Isolation and characterization of two novel peptide amides originating from myelin basic protein in bovine brain

During a systematic search for peptides that possess the C-terminal amide structure, two novel peptide amides, one with a tyrosine amide and the other with an alanine amide were isolated from bovine brain by acid extraction and sequential steps of reversed phase HPLC. Microsequence, amino acid and mass spectral analyses revealed the structures: Ac-Ala-Ala-Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-amide and Ac-Ala-Ala-Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-Leu-Ala-Ser-Ala-amide. These 12 and 16 residues peptides had the primary structure identical to the N-terminal fragment of myelin basic protein (MBP). The peptides were therefore designated myelin peptide amide-12 (MPA-12) and-16 (MPA-16). Unlike other amidated peptides, MPA might be generated from MBP by hydroxyl radicals produced via a Fenton reaction in situ. However, this unique amidation seems to occur exclusively to MBP in a site specific manner in the brain.     

Fmoc-based synthesis of the human CC chemokine CCL14/HCC-1 by SPPS and native chemical ligation

The CC chemokine CCL14/HCC-1(9-74), a 66-residue polypeptide containing two disulfide bonds, was recently discovered from a human hemofiltrate peptide library as a high-affinity ligand of the chemokine receptors CCR1 and CCR5. It has been shown to inhibit HIV infection by blocking CCR5. Using Fmoc methodology, we report the chemical synthesis of CCL14/HCC-1 by conventional stepwise solid-phase peptide synthesis (SPPS) and, alternatively, native chemical ligation. To optimize SPPS of CCL14/HCC-1, difficult sequence regions were identified by mass spectrometry, in order to obtain a crude tetrathiol precursor suitable for oxidative disulfide formation. For synthesis of CCL14/HCC-1 by native chemical ligation, the peptide was divided into two segments, CCL14/HCC-1(9-39) and CCL14/HCC-1(40-74), the latter containing a cysteine residue at the amino-terminus. The synthesis of the thioester segment was carried out comparing a thiol linker with a sulfonamide safety-catch linker. While the use of the thiol linker led to very low overall yields of the desired thioester, the sulfonamide linker was efficient in obtaining the 31-residue thioester of CCL14/HCC-1(9-39), suggesting a superior suitability of this linker in generating larger thioesters using Fmoc chemistry. The thioester of CCL14/HCC-1 was subsequently ligated with the cysteinyl segment to the full-length chemokine. Disulfides were introduced in the presence of the redox buffer cysteine/cystine. The products of both SPPS and native chemical ligation were identical. The use of a sulfonamide safety-catch linker enables the Fmoc synthesis of larger peptide thioesters, and is thus useful to generate arrays of larger polypeptides.     

Amidation of growth hormone releasing factor (1-29) by serine carboxypeptidase catalysed transpeptidation

The applicability of serine carboxypeptidase catalysed transpeptidation reactions, using amino acid amides as nucleophiles, for C-terminal amidation of peptides has been investigated. With the aim of converting an unamidated precursor into GRF(1-29)-NH2, an interesting biologically active derivative of growth hormone releasing factor, a number of model reactions were initially investigated. In such a transpeptidation reaction, where the C-terminal amino acid is replaced by the amino acid amide, used as nucleophile, the C-terminal amino acid residue of the substrate can be chosen freely since it functions as leaving group and does not constitute part of the product. Since the C-terminal sequence of GRF(1-29)-NH2 is -Met-Ser-Arg-NH2 the model reactions Bz-Met-Ser-X-OH (X = Ala, Leu, Arg) + H-Arg-NH2----Bz-Met-Ser-Arg-NH2 + H-X-OH were first studied. With carboxypeptidase Y and X = Ala or Leu the amidated product could be obtained of 98% and 41%, respectively. With carboxypeptidase W-II and X = Arg a yield of no more than 72% could be obtained. The choice of Ala as leaving group in combination with carboxypeptidase Y therefore appeared optimal. With the longer peptide Bz-Leu-Gln-Asp-Ile-Met-Ser-Ala-OH the amidated product could be obtained in a yield of 78%, using carboxypeptidase Y, the only other product being Bz-Leu-Gln-Asp-Ile-Met-Ser-OH, formed due to the competing hydrolysis reaction. The full length peptide GRF(1-28)-Ala-OH was synthesized by the continuous flow polyamide solid-phase method.(ABSTRACT TRUNCATED AT 250 WORDS)     

Making Ends Meet: Microwave-Accelerated Synthesis of Cyclic and Disulfide Rich Proteins Via In Situ Thioesterification and Native Chemical Ligation

The development of synthetic methodologies for cyclic peptides is driven by the discovery of cyclic peptide drug scaffolds such as the plant-derived cyclotides, sunflower trypsin inhibitor 1 (SFTI-1) and the development of cyclized conotoxins. Currently, the native chemical ligation reaction between an N-terminal cysteine and C-terminal thioester group remains the most robust method to obtain a head-to-tail cyclized peptide. Peptidyl thioesters are effectively generated by Boc SPPS. However, their generation is challenging using Fmoc SPPS because thioester linkers are not stable to repeated piperidine exposure during deprotection. Herein we describe a Fmoc-based protocol for synthesizing cyclic peptides adapted for microwave assisted solid phase peptide synthesis. The protocol relies on the linker Di-Fmoc-3,4-diaminobenzoic acid, and we demonstrate the use of Gly, Ser, Arg and Ile as C-terminal amino acids (using HBTU and HATU as coupling reagents). Following synthesis, an N-acylurea moiety is generated at the C-terminal of the peptide; the resin bound acylurea peptide is then deprotected and cleaved from the resin. The fully deprotected peptide undergoes thiolysis in aqueous buffer, generating the thioester in situ. Ultimately, the head-to-tail cyclized peptide is obtained via native chemical ligation. Two naturally occurring cyclic peptides, the prototypical Möbius cyclotide kalata B1 and SFTI-1 were synthesized efficiently, avoiding potential branching at the diamino linker, using the optimized protocol. In addition, we demonstrate the possibility to use the approach for the synthesis of long and synthetically challenging linear sequences, by the ligation of two truncated fragments of a 50-residue long plant defensin.     

Fmoc-based synthesis of the human CC chemokine CCL14/HCC-1 by SPPS and native chemical ligation

Summary The CC chemokine CCL14/HCC-1(9–74), a 66-residue polypeptide containing two disulfide bonds, was recently discovered from a human hemofiltrate peptide library as a high-affinity ligand of the chemokine receptors CCR1 and CCR5. It has been shown to inhibit HIV infection by blocking CCR5. Using Fmoc methodology, we, report the chemical synthesis of CCL14/HCC-1 by conventional stepwise solid-phase peptide synthesis (SPPS) and, alternatively, native chemical ligation. To optimize SPPS of CCL14/HCC-1, difficult sequence regions were identified by mass spectrometry, in order to obtain a crude tetrathiol precursor suitable for oxidative disulfide formation. For synthesis of CCL14/HCC-1 by native chemical ligation, the peptide was divided into two segments, CCL14/HCC-1(9–39) and CCL14/HCC-1(40–74), the latter containing a cysteine residue at the amino-terminus. The synthesis of the thioester segment was carried out comparing a thiol linker with a sulfonamide safety-catch linker. While the use of the thiol linker led to very low overall yields of the desired thioester, the sulfonamide linker was efficient in obtaining the 31-residue thioester of CCL14/HCC-1(9–39), suggesting a superior suitability of this linker in generating larger thioesters using Fmoc chemistry. The thioester of CCL14/HCC-1 was subsequently ligated with the cysteinyl segment to the full-length chemokine. Disulfides were introduced in the presence of the redox buffer cysteine/cystine. The products of both SPPS and native chemical ligation were identical. The use of a sulfonamide safety-catch linker enables the Fmoc synthesis of larger peptide thioesters, and is thus useful to generate arrays of larger polypeptides.     

Investigation of peptide thioester formation via N→Se acyl transfer

Native chemical ligation is widely used for the convergent synthesis of proteins. The peptide thioesters required for this process can be challenging to produce, particularly when using Fmoc‐based solid‐phase peptide synthesis. We have previously reported a route to peptide thioesters, following Fmoc solid‐phase peptide synthesis, via an N→S acyl shift that is initiated by the presence of a C‐terminal cysteine residue, under mildly acidic conditions. Under typical reaction conditions, we occasionally observed significant thioester hydrolysis as a consequence of long reaction times (~48 h) and sought to accelerate the reaction. Here, we present a faster route to peptide thioesters, by replacing the C‐terminal cysteine residue with selenocysteine and initiating thioester formation via an N→Se acyl shift. This modification allows thioester formation to take place at lower temperatures and on shorter time scales. We also demonstrate how application of this strategy also accelerates peptide cyclization, when a linear precursor is furnished with an N‐terminal cysteine and C‐terminal selenocysteine. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Sequential processing reactions in the formation of hormone amides

The substrate specificity of an enzyme with amidating activity, present in porcine pituitary, was investigated by examining its ability to convert the synthetic peptides D-Tyr-Val-Gly and D-Tyr-Val-Gly-Lys-Arg to the dipeptide amide D-Tyr-Val-CONH2. The purified enzyme catalysed the amidation reaction with the tripeptide but did not accept the pentapeptide as a substrate. With the mixture of enzymes present in a membrane fraction from porcine pituitary or the enzymes in a secretory granule fraction, both the tripeptide and pentapeptide substrates gave rise to D-Tyr-Val amide; the formation of dipeptide amide from the pentapeptide, however, involved a latency period after which amidation occurred at a similar rate with the two substrates. Evidence was obtained that arginine and lysine were released from the C terminus of the pentapeptide before amidation took place since the rate of formation of dipeptide amide was reduced at pH values that were compatible with amidation but unfavourable to the action of carboxypeptidase H. In addition formation of the dipeptide amide from the pentapeptide was blocked by guanidinoethylmercaptosuccinic acid and glycylarginine, which are inhibitors of carboxypeptidase enzymes. The experiments demonstrate that removal of basic residues from the C terminus of a peptide and amidation at C-terminal glycine are reactions that take place consecutively. These prohormone-processing reactions, which are intrinsic to the formation of hormone amides, did not synergise.     

Isolation and characterization of a novel peptide amide from porcine brain.

Peptide with C-terminal tyrosine amide was isolated from porcine brain by acid extraction and sequential steps of reverse phase HPLC. Microsequence, amino acid and mass spectral analyses revealed the structure: Ac-Ala-Ser-Glu-Lys-Arg-Pro-Ser-Glu-Arg-His-Gly-Ser-Lys- Tyr-amide. Since this peptide had the identical sequence to N-terminus of porcine myelin basic protein (pMBP) 1-14, we have designated porcine myelin peptide amide 14 (pMPA14). The final HPLC step yielded 20 micrograms of homogeneous peptide preparation from 20 kg brain tissue. Unlike other amidated peptides, pMPA14 may be produced by non enzymatic mechanism or unknown amidating enzyme. This unique amidation seems to occur exclusively to MBP in the brain.     

C-Terminus of the B-Chain of Relaxin-3 Is Important for Receptor Activity

Human relaxin-3 is a neuropeptide that is structurally similar to human insulin with two chains (A and B) connected by three disulfide bonds. It is expressed primarily in the brain and has modulatory roles in stress and anxiety, feeding and metabolism, and arousal and behavioural activation. Structure-activity relationship studies have shown that relaxin-3 interacts with its cognate receptor RXFP3 primarily through its B-chain and that its A-chain does not have any functional role. In this study, we have investigated the effect of modification of the B-chain C-terminus on the binding and activity of the peptide. We have chemically synthesised and characterized H3 relaxin as C-termini acid (both A and B chains having free C-termini; native form) and amide forms (both chains’ C-termini were amidated). We have confirmed that the acid form of the peptide is more potent than its amide form at both RXFP3 and RXFP4 receptors. We further investigated the effects of amidation at the C-terminus of individual chains. We report here for the first time that amidation at the C-terminus of the B-chain of H3 relaxin leads to significant drop in the binding and activity of the peptide at RXFP3/RXFP4 receptors. However, modification of the A-chain C-terminus does not have any effect on the activity. We have confirmed using circular dichroism spectroscopy that there is no secondary structural change between the acid and amide form of the peptide, and it is likely that it is the local C-terminal carboxyl group orientation that is crucial for interacting with the receptors.     

Synthesis of peptide thioesters via an N–S acyl shift reaction under mild acidic conditions on an N‐4,5‐dimethoxy‐2‐mercaptobenzyl auxiliary group

An efficient method of peptide thioester synthesis is described. The reaction is based on an N‐4,5‐dimethoxy‐2‐mercaptobenzyl (Dmmb) auxiliary‐assisted N‐S acyl shift reaction after assembling a peptide chain by Fmoc‐solid phase peptide synthesis. The Dmmb‐assisted N‐S acyl shift reaction proceeded efficiently under mildly acidic conditions, and the peptide thioester was obtained by treating the resulting S‐peptide with sodium 2‐mercaptoethanesulfonate. No detectable epimerization of the amino acid residue adjacent to the thioester moiety in the case of Leu was found. The reactions were also amenable to the on‐resin preparation of peptide thioesters. The utility was demonstrated by the synthesis of a 41‐mer peptide thioester, a phosphorylated peptide thioester and a 33‐mer peptide thioester containing a trimethylated lysine residue. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

The enzymatic formation of novel bile acid primary amides

Bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the copper-, ascorbate-, and O(2)-dependent cleavage of C-terminal glycine-extended peptides and N-acylglycines to the corresponding amides and glyoxylate. The alpha-amidated peptides and the long-chain acylamides are hormones in humans and other mammals. Bile acid glycine conjugates are also substrates for PAM leading to the formation of bile acid amides. The (V(MAX)/K(m))(app) values for the bile acid glycine conjugates are comparable to other known PAM substrates. The highest (V(MAX)/K(m))(app) value, 3.1 +/- 0.12 x 10(5) M(-1) s(-1) for 3-sulfolithocholylglycine, is 6.7-fold higher than that for d-Tyr-Val-Gly, a representative peptide substrate. The time course for O(2) consumption and glyoxylate production indicates that bile acid glycine conjugate amidation is a two-step reaction. The bile acid glycine conjugate is first converted to an N-bile acyl-alpha-hydroxyglycine intermediate which is ultimately dealkylated to the bile acid amide and glyoxylate. The enzymatically produced bile acid amides and the carbinolamide intermediates were characterized by mass spectrometry and two-dimensional (1)H-(13)C heteronuclear multiple quantum coherence NMR.     

An o-nitrobenzyl scaffold for peptide ligation: synthesis and applications

Chemical ligation approaches facilitate the chemoselective assembly of unprotected peptides in aqueous solution. Here, two photolabile auxiliaries are described that enlarge the applicability of native chemical ligation to non-cysteine targets. The auxiliaries, designed to allow reaction with thioester peptides, generate an amide bond between the two initial fragments. The o-nitrobenzyl tertiary benzylamide that is formed at the ligation junction can be transformed into a native amide group under mild photolysis conditions. The veratryl auxiliary was found to be excessively labile during peptide purification and ligation. However, the auxiliary based on the o-nitrobenzyl group shows all the necessary properties for a general application in routine peptide and protein synthesis. In addition, the auxiliary linked to the N-terminus can be efficiently photolyzed, suggesting a new approach for the generation of photocaged amines. Synthesis, solid phase introduction onto peptide chains, ligation properties and photolysis in water are described, and a careful study of compatibility of the method with potentially fragile peptide side chains is reported.     

Fmoc-Protein Synthesis: Preparation of Peptide Thioesters Using a Side-Chain Anchoring Strategy

A side-chain anchoring approach for preparation of peptide thioesters by Fmoc SPPS is reported. This strategy involves the side-chain anchoring of trifunctional amino acids, such as Lys, Glu, Gln, Asp and Asn, for peptide elongation and the post-chain assembly introduction of thioester functionality. This approach allows for the use of standard nucleophilic Fmoc peptide synthesis cycles, which are generally incompatible with thioester-based resin-linkages. The strategy was successfully demonstrated by the straightforward Fmoc syntheses of a model RANTES(1--33) thioester peptide. The Fmoc prepared RANTES(1--33) thioester peptide was then ligated to RANTES(34--68), folded and purified to give the RANTES protein.     

Peptide Thioester Synthesis via an Auxiliary-Mediated N–S Acyl Shift Reaction in Solution

The 4,5-dimethoxy-2-mercaptobenzyl (Dmmb) group attached to a main chain amide in a peptide is easily transformed into an S-peptide via an intramolecular N–S acyl shift reaction under acidic conditions, and the S-peptide produces a peptide thioester through an intermolecular thiol–thioester exchange reaction. In order to develop a method for efficiently preparing peptide thioesters based on the N–S acyl shift reaction, the factors involved in this process were analyzed in detail. The general features of the transformation at the Dmmb group attached amide bond in a trifluoroacetic acid (TFA) solution and the generation of a peptide thioester were examined by ¹³C-NMR spectral measurements, reversed-phase (RP) HPLC analyses, mass measurements, and amino acid analyses. The methoxy group of the Dmmb group was not essential for the N–S acyl shift reaction, but played a role in stabilizing the thioester form. The addition of water to the TFA solution accelerated the N–S acyl shift reaction mediated by the Dmmb group and also suppressed the acid-catalyzed cleavage of the Dmmb group. A peptide thioester was produced from the S-peptide via an intermolecular thiol–thioester exchange reaction with minimal epimerization of the amino acid residue that constituted the thioester bond. Undesirable side reactions, such as the hydrolysis of the thioester bond and an S–N acyl shift reaction occurred during the synthetic process, which is a subject of further investigation.     

Native N-glycopeptide thioester synthesis through N→S acyl transfer

Peptide thioesters are important tools for the total synthesis of proteins using native chemical ligation (NCL). Preparation of glycopeptide thioesters, that enable the assembly of homogeneously glycosylated proteins, is complicated by the perceived fragile nature of the sugar moiety. Herein, we demonstrate the compatibility of thioester formation via N→S acyl transfer with native N-glycopeptides and report observations that will aid in their preparation.     

Epimerization in peptide thioester condensation

Peptide segment couplings are now widely utilized in protein chemical synthesis. One of the key structures for the strategy is the peptide thioester. Peptide thioester condensation, in which a C‐terminal peptide thioester is selectively activated by silver ions then condensed with an amino component, is a powerful tool. But the amino acid adjacent to the thioester is at risk of epimerization. During the preparation of peptide thioesters by the Boc solid‐phase method, no substantial epimerization of the C‐terminal amino acid was detected. Epimerization was, however, observed during a thioester–thiol exchange reaction and segment condensation in DMSO in the presence of a base. In contrast, thioester–thiol exchange reactions in aqueous solutions gave no epimerization. The epimerization during segment condensation was significantly suppressed with a less polar solvent that is applicable to segments in thioester peptide condensation. These results were applied to a longer peptide thioester condensation. The epimer content of the coupling product of 89 residues was reduced from 27% to 6% in a condensation between segments of 45 and 44 residues for the thioester and the amino component, respectively. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Design,synthesis and biological evaluation of a novel class of anticancer agents: Anthracenylisoxazole lexitropsin conjugates

The synthesis and in vitro anti-tumor 60 cell lines screen of a novel series of anthracenyl isoxazole amides (AIMs) (While not a strict acronym, the designation AIM is in honor of the memory of Professor Albert I. Meyers.) (22–33) are described. The molecules consist of an isoxazole that pre-organizes a planar aromatic moiety and a simple amide and/or lexitropsin-oligopeptide. The new conjugate molecules were prepared via doubly activated amidation modification of Weinreb’s amide formation technique, using SmCl₃ as an activating agent which produces improved yields for sterically hindered 3-aryl-4-isoxazolecarboxylic esters. The results of the National Cancer Institute’s (NCI) 60 cell line screening assay show a distinct structure activity relationship (SAR), wherein a trend of the highest activity for molecules with one N-methylpyrrole peptide. Evidence consistent with a mechanism of action via the interaction of these compounds with G-quadruplex (G4) DNA and a structural based rational for the observed selectivity of the AIMs for G4 over B-DNA is presented.     

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.     

The importance of the amide bond nearest the thiol group in enzymatic reactions of coenzyme A

Analogues of coenzyme A (CoA) and of CoA thioesters have been prepared in which the amide bond nearest the thiol group has been modified. An analogue of acetyl-CoA in which this amide bond is replaced with an ester linkage was a good substrate for the enzymes carnitine acetyltransferase, chloramphenicol acetyltransferase, and citrate synthase, with K(m) values 2- to 8-fold higher than those of acetyl-CoA and V(max) values from 14 to >80% those of the natural substrate. An analogue in which an extra methylene group was inserted between the amide bond and the thiol group showed less than 4-fold diminished binding to the three enzymes but exhibited less than 1% activity relative to acetyl-CoA with carnitine acetyltransferase and no measurable activity with the other two enzymes. Analogues of several CoA thioesters in which the amide bond was replaced with a hemithioacetal linkage exhibited no measurable activity with the appropriate enzymes. The results indicate that some aspects of the amide bond and proper distance between this amide and the thiol/thioester moiety are critical for activity of CoA ester-utilizing enzymes.     

3-Thiopropionic acid as a highly versatile multidetachable thioester resin linker

This paper describes a practical new use of3-mercaptopropionic acid as a highly versatilemultidetachable linker for solid-phase synthesis. Ourapproach is based on the stability of thealkylthioester functionality to optimized Boc-SPPSprotocols and HF treatment, as well as on the mildactivation of the thioester functionality towardnucleophilic or reductive displacement. This allowsseveral C-terminal modifications to be introduced intoa synthetic molecule during the cleavage step. We haveshown that unprotected peptides can be efficientlycleaved from a propyl thioester-polyethyleneglycol-poly-(N,N-dimethylacrylamide) copolymerresin using a great variety of nucleophiles to givethe corresponding C-terminally modified peptides(esters, thioesters, carboxylic acids, thioacids,amides, hydroxamic acids, hydrazides, alcohols). Thenucleophilic cleavage reaction is both rapid andexceptionally clean in all the cases tested.