Evaluation of new base‐labile2‐(4‐nitrophenylsulfonyl)ethoxycarbonyl (Nsc)‐amino acids for solid‐phase peptide synthesis

Abstract: The 2‐(4‐nitrophenylsulfonyl)ethoxycarbonyl (Nsc) group is a new base‐labile protecting group for solid‐phase peptide synthesis, completely interchangeable with the fluorenylmethoxycarbonyl (Fmoc) protecting group, but with certain advantages. In this paper, we report a methodology with N^α‐Nsc‐protected amino acids for the synthesis of some melanotropins important to our research, namely, γ‐melanocyte‐stimulating hormone (γ‐MSH), its [Nle³]‐analogue, and a cyclic α‐MSH/β‐MSH hybrid. We developed an efficient protocol for the synthesis of the cyclic MSH analogue that yielded this peptide in > 98% purity. The γ‐MSH synthesis, which gave problems with both the Boc and Fmoc strategies, yielded the desired peptide by Nsc‐chemistry but was accompanied by side products. Finally, the Nle³‐γ‐MSH analogue was synthesized more efficiently using the Fmoc strategy, suggesting that Nsc‐chemistry might not be the best methodology for certain sequences.     

Solid-phase synthesis of a range of O-phosphorylated peptides by post-assembly phosphitylation and oxidation

A completely general method for the O-phosphorylation of peptides of any given composition using solid-phase methodology is described. Peptides were assembled using Fmoc amino acid active esters, with base used for Fmoc deprotection. Unprotected amino acid side chain hydroxyl groups were phosphitylated and oxidised at the end of the assembly using bis (benzyloxy)(diisopropylamino)phosphine and tert.-butylhydroperoxide respectively. TFA was used for final deprotection of the amino acid side chains and for simultaneous cleavage from the resin. The synthesis of O-phosphopeptides of up to 15 residues in length is described.     

Synthesis of a tripeptide with a C-terminal nitrile moiety and the inhibition of proteinases

The synthesis of the tripeptide d -Phe-Pro-Arg with the nitrite group instead of the carboxylgroup is described. Initially, the corresponding peptide amide was synthesized by conventional methods in solution using Boc and Fmoc as the protecting group for d -Phe. The dehydration in order to create the nitrite moiety was achieved by treating the peptide amide with phosphorus oxichloride or trifluoroacetic anhydride. Best results were obtained by the use of phosphorus oxichloride in pyridine as the solvent in the presence of imidazole. After deprotection of the N-terminal amino acid the crude product was purified by chromatography on Butyl-Fractogel® HW-40 (S). The purity of the final product was checked on a RP18 phase by hplc. The existence of the nitrite group was demonstrated by i.r. and ¹³C-n.m.r. spectra. The peptide nitrite exhibited a strong inhibition of thrombin compared to the tripeptide amide.     

Carbopeptides: carbohydrates as potential templates for de novo design of protein models

Abstract: De novo design of proteins has evolved into a powerful approach for studying the factors governing protein folding and stability. Among the families of structures frequently studied is the ‘four‐helix bundle’ in which four α‐helical peptide strands, linked by loops, form a hydrophobic core. Assembly of protein models on a template has been suggested as a way to reduce the entropy of folding. Here we describe the potential use of a carbohydrate as such a template. The monosaccharide d ‐galactose was per‐O‐acylated with (N^β‐Fmoc‐βAla)₂O to give a penta‐substituted derivative, which was converted to the corresponding glycosyl bromide and used for the glycosylation of 4‐hydroxymethylbenzoic acid pentafluorophenyl ester (HMBA‐OPfp). The β‐glycosidic carbohydrate template (N^β‐Fmoc‐βAla)₄‐β‐d ‐Galp‐(1‐O)‐MBA‐OPfp thus obtained was coupled to a PAL‐PEG‐PS resin and simultaneously extended at the four arms to yield, after cleavage from the solid support, a carbopeptide with four identical peptide strands. Extension of this concept to, for example, synthesis of novel multiple antigenic peptides (MAPs) and synthesis of carbohydrate clusters can be easily envisioned. The ability to efficiently synthesize such structures sets the stage for further studies to test whether the carbohydrate templates do indeed nucleate folding.     

胸腺五肽合成工艺的研究

采用固相合成法进行了胸腺五肽的合成研究,以Fmoc-Tyr(But)-Wang Resin为载体,所有氨基酸衍生物都采用Fmoc氨基酸保护系统,并以BOP-HOBT活化酯法偶联形成肽键,以20%哌啶/DMF为脱Fmoc试剂,以三氟乙酸(TFA)为脱氨基酸衍生物侧链保护基团和从树脂上切下肽试剂.经冷乙醚沉淀得粗品,以RP-HPLC纯化后得TFA型TP-5,经醋酸转型后冷冻干燥行TP-5白色粉末状物质.     

Synthesis of polyamide supports for use in peptide synthesis and as peptide-resin conjugates for antibody production

We have synthesized beaded, hydrophilic cross-linked, aminoalkyl polydimethylacrylamide supports upon which peptides have been assembled using standard Boc or Fmoc chemistry in automated equipment. The resins were prepared by the free radical-initiated co-polymerization of N,N-dimethylacrylamide, N,N′-bisacrylyl-1,3-diaminopropane, and a functional monomer which were contained in a reverse-phase, detergent-emulsified suspension. The functional monomers used were N-(2-(methylsulfonyl)ethyloxycarbonyl)-allyl-amine (MSC-allylamine), N-acrylyl-1,6-diaminohexane hydrochloride or N-methacrylyl-1,3-diaminopropane hydrochloride. The MSC protecting group was removed by treatment of the resin with methanolic base during workup. After coupling of N-α-t-butyloxycarbonyl-alanine (Boc-alanine), amino acid analyses gave resin loading capacities between 0.15 mmol/g and 1.4 mmol/g, depending on the concentration and composition of the functional monomer. The resulting polymers were highly swollen by polar solvents including aqueous buffers. Peptides were synthesized on these supports after attaching the first amino acid directly or through a cleavable ester linker. When the carboxyl-terminal amino acid was coupled as the 4-oxymethylbenzoic acid derivative, the peptide could be deprotected and remain attached to the hydrophilic polymer since the peptide-benzyl ester bond was stable to HF deprotection at 0° in the presence of 10% anisole and 1% ethanedithiol. The resulting peptidyl-resin could be swollen in aqueous buffers and injected into animals for the production of antibodies.     

Solid-phase synthesis of [Ala1,3,11,15]-endothelin-1 by a modified double-coupling procedure

[Ala^1,3,11,15]-Endothelin-1, a linear analogue of endothelin-1 in which alanines replace the cystine residues, has been prepared by solid-phase synthesis in approximately 17% yield. Fmoc amino acids were coupled using an economical modified double-coupling (symmetrical anhydride followed by O-benzotriazolyl ester) procedure under fully automated conditions. Conditions for synthesis and purification were closely monitored and should be applicable to other endothelin analogues.     

Solid-phase synthesis of phosphopeptides

We report the solid-phase synthesis of peptides containing O-phosphoserine. Coupling was with commercially available Fmoc-amino acid pentafluorophenyl esters, with base used at each cycle to cleave Fmoc. Phosphorylation of those serine residues left unprotected on the peptide-resin was achieved with dibenzylphosphochloridate, and finally trifluoroacetic acid was used to remove side-chain protecting groups (including the benzyl groups used for the phosphate), and to cleave the peptide from the resin in the same step. This synthetic strategy enables the preparation of peptides with individual, selectively phosphorylated residues. Alternative approaches to introduce protected phosphate and continue with coupling of further amino acids were less advantageous due to the lability of the phosphate group to base and to steric hindrance.     

Preparation of ‘side‐chain‐to‐side‐chain’ cyclic peptides by Allyl and Alloc strategy: potential for library synthesis

Abstract: Automated and manual deprotection methods for allyl/allyloxycarbonyl (Allyl/Alloc) were evaluated for the preparation of side‐chain‐to‐side‐chain cyclic peptides. Using a standard Allyl/Alloc deprotection method, a small library of cyclic peptides with lactam bridges (with seven amino acids) was prepared on an automatic peptide synthesizer. We demonstrate that the Guibé method for removing Allyl/Alloc protecting groups under specific neutral conditions [Pd(PPh₃)₄/PhSiH₃)/DCM] can be a useful, efficient and reliable method for preparing long cyclic peptides on a resin. We have also manually synthesized a cyclic glucagon analogue containing 24 amino acid residues. These results demonstrated that properly controlled palladium‐mediated deprotection of Allyl/Alloc protecting groups can be used to prepare cyclic peptides on the resin using an automated peptide synthesizer and cyclic peptides with a long chain.     

Applications of BOP reagent in solid phase synthesis

Using a variety of activating agents, a kinetic study was carried out to evaluate the rate of solid phase side-chain to side-chain cyclization of Asp³ to Lys¹² in the model peptide-resin, [Ala¹⁵]-GRF(1–29)-BHA-resin. Asp³ and Lys¹² were introduced in the peptide chain by using N^α-Boc amino acids in conjunction with the OFm/Fmoc side-chain protection scheme. The OFm and Fmoc side-chain protecting groups were shown to be stable to diisopropylethylamine and selectively deprotected on treatment with 20% piperidine in DMF. Solid phase side-chain to side-chain cyclization (lactamization) was shown to proceed to completion within 2 h using benzotriazol-1-yl-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate (BOP reagent) while the reaction was only 55% completed in 24h using DCC/HOBt. Solid phase side-chain to side-chain cyclization by the BOP procedure not only proceeded more rapidly but also gave a purer cyclic product.     

Synthesis of peptide sequences related to thrombospondin: factors affecting aspartimide by‐product formation

Abstract: Aspartimide formation is one of the most common secondary reactions on solid phase peptide synthesis. In the present work, we describe the optimization of the synthesis of two thrombospondin fragments containing an Asp‐Gly sequence that show a strong tendency to form cyclic aspartimide derivatives in an unusual high percentage. Several different strategies were applied changing type of resin, Fmoc‐deprotection reagents, coupling additives, resin cleavage cocktails and the use of Hmb‐Gly derivative to minimize the extension of this byproduct. Best results were obtained with cross‐linked ethoxylate acrylate (CLEAR^®‐cross‐linked ethoxylate Acrylate Resin)‐type resin and pip/dimethylformamide deprotection. Besides, as in biological assays the aspartimide containing sequence resulted to be more active than the linear one, the optimization of its synthesis was also carried out.     

STABLE ISOLATED SYMMETRICAL ANHYDRIDES OF Nα-9-FLUORENYLMETHYLOXYCARBONYLAMINO ACIDS IN SOLID-PHASE PEPTIDE SYNTHESIS:

The synthesis and isolation of symmetrical anhydrides of N^α-9-fluorenylmethyloxycarbonyl (Fmoc) amino acids using water soluble carbodiimide is described. These compounds were used in a solid phase peptide synthesis of methionine-enkephalin on a p-benzyloxybenzyl ester polystyrene 1% divinylbenzene resin support. Homogeneous free pentapeptide was obtained in 42% overall yield. The Fmoc amino acid symmetrical anhydrides were stable during prolonged storage (2 years at 0°) and offer advantages over present “Fmoc solid phase” methods which use anhydrides formed in situ.     

Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids

9-Fluorenylmethoxycarbonyl (Fmoc) amino acids were first used for solid phase peptide synthesis a little more than a decade ago. Since that time, Fmoc solid phase peptide synthesis methodology has been greatly enhanced by the introduction of a variety of solid supports, linkages, and side chain protecting groups, as well as by increased understanding of solvation conditions. These advances have led to many impressive syntheses, such as those of biologically active and isotopically labeled peptides and small proteins. The great variety of conditions under which Fmoc solid phase peptide synthesis may be carried out represents a truly “orthogonal” scheme, and thus offers many unique opportunities for bioorganic chemistry.     

Amino acid–azetidine chimeras: synthesis of enantiopure 3‐substituted azetidine‐2‐carboxylic acids

Abstract: Azetidine‐2‐carboxylic acid (Aze) analogs possessing various heteroatomic side chains at the 3‐position have been synthesized by modification of 1‐9‐(9‐phenylfluorenyl) (PhF)‐3‐allyl‐Aze tert‐butyl ester (2S,3S)‐ 1 . 3‐Allyl‐Aze 1 was synthesized by regioselective allylation of α‐tert‐butyl β‐methyl N‐(PhF)aspartate 13 , followed by selective ω‐carboxylate reduction, tosylation, and intramolecular N‐alkylation. Removal of the PhF group and olefin reduction by hydrogenation followed by Fmoc protection produced nor‐leucine–Aze chimera 2 . Regioselective olefin hydroboration of (2S,3S)‐ 1 produced primary alcohol 23 , which was protected as a silyl ether, hydrogenated and N‐protected to give 1‐Fmoc‐3‐hydroxypropyl‐Aze 26 . Enantiopure (2S,3S)‐3‐(3‐azidopropyl)‐1‐Fmoc‐azetidine‐2‐carboxylic acid tert‐butyl ester 3 was prepared as a Lys–Aze chimera by activation of 3‐hydroxypropyl–Aze 26 as a methanesulfonate and displacement with sodium azide. Moreover, orthogonally protected azetidine dicarboxylic acid 4 was synthesized as an α‐aminoadipate–Aze chimera by oxidation of alcohol 26 . These orthogonally protected amino acid–Aze chimeras are designed to serve as tools for studying the influence of conformation on peptide activity.     

PHOTOLABILE MULTI-DETACHABLE p-ALKOXYBENZYL ALCOHOL RESIN SUPPORTS FOR PEPTIDE FRAGMENT OR SEMI-SYNTHESIS

Two photolabile multi-detachable alkoxybenzyl alcohol resins, 2-[4-(oxymethyl)phenoxy]propionyl-resin 4 and 4-[4-(oxymethyl)phenoxymethyl]-3-nitrobenzamidomethyl-resin 5 have been synthesized. Bpoc-peptide attached to resin 4 or 5 when treated with 50% trifluoroacetic acid provided the free, unprotected peptide, but on photolysis gave Bpoc-peptide p-hydroxybenzyl ester. Removal of the p-hydroxybenzyl ester in aqueous base or by oxidative work up gave a protected Bpoc-peptide suitable for fragment synthesis at its C-terminus. However, methylation of the ester to Bpoc-peptide p-methoxybenzyl ester followed by removal of the Bpoc-group gave a protected peptide p-methoxybenzyl ester suitable for fragment coupling at its N-terminus. The efficacies of these resins were evaluated in the syntheses of a model tetrapeptide and an octapeptide by using N^α-Bpoc-, Fmoc- and Nps-amino acids. The use of 2-thiopyridine with pyridinium hydrochloride as a new and efficient thiolytic reagent for the deprotection of the Nps-group was studied.     

Susceptibility of glycans to β-elimination in Fmoc-based O-glycopeptide synthesis

order to investigate the possible extent of β-elimination occuring in Fmoc-based continuous-flow solid-phase glycopeptide synthesis, the influence of the pK_b of the base used for N^α-deprotection has been studied. A glycosylated pentapeptide as synthesized using 50%morpholine, 10%, piperidine or 2% DBU, respectively, in DMF for deprotection. The dehydropentapeptide N^α-.Ac-Thr-Thr-δAba-Val-Thr-NH₂, which would be formed in the case of β-elimination, was prepared independently and used as a control in HPLC analysis; however, this product was not formed under any of the deprotection conditions applied. Furthermore, a 23 amino acid long glycopeptide from human intestinal mucin was prepared using 2% DBU as a base for Fmoc cleavage, and similarly no β-elimination was observed. The glycopeptide products were subjected to a prolonged treatment with sodium hydroxide in methanol/water without significant formation of byproducts, and the pure glycopeptides were isolated and characterized by ¹H-NMR spectroscopy.     

Synthesis of peptide amides by Fmoc-solid-phase peptide synthesis and acid labile anchor groups

The preparation and use of new anchor groups for the synthesis of peptide amides by solid-phase peptide synthesis employing the Fmoc-method is described. Based on the structure of the 4,4′-dimethoxybenzhydryl group (Mbh) handles were developed, which could be cleaved by mild acid treatment to give carboxamides. The syntheses and application of Fmoc-amino-acid-(4-carboxylatomethyloxyphenyl-4′-methoxyphenyl) methyl amide and Fmoc-(4-carboxylatopropyloxyphenyl-4′-methoxyphenyl) methyl amide are described in detail. These handles were coupled to resins and a stepwise elongation of peptide chains proceeded smoothly with N^x-9-fluorenylmethoxycarbonyl (Fmoc) amino acid derivatives using a carbodiimide/HOBt mediated reaction. The final cleavage of side-chain protecting groups and the release of the C-terminal amide moiety was achieved by the treatment with trifluoroacetic acid, dichloromethane in the presence of scavengers. Various peptides, such as the Leu-enkephalin amide and Leu-Gly-Gly-Gly-Gln-Gly-Lys-Val-Leu-Gly-NH₂, which is a good substrate for F XIII, were prepared in high yields and purities.     

Application of arylsulphonyl side-chain protected arginines in solid-phase peptide synthesis based on 9- fluorenylmethoxycarbonyl amino protecting strategy

One of the main problems still hampering solid-phase peptide synthesis using orthogonal protection strategies based on the 9-fluorenylmethoxycarbonyl amino protecting group is the difficult removal of currently used arginine arylsulphonyl guanidino protecting groups. Poor acid lability of 4-methoxy-2,3,6-trimethylbenzenesulphonyl-protected arginine has led to the popularity of the newer 2,2,5,7,8-pentamethylchroman-6-sulphonyl guanidino protecting group. This group was initially believed to have lability to trifluoroacetic acid, the reagent commonly used to simultaneously deprotect peptides and detach them from the synthesis resin, comparable to tert.-butyl and trityl type protecting groups used for the protection of other peptide side-chain functionalities. In a comparison of three established cleavage/deprotection mixtures we have shown that this is not always the case, particularly in multiple arginine peptides. We have found that only hard-acid deprotection with trimethylsilyl bromide reliably removed both arylsulphonyl guanidino protecting groups from a variety of arginine-containing peptides.     

An efficient and highly selective deprotection of N‐Fmoc‐α‐amino acid and lipophilic N‐Fmoc‐dipeptide methyl esters with aluminium trichloride and N,N‐dimethylaniline

Abstract: A novel procedure for the deprotection of the carboxyl group of amino acid methyl esters is presented. The process is carried out by the reagent system aluminium trichloride/N,N‐dimethylaniline that can successfully be applied to unblock the carboxyl moiety either of N‐Fmoc‐protected amino acid methyl esters and N‐Fmoc‐protected short dipeptide methyl esters. The chiralities of the optically pure amino acid or peptide precursors are maintained totally unchanged.     

Improved synthesis of 5‐hydroxylysine (Hyl) derivatives*

Abstract: The synthesis of 5‐hydroxylysine (Hyl) derivatives for incorporation by solid‐phase methodologies presents numerous challenges. Hyl readily undergoes intramolecular lactone formation, and protected intermediates often have poor solubilities. The goals of this work were twofold: first, develop a convenient method for the synthesis of O‐protected Fmoc‐Hyl; secondly, evaluate the efficiency of methods for the synthesis of O‐glycosylated Fmoc‐Hyl. The 5‐O‐tert‐butyldimethylsilyl (TBDMS) fluoren‐9‐ylmethoxycarbonyl‐Hyl (Fmoc‐Hyl) derivative was conveniently prepared by the addition of tert‐butyldimethylsilyl trifluoromethanesulfonate to copper‐complexed Hyl[?‐tert‐butyloxycarbonyl (Boc)]. The complex was decomposed with Na⁺ Chelex resin and the Fmoc group added to the α‐amino group. Fmoc‐Hyl(?‐Boc, O‐TBDMS) was obtained in 67% overall yield and successfully used for the solid‐phase syntheses of 3 Hyl‐containing peptides. The preparation of Fmoc‐Hyl[?‐Boc, O‐(2,3,4,6‐tetra‐O‐acetyl‐β‐d ‐galactopyranosyl)] was compared for the thioglycoside, trichloroacetimidate and Koenigs–Knorr methods. The most efficient approach was found to be Koenigs–Knorr under inverse conditions, where Fmoc‐Hyl(?‐Boc)‐OBzl and peracetylated galactosyl bromide were added to silver trifluoromethanesulfonate in 1,2‐dichloroethane, resulting in a 45% isolated yield. Side‐reactions that occurred during previously described preparations of glycosylated Hyl derivatives, such as lactone formation, loss of side‐chain protecting groups, orthoester formation, or production of anomeric mixtures, were avoided here. Research on the enzymology of Lys hydroxylation and subsequent glycosylation, as well as the role of glycosylated Hyl in receptor recognition, will be greatly aided by the convenient and efficient synthetic methods developed here.