both chains as the C-terminal amide) and one analog with the C-terminus of its A-chain as the amide and the C-terminus of the B-chain as the acid. The results showed that C-terminus of the B-chain is more important than that of the A-chain for RXFP4 binding and activity. Amidation of the A-chain C-terminus does not have any effect on the INSL5 activity. The difference in RXFP4 binding and activation between the three peptides is believed to be due to electrostatic interaction of the free carboxylate of INSL5 with a positively charged residue (s), either situated within the INSL5 molecule itself or in the receptor extracellular loops.

Confocal laser scanning microscopy (CLSM) is the most popular technique for mapping the subcellular distribution of a fluorescent molecule and is widely used to investigate the penetration properties of exogenous macromolecules, such as cell-penetrating peptides (CPPs), within cells. Despite the membrane-association propensity of all these CPPs, the signal of the fluorescently labeled CPPs did not colocalize with the plasma membrane. We studied the origin of this fluorescence extinction and the overall consequence on the interpretation of intracellular localizations from CLSM pictures. We demonstrated that this discrepancy originated from fluorescence self-quenching. The fluorescence was unveiled by a “dilution” protocol, i.e. by varying the ratio fluorescent/non-fluorescent CPP. This strategy allowed us to rank with confidence the subcellular distribution of several CPPs, contributing to the elucidation of the penetration mechanism. More generally, this study proposes a broadly applicable and reliable method to study the subcellular distribution of any fluorescently labeled molecules.

Intramolecular acyl transfer equilibrium in peptides and proteins has stimulated the development of new methodologies for ligation, aggregation suppression or difficult peptide synthesis. Native chemical ligation or aggregation suppression methodologies are based on an X-to-N acyl transfer of a peptide chain (X = S, O). The reverse reaction from N-to-X has led to exciting developments in solving key synthetic problems such as peptide thioester preparation using Fmoc/tBu strategy. Depending on the target peptide or protein, variations of these methods, which are also based on acyl transfer equilibriums, are now available. In this review, we provide a detailed overview of development and utility of methodologies in peptide chemistry that are based on the control of intramolecular equilibrium. To this end, we outline the scaffolds that are favorable for acyl transfer, the conditions for controlling both sides of the acyl transfer equilibrium and their applications to peptide and protein chemistry. Additional new methodologies have been developed for the synthesis of difficult peptides such as peptide alcohols or head-to-tail cyclic peptides. Promising new applications of intramolecular acyl transfer reactions are also highlighted. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

An enhanced synthesis of 1,4,8-triazaspiro[4.5]decan-2-one derivatives on SynPhase lanterns using microwave irradiation is reported. In comparison to the conventional heating procedure, microwave irradiation considerably accelerated the formation of the spiroimidazolidinone system.

Intramolecular acyl transfer equilibrium in peptides and proteins has stimulated the development of new methodologies for ligation, aggregation suppression or difficult peptide synthesis. Native chemical ligation or aggregation suppression methodologies are based on an X-to-N acyl transfer of a peptide chain (X = S, O). The reverse reaction from N-to-X has led to exciting developments in solving key synthetic problems such as peptide thioester preparation using Fmoc/tBu strategy. Depending on the target peptide or protein, variations of these methods, which are also based on acyl transfer equilibriums, are now available. In this review, we provide a detailed overview of development and utility of methodologies in peptide chemistry that are based on the control of intramolecular equilibrium. To this end, we outline the scaffolds that are favorable for acyl transfer, the conditions for controlling both sides of the acyl transfer equilibrium and their applications to peptide and protein chemistry. Additional new methodologies have been developed for the synthesis of difficult peptides such as peptide alcohols or head-to-tail cyclic peptides. Promising new applications of intramolecular acyl transfer reactions are also highlighted.

The increasing resistance of pathogens to antibiotics causes a huge clinical burden that places great demands on academic researchers and the pharmaceutical industry for resolution. Antimicrobial peptides, part of native host defense, have emerged as novel potential antibiotic alternatives. Among the different classes of antimicrobial peptides, proline-rich antimicrobial peptides, predominantly sourced from insects, have been extensively investigated to study their specific modes of action. In this review, we focus on recent developments in these peptides. They show a variety of modes of actions, including mechanism shift at high concentration, non-lytic mechanisms, as well as possessing different intracellular targets and lipopolysaccharide binding activity. Furthermore, proline-rich antimicrobial peptides display the ability to not only modulate the immune system via cytokine activity or angiogenesis but also possess properties of penetrating cell membranes and crossing the blood brain barrier suggesting a role as potential novel carriers. Ongoing studies of these peptides will likely lead to the development of more potent antimicrobial peptides that may serve as important additions to the armoury of agents against bacterial infection and drug delivery.