Progress in the preparation of peptide aldehydes via polymer supported IBX oxidation and scavenging by threonyl resin.

Peptide aldehydes are of interest due to their inhibitory properties toward numerous classes of proteolytic enzymes such as caspases or the proteasome. A novel access to peptide aldehydes is described using a combination of solid phase peptide synthesis with polymer-assisted solution phase synthesis based on the oxidation of peptide alcohols with a mild and selective polymer-bound IBX derivative. The oxidation is followed by selective purification via scavenging the peptide aldehyde in a capture-release procedure using threonine attached to an aminomethyl resin. Peptide aldehydes are obtained in excellent purity and satisfying yield. The optical integrity of the C-terminal residue is conserved in a high degree. The procedures are compatible with the use of common side-chain protecting groups. The potential for using the method in parallel approaches is very advantageous. A small collection of new and known peptide aldehydes has been tested for inhibitory activity against caspases 1 and 3.     

Synthesis of peptide aldehydes via enzymatic acylation of amino aldehyde derivatives

Two ways for semi-enzymatic preparation of the peptide aldehydes are proposed: (1) enzymatic acylation of amino alcohols with acyl peptide esters and subsequent chemical oxidation of the resulting peptide alcohols with DMSO/acetic anhydride mixture or (2) enzymatic acylation of the preliminarily obtained by a chemical route amino aldehyde semicarbazones. Subtilisin 72, serine proteinase with a broad specificity, distributed over macroporous silica, was used as a catalyst in both cases. Due to the practical absence of water in the reaction mixtures the yields of the products in both enzymatic reactions were nearly quantitative. The second way seems to be more attractive because all chemical stages were carried out with amino acid derivatives, far less valuable compounds than peptide ones. A series of peptide aldehydes of general formula Z-Ala-Ala-Xaa-al (where Xaa-al=leucinal, phenylalaninal, alaninal, valinal) was obtained. The inhibition parameters for these compounds, in the hydrolysis reactions of corresponding chromogenic substrates for subtilisin and -chymotrypsin, were determined.     

An efficient gel-phase synthesis of peptides on a high capacity flexible crosslinked polystyrene support: comparison with Merrifield resin.

A highly solvating copolymer was prepared in high yield by introducing a flexible crosslinker, 1,4-butanedioldimethacrylate, into the polystyrene matrix by a free radical aqueous suspension polymerization. A 2 mol% crosslinked resin showed rigidity and mechanical characteristics comparable to those of divinylbenzene-crosslinked polystyrene (Merrifield resin, DVB-PS) support. Swelling and solvation characteristics of the new resin, BDDMA-PS, were much higher than DVB-PS support in all solvents used for solid phase peptide synthesis. The diacrylate crosslinks in the resin network were found to be highly stable even after 48 h treatment with neat TFA, 6 N HCl and 6 N KOH at 110 degrees C. To demonstrate the usefulness of the new resin in high capacity peptide synthesis, a typical difficult peptide, acyl carrier protein (ACP) fragment (65-74), was synthesized on commercially available 1 mol% crosslinked DVB-PS and 2 mol% crosslinked BDDMA-PS resins under identical conditions. A protocol using NMP/DMSO mediated coupling was employed for chain assembly. The yield and purity of the product from BDDMA-PS resin was higher than when the DVB-PS resin was used. The mechanistic reason behind the synthetic efficiency of the new resin was found to be its ability to induce random coil conformation to the growing peptide chains.     

Development of affinity chromatography using a bioactive peptide as a ligand

By repeatedly introducing hydrophilic polyethylene glycol (PEG) spacer (2) onto affinity resin bearing a bioactive peptide (1/2 secretory leukocyte protease inhibitor, 1/2SLPI) as a ligand, the adsorption of nonspecific binding proteins was effectively reduced and the purification efficacy of elastase, which is one of the target molecules for 1/2SLPI, from a protein mixture was improved. Moreover, using this resin, we also successfully detected L-plastin, as an endogenous target molecule for SLPI, from HL-60 cell lysate.     

Preparation of protected peptide amides using the Fmoc chemical protocol. Comparison of resins for solid phase synthesis.

Different resins were examined for their potential use in the solid phase synthesis of protected peptide amides using the 9-fluorenylmethoxycarbonyl (Fmoc) chemical protocol. The model protected peptide amide BocTyr-Gly-Gly-Phe-Leu-Arg(Pmc)NH2 (1) was synthesized on both the acid-labile 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin (Rink amide resin) (2) and on resins containing the base-labile linker 4-hydroxymethylbenzoic acid. Of the resins examined only the methylbenzhydrylamine resin containing the 4-hydroxymethylbenzoic acid linkage, which was cleaved by ammonolysis in isopropanol, gave the model peptide 1 in good overall yield (53% including functionalization). Thus the synthesis of protected peptide amides by solid phase synthesis using Fmoc-protected amino acids with t-butyl-type side chain protecting groups is feasible. The choice of peptide-resin linkage and its cleavage conditions, however, are critical to the success of such syntheses. The potential application of this synthetic strategy to the preparation of novel peptide amides is discussed.     

Syntheses, characterization and application of cross-linked polystyrene-ethyleneglycol acrylate resin (CLPSER) as a novel polymer support for polypeptide syntheses.

Cross-linked polystyrene-ethyleneglycol acrylate resin (CLPSER) was developed for the solid-phase synthesis of peptide by introducing a cross-linker, O,O'-bis(2-acrylamidopropyl)polyethylene glycol(1900) (Acr(2)PEG), into polystyrene. The cross-linker was prepared by treating acryloyl chloride with O,O'-bis(2-aminopropyl) polyethylene glycol(1900) [(NH(2))(2)PEG] in the presence of diisopropylethylamine. The copolymer was prepared either by bulk or inverse suspension copolymerization of Acr(2)PEG(1900) and styrene using sorbitan monolaurate as the suspension stabilizer, and a mixture of ammonium peroxodisulfate and benzoyl peroxide as the radical initiators. The resin was characterized using gel-phase (13)C NMR, infrared (KBr) spectroscopic techniques and the morphological features of the resin were investigated using scanning electron microscopy photographs. CLPSER showed excellent swelling in a broad range of solvents and was found to be chemically inert to various reagents and solvents used in solid-phase peptide synthesis. To demonstrate the usefulness of the new resin in polypeptide synthesis, the support was derivatized with an 'internal reference' amino acid (norleucine) and a handle 4-(4-hydroxymethyl-3-methoxy)butyric acid. The new resin was compared with commercial supports such as Merrifield and Sheppard resins by synthesizing an acyl carrier protein (65-74) fragment under the same experimental conditions. HPLC profiles revealed the high efficiency of the newly developed support. Resin capability in peptide synthesis was further demonstrated by the solid phase synthesis of a 25-residue peptide from the E2/NS1 region hepatitis C viral polyprotein.     

Solvent effects on coupling yields during rapid solid-phase synthesis of CGRP(8-37) employing in situ neutralization.

The success of solid-phase peptide synthesis is often dependent upon solvation of the resin and the growing resin-bound peptide chain. We investigated the relationship between solvent properties and solvation of the resin and peptide-resin in order to obtain satisfactory coupling yields for the rapid solid-phase peptide synthesis, using butyloxycarbonyl-(Boc)-amino acid derivatives, of human-alpha-calcitonin gene-related peptide(8-37) (CGRP(8-37)). Solvation of (p-methylbenzhydrylamine)copoly(styrene-1% divinylbenzene (DVB) (resin) and resin covalently bound to the fully protected amino acid sequence of CGRP(8-37) (peptide-resin) was correlated to solvent Hildebrand solubility (delta) and hydrogen-bonding (delta(h)) parameters. Contour solvation plots of delta(h) vs. delta revealed maximum solvation regions of resin and peptide-resin. Maximum resin solvation occurred with N-methylpyrrolidinone (NMP), NMP : dimethylsulfoxide (DMSO) (8 : 2) and DMSO. Inefficient solvation of the peptide-resin occurred with these solvents and resulted in poor syntheses with average coupling yields of 78.1, 88.9 and 91.8%, respectively. Superior peptide-resin solvation was obtained using dimethylacetamide (DMA) and dimethylformamide (DMF), resulting in significantly higher average coupling yields of 98.0 and 99.5%, respectively. Thus, the region of maximum peptide-resin solvation shifts to solvents with higher delta(h) values. DMF provided the most effective peptide-resin solvation and was the only solvent from which CGRP(8-37) was obtained as a single major product in the crude cleaved material.     

Solid Phase Synthesis of a Hydroxypyrrolidine Derivative and its Use in Solid Phase Peptide Synthesis as Constrained Statine Mimic

As part of our efforts to design constrained peptide mimics and introduce them in peptide sequences, we set up the synthesis of racemic N-Fmoc protected hydroxypyrrolidine by reduction of the corresponding oxopyrroline. Hydroxypyrrolidines are synthesized using amino acid building block and β-ketoester via a 4-steps solid supported route on Wang resin beads. The hydroxypyrrolidine template can be seen as a constrained mimic of statine. As proof of concept, the pseudopeptide JMV 2776, incorporating this new statine mimic has been synthesized. We replaced the phenyl statine building block in the sequence of known BACE 1/2 inhibitors by 5-benzyl 2-methyl 4-hydroxypyrrolidine, using conventional Fmoc SPPS on Rink amide PS resin. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Optimization of butanediol dimethacrylate crosslinked polystyrene: A novel polymeric support for solid phase peptide synthesis

Studies leading to optimization of butanedioldimethacrylate-crosslinked polystyrene supports (BDDMA–PS) forsolid phase peptide synthesis are delineated. BDDMA–PScopolymers with different crosslink densities were prepared andfunctionalised with chloromethyl groups. The reactivity of theLys(2-Cl-Z)-OH residue bound to these polymers through a benzylester linkage was investigated by following the kinetics ofacylation by the HOBt active ester of Boc-Alanine. From theresults it was observed that the rate of peptide bond formationwas maximum for a 2% BDDMA crosslinked resin. This resin wascompared with a 2% DVB-crosslinked polystyrene resin (DVB–PS). Synthesis of an extremely insoluble, hydrophobic,antiparallel -sheeted difficult sequencepeptide LMVGGVVIA ( 34–42), C-terminal fragment of -amyloid protein, (1–42), wascarried out on both 2% DVB–PS and 2% BDDMA-crosslinkedpolystyrene supports. The synthesis of the peptide was carriedout using Boc amino acid strategy. Greater extent of swellingof the resino peptide, increased coupling efficiency during theassembly of amino acids and relatively high purity of synthesised peptide were observed in the case of 2% BDDMA–PS polymer.     

Design and synthesis of peptide inhibitors of blood coagulations

Inhibition of blood coagulation by peptide aldehydes has been studied. Amino acid sequences were assembled from the P1-P2 portion of the cleavage sites(s) of clotting factors and residues selected experimentally. The thrombin-fibrinogen reaction could effectively be inhibited by D-Phe-Pro-Arg-H (GYKI-14,166) and Boc-D-Phe-Pro-Arg-H (GYKI-14,451). Plasmin digestion of fibrin could be retarded by Boc-Gln-Phe-Lys-H (GYKI-14,605) derived from a susceptible fragment, i.e. Asn-Phe-Lys decreases to Ser. However, such peptides could not retard the zymogen activations proceeding in Ca++ complexes (which seemed to be uneffected by heparin-antithrombin III, too). Inhibition of enzymes by peptide aldehydes showed marked substrate dependence.     

Morphiceptin and beta-casomorphin-5 analogues containing a reduced peptide bond: selective mu-receptor agonists and a novel mu antagonist, H-Tyr-Pro psi (CH2-NH)Phe-Pro-Gly-OH.

In order to prevent enzymatic degradation of beta-casomorphin-5 (1) and morphiceptin, reduced peptide bonds were incorporated at the 2-3 and 3-4 bonds, respectively. The analogues were synthesized by a combination of solid phase methodology and reductive alkylation of resin-bound peptide amines with Boc-amino acid aldehydes (Boc: tert-butyloxycarbonyl) in the presence of NaBH3CN. During reversed phase high pressure liquid chromatography purification, peak shape distortions could be observed. Epimerization was excluded, based on gas chromatography/mass spectroscopy analysis, which indicated acceptable levels of racemization (less than 3%) in the crude product. Instead, the phenomena could be attributed to slow cis/trans isomerizations originating from the Xxx-Pro bonds in the sequence. The presence of different conformational isomers was also established by 1H-nmr spectroscopy in DMSO-d6. All analogues showed high stability in blood plasma, enhanced binding affinity for the mu receptor, and very low binding to the delta receptor. While the Phe 3 psi(CH2-N)Pro4 analogues (3) and (5) displayed agonist activity, the Pro 2 psi(CH2-NH)Phe3 modified analogue (2) showed antagonist activity comparable to D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2.     

Systematic solid-phase synthesis of linear pseudooligolysines containing multiple adjacent CH(2)NH amide bond surrogates: potential agents for gene delivery.

Solid-phase methodology was used to synthesize a series of fully reduced linear oligolysines (pseudooligolysines, abbreviated herein as PLs) containing up to five adjacent CH2NH peptide bond isosteres. The reduced peptide bonds were introduced by the reductive alkylation reaction between Fmoc-Lys-(Boc)-al and a free alpha-amine moiety on the pseudopeptidyl resin, using sodium cyanoborohydride in an acidified mixture of NMP/CH3OH (1 : 1 v/v). The oligomeric molecules, which can be regarded as polyethylene imine and spermine analogs, possess multiple positive charges under physiological conditions and form tight complexes with plasmid DNA. These characteristics and the increased resistance to hydrolysis by trypsin make these molecules potential candidates for future use as DNA carriers in gene delivery.     

Application of 2-chlorotrityl resin in solid phase synthesis of (Leu15)-gastrin I and unsulfated cholecystokinin octapeptide. Selective O-deprotection of tyrosine.

The carboxyl terminal dipeptide amide, Fmoc-Asp-Phe-NH2, of gastrin and cholecystokinin (CCK) has been attached in high yield through its free side chain carboxyl group to the acid labile 2-chlorotrityl resin. The obtained peptide resin ester has been applied in the solid phase synthesis of partially protected (Leu15)-gastrin I utilising Fmoc-amino acids. Quantitative cleavage of this peptide from resin, with the t-butyl type side chain protection intact is achieved using mixtures of acetic acid/trifluoroethanol/dichloromethane. Under the same conditions complete detritylation of the tyrosine phenoxy function occurs simultaneously. Thus, the solid-phase synthesis of peptides selectively deprotected at the side chain of tyrosine is rendered possible by the use of 2-chlorotrityl resin and Fmoc-Tyr(Trt)-OH. The efficiency of this approach has been proved by the subsequent high-yield synthesis of three model peptides and the CCK-octapeptide.     

Direct synthesis of phosphinopeptides containing C-terminal α-aminoalkylphosphinic acids

A series of phosphinopeptides containing C-terminal α-aminoalkylphosphinic acids were prepared in good yields directly in one-pot reactions of 2-(N-benzoxycarbonylamino)alkanamides/peptide amides, aldehydes, and aryldichlorophosphines, followed by hydrolysis. In the current method, the peptide bond was formed in a Mannich-type reaction.     

A general method for preparation of peptides biotinylated at the carboxy terminus.

A method for the preparation of a biotinylated resin that can be elongated by standard methods of solid-phase peptide synthesis to give peptides biotinylated at the carboxy terminus is described. This methodology is particularly important for the preparation of biotinylated peptides in which a free amino terminus is required. Coupling of N epsilon-9-fluorenylmethoxycarbonyl-(Fmoc)-N alpha-tert-butyloxycarbonyl(Boc)-L- lysine to p-methylbenzhydrylamine resin, followed by removal of the Fmoc protecting group and reaction with (+)-biotin-4-nitrophenyl ester yielded N alpha-Boc-biocytin-p-methyl-benzhydrylamine resin. The utility of this resin was tested by the synthesis of a biotinylated peptide, Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg-biocytin-NH2, for use as an in vitro substrate for myristoyl-CoA:protein N-myristoyltransferase (NMT), the enzyme that catalyzes protein N-myristoylation. Analysis of the peptide derivative by HPLC and mass spectrometry revealed a single major product of the expected mass, indicating that the biotin group survived cleavage and deprotection with HF. The biotinylated peptide served as a substrate for NMT, and the resulting myristoylated peptide could be quantitatively recovered by adsorption to immobilized avidin.     

Chemistry of alpha-hydroxymethylserine: problems and solutions

Further improvements related to the synthesis of peptides containing HmS are presented. Efficient synthetic protocols have been developed to synthesize "difficult" sequences containing a C-terminal HmS residue, MeA-HmS or consecutive HmS. Preparative methods for orthogonal N- and/or C-protected HmS(Ipr) derivatives are described. Their compatibility with standard solution or solid-phase peptide chemistry protocols allows synthetic flexibility toward HmS-containing peptides. In the synthesis of the sterically hindered dipeptides with the C-terminal HmS(Ipr) residue, HATU proves the highest efficiency, as compared with the fluoride and PyBroP/DMAP coupling methods. The HATU method also outperforms the fluoride activation in the solid-phase assembly of HmS homosequence. Specific protocols are described to overcome an undesired cyclization to diketopiperazines that occurs during the removal of Fmoc from dipeptides with the C-terminal HmS(Ipr) or HmS residues, thus precluding their C-->N elongation. The successful protocols involve: (i) the 2+1 condensation using mixed anhydride activation yielding the desired product with the highest optical integrity or (ii) use of the 2-chlorotrityl resin as a solid support sterically suppressing the undesired cleavage due to diketopiperazine formation. The latter approach allows the mild conditions of peptide cleavage from solid support, preserving the isopropylidene protection and minimizing the undesired N-->O-acyl migration that was observed under prolonged acid treatment used for cleaving the HmS peptide from the Wang resin.     

Comparison of procedures for directly obtaining protected peptide acids from peptide-resins.

The preparation of small-sized protected peptide acids related to cholecystokinin and gomesin was attempted using peptide-Kaiser oxime resins (KOR) as starting materials. For comparison, peptide-2-Cl-trityl resin (CLTR) was also employed. While peptide detachment from KOR was achieved through the oxime ester bond hydrolysis mediated by DBU, hydroxide ion or Ca(+2) ion, peptide release from CLTR was accomplished through acid-catalysed hydrolysis of the peptide-resin ester linkage. Amino acid analysis of the peptide-resins before and after peptide release allowed the calculation of the reaction yields. RP-HPLC and LC/ESI-MS of the resulting crude peptides allowed estimation of their quality. The data collected indicated that: (i) among the procedures used for peptide displacement from KOR, the one employing DBU was the most efficient since it furnished all model protected peptide acids with the highest quality in a very short time; (ii) although slow, Ca(+2)-assisted peptide detachment from KOR was selective and was suitable for generating high-quality protected peptide acids containing up to five residues; (iii) even though the protocols employed for peptide release from CLTR have shown to be appropriate, the one employing 1% TFA/DCM was the most productive; (iv) in terms of product quality, DBU-catalysed peptide detachment from KOR was similar to TFA-catalysed peptide release from CLTR although the latter was more productive. These findings are relevant to peptide chemists in general, but especially to those interested in preparing acyl donors for convergent peptide syntheses by the t-Boc chemistry.     

多肽固相序列分析载体的合成及应用

用合成的聚合物作载体,与模型15肽(纯品)偶联,使用Edman降解法进行多肽序列分析。比较了各种类型载体对肽的偶联效果。实验结果表明合成的大孔型四次乙基五胺类树脂载体已经达到国外同类产品水平。     

Optimization of butanediol dimethacrylate crosslinked polystyrene: A novel polymeric support for solid phase peptide synthesis

Summary Studies leading to optimization of butanediol dimethacrylate-crosslinked polystyrene supports (BDDMA-PS) for solid phase peptide synthesis are delineated. BDDMA-PS copolymers with different crosslink densities were prepared and functionalised with chloromethyl groups. The reactivity of the Lys(2-Cl−Z)−OH residue bound to these polymers through a benzyl ester linkage was investigated by following the kinetics of acylation by the HOBt active ester of Boc-Alanine. From the results it was observed that the rate of peptide bond formation was maximum for a 2% BDDMA crosslinked resin. This resin was compared with a 2% DVB-crosslinked polystyrene resin (DVB-PS). Synthesis of an extremely insoluble, hydrophobic, antiparallel β-sheeted difficult sequence peptide LMVGGVVIA (β 34–42), C-terminal fragment of β-amyloid protein, β (1–42), was carried out on both 2% DVB-PS and 2% BDDMA-crosslinked polystyrene supports. The synthesis of the peptide was carried out using Boc amino acid strategy. Greater extent of swelling of the resino peptide, increased coupling efficiency during the assembly of amino acids and relatively high purity of synthesised peptide were observed in the case of 2% BDDMA-PS polymer.     

Polyethyleneglycol graft copoly (styrene-1,4-butanediol dimethacrylate) resin for gel-phase peptide synthesis

Summary Synthesis and characterization of a flexible crosslinked polystyrene grafted polyethyleneglycol (PEG) resin which allows for efficient synthesis of aggregating peptides in high yield and purity has been described. The resin showed rigidity, mechanical and chemical stability, and improved swelling and solvation characteristics essential for the successful synthesis of peptides. To demonstrate the usefulness of the new resin in polypeptide synthesis, a 4-(hydroxymethyl)phenoxyacetic acid (HMPA) handle was anchored to the free terminus of PEG and a typical hydrophobic peptide, Alzheimer’s β-amyloid plaque protein (33–42) fragment, was synthesized using Fmoc/tBu tactics. The new resin was compared with commercially available 1 mol% divinylbenzene (DVB)-crosslinked Tentagel resin under identical conditions. HPLC profiles and LC/MS analyses of the crude products revealed the high synthetic efficiency of the newly developed support. Efficiency of the resin was further illustrated by the gel-phase synthesis of a 15-residue peptide, (28–42) fragment of β-amyloid protein.