Proteolytic excision and in situ cyclization of a bioactive loop from an REI-VL presentation scaffold.

Covalent cyclization of peptides is an important tool in structure-function analysis of bioactive peptides, because it constrains the molecule to enrich or exclude the receptor-bound conformation. Previously we described a 2-step procedure for cyclizing purified, native peptides in aqueous solution by reacting a Met or Lys side chain with an iodoacetylated N-terminus (Wood SJ, Wetzel R, 1992a, Int J Pept Protein Res 39:533-539). We show here that the cyclization reaction scheme can be extended to peptides excised from proteins by endo-LysC proteolysis, which generates fragments terminating with Lys. To illustrate the method, we used an immunoglobulin VL domain (REI-VL) with an RGD-containing sequence engineered into its CDR3 and flanked by Lys residues. This REI-VL/RGD hybrid displayed an IC50 of 24 nM for ligand competition at the platelet fibrinogen receptor alpha IIb beta 3. The RGD-containing peptide excised by endo-LysC from the REI-VL presentation scaffold exhibited an IC50 of about 50 nM, and the corresponding cyclized peptide, and IC50 of about 10 nM. Significantly, both the N alpha-acylation and the cyclization reactions occur efficiently even in the context of the other endo-LysC fragments of REI-VL, which suggests that the reaction may prove useful in converting mixtures of endo-LysC products of many proteins into the corresponding cyclic peptides in situ.     

Backbone cyclization: A new method for conferring conformational constraint on peptides.

This article describes a new concept of medium- and long-range cyclization of peptides through "backbone cyclization." In this approach, conformational constraints are conferred on a peptide by linking omega-substituted alkylidene chains replacing N(alpha) or C(alpha) hydrogens in a peptidic backbone. Backbone cyclization, which is divided into N-backbone and C-backbone cyclizations, allow for new modes of cyclization in addition to the classical ones that are limited to cyclization through the side chains and/or the amino or carboxyl terminal groups. The article also describes the application of the N-backbone cyclization to the active region of substance P. Conformational constraints of this peptide by the classical cyclization modes led to inactive analogues whereas N-backbone cyclization provided an active, selective, and metabolically stable analogue.     

Generality of peptide cyclization catalyzed by isolated thioesterase domains of nonribosomal peptide synthetases

The C-terminal thioesterase (TE) domains from nonribosomal peptide synthetases (NRPSs) catalyze the final step in the biosynthesis of diverse biologically active molecules. In many systems, the thioesterase domain is involved in macrocyclization of a linear precursor presented as an acyl-S-enzyme intermediate. The excised thioesterase domain from the tyrocidine NRPS has been shown to catalyze the cyclization of a peptide thioester substrate which mimics its natural acyl-S-enzyme substrate. In this work we explore the generality of cyclization catalyzed by isolated TE domains. Using synthetic peptide thioester substrates from 6 to 14 residues in length, we show that the excised TE domain from the tyrocidine NRPS can be used to generate an array of sizes of cyclic peptides with comparable kinetic efficiency. We also studied the excised TE domains from the NRPSs which biosynthesize the symmetric cyclic decapeptide gramicidin S and the cyclic lipoheptapeptide surfactin A. Both TE domains exhibit expected cyclization activity: the TE domain from the gramicidin S NRPS catalyzes head-to-tail cyclization of a decapeptide thioester to form gramicidin S, and the TE domain from the surfactin NRPS catalyzes stereospecific cyclization to form a macrolactone analogue of surfactin. With an eye toward generating libraries of cyclic molecules by TE catalysis, we report the solid-phase synthesis and TE-mediated cyclization of a small pool of linear peptide thioesters. These studies provide evidence for the general utility of TE catalysis as a means to synthesize a wide range of macrocyclic compounds.     

Synthesis of carba vasopressin analogues by solid-phase cyclization

A method is described for the solid-phase cyclization of analogues of arginine vasopressin (AVP) in which one of the sulfur atoms of the disulfide bridge is formally replaced by a methylene group and in which the terminal amino group is formally replaced by a hydrogen atom. The linear precursors of these vasopressin analogues were assembled by a standard Merrifield solid-phase procedure and were cyclized by intramolecular peptide bond formation while the peptide was still attached to the resin support; then the final products were simultaneously deprotected and released from the polymeric support by treatment with liquid hydrogen fluoride. The products of this synthetic procedure were isolated by chromatography and exhibited high biological activities. This method for cyclization of resin-bound peptide is being applied in the synthesis of many other cyclopeptides for conformation and biological studies.     

Chain reversals in model peptides: studies of cystine-containing cyclic peptides. II. Effects of valyl residues and possible i-to-(i + 3) attractive ionic interactions on cyclization of [Cys1], [Cys6] hexapeptides

The synthesis of four N-acetyl N'-methylamide cystine-containing hexapeptides, CVPGVC, CGVVGC, CKPGEC, and CEPGKC, is described. These were used in disulfide-exchange reactions with the peptide CVPGGC as the formal oxidant. The relative propensities for peptide cyclization were thus deduced, and the tendency toward the formation of chain-reversal conformations was established quantitatively. An additional peptide, CVVVVC, was prepared but was never obtained as the cyclic monomer, demonstrating that the formation of chain-reversals in this peptide was of very low probability. Incorporation of pairs of valyl residues decreased the ease of cyclization, but it appeared that conformational flexibility in the cystine-containing hexapeptides may have compensated for substitutions which would have been expected to hinder the adoption of certain beta-turn conformations. The peptides containing ionic residues were cyclized more readily than expected, and this process was relatively insensitive to salt concentration. This observation is discussed with regard to the stabilization of beta-turns by i-to-(i + 3) ionic interactions in peptides and proteins. A method for blocking thiols was introduced as an improvement in the analysis of the equilibrium mixtures.     

Mutants of the zinc ligands of lacticin 481 synthetase retain dehydration activity but have impaired cyclization activity

Lantibiotics are ribosomally synthesized and post-translationally modified peptide antibiotics. The modifications involve dehydration of Ser and Thr residues to generate dehydroalanines and dehydrobutyrines, followed by intramolecular attack of cysteines onto the newly formed dehydro amino acids to produce cyclic thioethers. LctM performs both processes during the biosynthesis of lacticin 481. Mutation of the zinc ligands Cys781 and Cys836 to alanine did not affect the dehydration activity of LctM. However, these mutations compromised cyclization activity when investigated with full length or truncated peptide substrates. Mutation of His725, another residue that is fully conserved in lantibiotic cyclases, to Asn resulted in a protein that still catalyzed dehydration of the substrate peptide and also retained cyclization activity, but at a decreased level compared to that of the wild type enzyme. Collectively, these results show that the C-terminal domain of LctM is responsible for cyclization, that the zinc ligands are critical for cyclization, and that dehydration takes place independently from the cyclization activity. Furthermore, these mutant proteins are excellent dehydratases and provide useful tools to investigate the dehydration activity as well as generate dehydrated peptides for study of the cyclization reaction by wild type LctM.     

A comparative study of backbone versus side chain peptide cyclization: application for HIV-1 integrase inhibitors

Peptide cyclization is an important tool for overcoming the limitations of linear peptides as drugs. Backbone cyclization (BC) has advantages over side chain (SC) cyclization because it combines N-alkylation for extra peptide stability. However, the appropriate building blocks for BC are not yet commercially available. This problem can be overcome by preparing SC cyclic peptide analogs of the most active BC peptide using commercially available building blocks. We have recently developed BC peptides that inhibit the HIV-1 integrase enzyme (IN) activity and HIV-1 replication in infected cells. Here we used this system as a model for systematically comparing the BC and SC cyclization modes using biophysical, biochemical and structural methods. The most potent SC cyclic peptide was active almost as the BC peptide and inhibited IN activity in vitro and blocked IN activity in cells even after 6 days. We conclude that both cyclization types have their respective advantages: The BC peptide is more active and stable, probably due to the N-alkylation, while SC cyclic peptides are easier to synthesize. Due to the high costs and efforts involved in preparing BC peptides, SC may be a more approachable method in many cases. We suggest that both methods are interchangeable.     

Intein-mediated ligation and cyclization of expressed proteins

Protein splicing is a posttranslational processing event that releases an internal protein sequence from a protein precursor. During the splicing process the internal protein sequence, termed an intein, embedded in the protein precursor self-catalyzes its excision and the ligation of the flanking protein regions, termed exteins. The dissection of the splicing pathway, which involves the precise cleavage and formation of peptide bonds, and the identification of key catalytic residues at the splice junctions have led to the modulation of the protein splicing process as a protein engineering tool. Novel strategies have been developed to use intein-catalyzed reactions for the production and manipulation of proteins and peptides. These new approaches have broken down the size limitation barrier of chemical synthetic methods and are less technically demanding. The purpose of this article is to describe how to use self-splicing inteins in protein semisynthesis and backbone cyclization. The first two sections of the article provide a brief review of the distinct chemical steps that underlie protein splicing and intein enabled technology.     

NMR studies on Ni(II) induced cyclization of a histidine-tagged peptide.

A linear decapeptide, HGASYQDLGH, was synthesized and used as a model to evaluate the effect of nickel addition upon non-covalent backbone cyclization. The NMR data, obtained for the peptide in the presence of the metal ion, support the existence of predominant folded structures in solution, where the two His residues are maintained close to each other. These results suggest that insertion of even a single His residue at each peptide terminus can be used efficiently to reduce peptide flexibility without any backbone modification.     

Effect of backbone cyclization on protein folding stability: chain entropies of both the unfolded and the folded states are restricted

Circular versions of a large number of proteins have been designed by connecting the N and C termini via peptide linkers. A motivation for these designs is the assumed enhancement in folding stability, because backbone cyclization reduces the chain entropy of the unfolded state. Here, it is recognized that backbone cyclization also reduces the chain entropy of a flexible peptide linker in the folded state. Specifically, the end-to-end distance of the linker is restricted to fluctuations around the average displacement between the N and C termini of the folded protein. The balance of the chain-entropy reductions in the folded and unfolded states is used to predict the change in the unfolding free energy, deltadeltaG(cycl), by backbone cyclization. Predicted values of deltadeltaG(cycl) are in quantitative agreement with results of a careful study on cyclizing the 34 residue PIN1 WW domain by linkers with two to seen residues. The experimental results of an optimal linker length l=4 and a maximum stabilization of 1.7 kcal/mol are reproduced. Calculations of deltadeltaG(cycl) for a broad selection of circular proteins suggest that the stabilizing effect of backbone cyclization is modest, reflecting entropy reductions in both the unfolded and the folded states.     

Chemical synthesis and folding pathways of large cyclic polypeptides: studies of the cystine knot polypeptide kalata B1.

Kalata B1 is a member of a new family of polypeptides, isolated from plants, which have a cystine knot structure embedded within an amide-cyclized backbone. This family of molecules are the largest known cyclic peptides, and thus, the mechanism of synthesis and folding is of great interest. To provide information about both these phenomena, we have synthesized kalata B1 using two distinct strategies. In the first, oxidation of the cysteine residues of a linear precursor peptide to form the correct disulfide bonds results in folding of the three-dimensional structure and preorganization of the termini in close proximity for subsequent cyclization. The second approach involved cyclization prior to oxidation. In the first method, the correctly folded peptide was produced only in the presence of partially hydrophobic solvent conditions. These conditions are presumably required to stabilize the surface-exposed hydrophobic residues. However, in the synthesis involving cyclization prior to oxidation, the cyclic reduced peptide folded to a significant degree in the absence of hydrophobic solvents and even more efficiently in the presence of hydrophobic solvents. Cyclization clearly has a major effect on the folding pathway and facilitates formation of the correctly disulfide-bonded form in aqueous solution. In addition to facilitating folding to a compact stable structure, cyclization has an important effect on biological activity as assessed by hemolytic activity.     

On-bead cyclization in a combinatorial library of 15,625 octapeptides

Combinatorial peptide libraries prepared by split-and-mix synthesis on solid support can be decoded by amino acid analysis (AAA) using the TAGSFREE method, which assigns variable amino acids to 'unique pair' positions. The method was used here to investigate on-bead cyclization in a library of 15,625 octapeptides X(8)X(7)X(6)X(5)X(4)-Lys-X(2)-glu(beta-Ala-beta-Ala-TentaGel Macrobead)-OAllyl, anchored via the side-chain carboxylate of the d-glutamate. Cyclization was carried out by amide bond formation between the free N-terminus and the alpha-carboxyl group of d-glutamate after selective removal of the Fmoc and allyl protecting groups, and followed using the TNBS test for free amines. Fast-cyclizing sequences often contained a turn element, in particular Ala-(Asp/Thr)-Pro at X(8)-X(7)-X(6), and phenylalanine at X(2). Slow-cyclizing sequences contained predominantly basic and polar residues, in particular Arg-His-Ser at X(7)-X(6)-X(5) and threonine at X(8). Fast-cyclizing sequences gave higher preparative yields of cyclic peptides (22-26% purified yields) than slow-cyclizing sequences (6-8%), showing that fast reaction is associated with efficient cyclization. This experiment demonstrates the first use of a TAGSFREE library of cyclic peptides.     

Synthesis of cyclopentapeptides and cycloheptapeptides by DEPBT and the influence of some factors on cyclization.

Three cyclic peptides - cyclo(GlyAlaTyrLeuAla), cyclo(GlyProTyrLeuAla) and cyclo(GlyTyrGlyGlyProPhePro) - isolated and identified from medicinal herbs were chosen as model cyclic peptides to study the influence of the linear precursors and coupling reagents on cyclization. The 17 linear precursors of these three cyclic peptides were synthesized and cyclized using 3-(diethoxyphosphoryloxy)-(1-3)-benzotriazin-4 (3H)-one (DEPBT) as the major coupling reagent. The present work shows that: (i) the effects of linear peptide precursors on the cyclization are complex but some guidelines for choosing suitable precursor for cyclization could be considered; and (ii) DEPBT results in a higher cyclization yield compared with other coupling reagents. In addition, it was confirmed that peptides containing alternating D and L residues favor cyclization.     

Excision of the epothilone synthetase B cyclization domain and demonstration of in trans condensation/cyclodehydration activity

The epothilones are potent anticancer natural products produced by a polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) hybrid involving proteins EpoA-F. The single NRPS module of the epothilone assembly line, EpoB, is a distinct subunit of approximately 160 kDa and consists of four successive domains: cyclization, adenylation, oxidation, and peptidyl carrier protein (Cy-A-Ox-PCP). The cyclization domain is responsible for introduction of the thiazoline heterocycle into the growing polyketide/nonribosomal peptide chain from the precursors malonyl-CoA and cysteine through the multiple steps of condensation, cyclization, and dehydration. This enzyme-bound thiazoline intermediate is subsequently oxidized to a thiazole by the EpoB Ox domain. The EpoB module was dissected to provide 57 kDa EpoB(Cy) and 102 kDa EpoB(A-Ox-PCP) as subunit fragments to evaluate Cy as a free-standing domain. EpoB was reconstituted by these fragments in trans to generate the methylthiazole product. Using this system, apparent kinetic constants for the upstream acyl donor EpoA(ACP) and EpoB(Cy) were determined, providing a measure of affinity for the naturally occurring interface of the amino terminus of EpoB and the EpoA carboxy terminus. Site-directed mutants in excised EpoB(Cy) were prepared and used to examine residues involved in condensation and heterocycle formation. This work demonstrates the ability to define a functional Cy domain by excision from its native NRPS module, and examine both its protein-protein interactions and mechanism of activity.     

Abietadiene synthase catalysis: conserved residues involved in protonation-initiated cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate

Abietadiene synthase catalyzes two sequential, mechanistically distinct cyclization reactions in the formation of a mixture of abietadiene double bond isomers as the committed step in resin acid biosynthesis. Each reaction is carried out at a separate active site residing in a structurally distinct domain, and the reactions are kinetically separable. The first cyclization reaction is initiated by protonation of the terminal double bond of the universal diterpene precursor, geranylgeranyl diphosphate. The pH dependence of the overall reaction is consistent with an acid-base catalytic mechanism, and a divalent metal ion plays a role in this reaction probably by binding the diphosphate moiety to assist in positioning the substrate for catalysis. A putative active site for the protonation-initiated cyclization was defined by modeling abietadiene synthase and locating the DXDD motif previously shown to be involved in this reaction. A number of charged and aromatic residues, which are highly conserved in mechanistically related diterpene cyclases, line the putative active site. Alanine substitutions were made for each of these residues, as were asparagine and glutamate substitutions for the aspartates of the DXDD motif. Kinetic evaluation confirmed the involvement of most of the targeted residues in the reaction, and analysis of mutational effects on the pH-activity profile and affinity for a transition state analogue suggested specific roles for several of these residues in catalyzing the cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate. A functional role was also suggested for the cryptic insertional element found in abietadiene synthase and other diterpene synthases that carry out similar protonation-initiated cyclizations.     

Influence of ions on cyclization of the amino terminal glutamine residues of tryptic peptides of streptococcal PepM49 protein. Resolution of cyclized peptides by HPLC and characterization by mass spectrometry

RPHPLC of the tryptic digest of lysine blocked group A streptococcal PepM49 protein (DHP-PepM49) consistently yielded, among others, two pairs of peptides which were well resolved, eluted in tandem, and had identical amino acid compositions. In each pair, the earlier eluting peptide was readily amenable to sequencing and yielded an amino-terminal glutamine whereas the later eluting peptide could not be sequenced. Mass spectral analysis revealed that each of these pairs of peptides differed in mass corresponding to the loss of ammonia. These data suggested that the later eluting peptide in each pair is a result of cyclization of the amino-terminal glutamine residue to pyroglutamic acid, which apparently leads to an increase in the hydrophobicity of the peptide. A kinetic analysis of the tryptic digestion of the DHP-PepM49 protein revealed that the cyclized form of the peptides were essentially absent during the initial time and increased with time of incubation, with a concomitant decrease in the uncyclized form. In 0.2 M ammonium bicarbonate at 37 degrees, nearly 44% conversion of the glutaminyl peptides to the pyroglutamyl peptides was observed in 24 h. This conversion was accelerated in sodium phosphate buffer relative to that in ammonium bicarbonate whereas it had a significantly lower rate in ammonium acetate buffer. The conversion was also temperature dependent, with essentially no cyclization at 0 degree, in all the three buffers. Thus, an extended digestion at 0 degree or a brief digestion at 37 degrees in ammonium acetate was found to be a suitable condition for limiting the cyclization of amino-terminal glutamine residues of PepM49 peptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solid-phase synthesis and cyclization of a large branched peptide from IgG Fc with affinity for Fc gammaRI.

A solid phase approach has been used to synthesize a large branched disulphide peptide from IgG Fc, Ac-F-C*-A-K-V-N-N-K-D-L-P-A-P-I-E-K(Ac-E-L-L-G-G-P-S-V-F)-C*-I-NH2. This peptide combines the lower hinge region of IgG and a proximal beta-hairpin loop, both implicated in binding to Fc gammaRI. Solid phase Tl(tfa)3 cyclization of the linear branched peptide resulted in a poor yield of cyclic hinge-loop peptide (11%) most likely due to steric hindrance caused by the branch. However, if addition of the branch was preceded by solid phase Tl(tfa)3 cyclization of the loop, the yield was excellent at 75%. Cyclic hinge-loop peptide was active in displacing IgG2a from Fc gammaRI expressed on monocyte cell lines with an IC50 of 40 microM, whereas the linear form of this peptide was inactive. The Fc hinge-loop peptide demonstrates the potential for a non-mAb high affinity, immunomodulatory ligand for Fc gammaRI.     

Novel cyclization chemistry especially suited for biologically derived, unprotected peptides.

A novel method is described for the cyclization of peptides--or segments of polypeptides--which requires a free N-terminal alpha-amino group and a distal amino acid residue containing a nucleophilic side chain. The reaction is conducted in two steps, both in the aqueous phase. The first step involves acylation of the N-terminal alpha-amino group with iodoacetic anhydride at pH 6. This acylation reaction has greater than 90% specificity for peptide alpha-amino groups and gives no alkylation of Arg, His, Lys or Met by the iodoacetate side product (R. Wetzel et al., Bioconjugate Chem., 1, 114-122, 1990). In the second step, the acylation reaction mixture or the isolated iodoacetyl-peptide is incubated at room temperature to give the cyclic peptide formed by reaction of the nucleophilic side chain with the iodoacetyl moiety. The pH dependence of the cyclization reaction by Met, Lys, Arg or His is consistent with the pKa of the nucleophilic side chain. Thus, peptides containing Met plus other nucleophilic amino acids should preferentially cyclize via Met at low pH. In this paper, preparation of cyclic peptides containing 3-6 amino acids is described; the full range of ring sizes and sequences which can undergo this cyclization has not been further explored. Preliminary results suggest that this method is also fairly general with respect to the amino acid sequence being cyclized. The reaction appears to be particularly suited for cyclization via Lys and Met side chains. All of the cyclized products are sufficiently stable for many biological applications.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and kinetics of cyclization of MHC class II-derived cyclic peptide vaccine for diabetes.

Conformationally constrained cyclic peptides are known to be better vaccines because of their ability to mimic the native structure of a protein against which an immune response is sought. To test the hypothesis of using conformationally constrained, disease-associated, MHC-derived peptides as vaccines for the prevention of type I diabetes, a 22 amino acid nonobese diabetic(NOD) mouse MHC class II-derived synthetic peptide was cyclized by the formation of end-to-end disulfide bonds and used to prevent diabetes and insulitis in NOD mice. The peptide was synthesized by Fmoc chemistry and cyclized using two methods: a commercially available cyclizing resin (Ekathiox) and air oxidation. When a 10 m excess of resin was used, the Ekathiox yielded a substantial amount of cyclic peptide with few or no side reactions. The kinetics of cyclization by air oxidation at different temperatures indicated that increasing both temperature and pH decreased the cyclization time significantly. Air oxidation at pH 10 at 37-55 degrees C yielded the desired product within 2 h.     

Selective and facile cyclization of N-chloroacetylated peptides from the C4 domain of HIV Gp120 in LiCl/DMF solvent systems.

Lithium salts have been reported to mediate the solubilization of peptides in organic solvents in 1989 (Seebach, D., Thaler, A. & Beck, A. K. Helv. Chim. Acta 1989; 72, 857-867). The use of Li salts in an organic solvent to influence cyclization of a reactive peptide that only polymerizes in an aqueous solvent, has not been reported. Here, the selective and facile cyclization of N-chloroacetylated, C-cysteine amide peptides from the C4 domain of HIV-1 gp120 in LiCl/DMF solvent systems is demonstrated. The addition of stoichiometric amounts of Tris base to 1 mg/mL peptide in LiCl/DMF solutions was sufficient to drive the cyclization to completion within 3 h at ambient temperatures. Cyclic peptides were the only detectable reaction products and these were confirmed using reversed-phase HPLC and mass spectrometric analyses of the final products. In aqueous solutions at pH 7.4, only polymers were obtained as judged by HPLC and SDS-PAGE. The method of using Li salts in an organic solvent to enhance the cyclization of unprotected amphipathic peptides may be useful in many situations beyond those described here.