抗肿瘤多肽蜂毒素的固相合成

目的 合成具有抗肿瘤活性的二十六肽蜂毒素.方法 采用Fmoc固相逐步化学合成,确立了以Wang树脂为固相载体,HOBt/ DCC为缩合剂的合成工艺.结果 通过本法能够顺利合成得到二十六肽蜂毒素,收率达32％.结论 该合成方法具有可行性强、操作简便、总收率高等特点,适用于长链多肽的合成.     

Solid-phase peptide synthesis: a silver anniversary report*

It has been a quarter of a century since Merrifield's initial report on solid-phase peptide synthesis. The field has matured significantly in recent years with a better understanding of the underlying chemistry. This is reflected by new, milder orthogonal protection schemes and more efficient coupling methods, some of which have been incorporated into automated systems. Advances in purification, especially high performance liquid chromatography, have had a major impact. The efficacy of these improvements has been demonstrated by an impressive litany of applications to biological problems.     

Solid-phase peptide synthesis without side-chain hydroxyl protection of threonine

A peptide containing four threonine residues was synthesised by the solid-phase method using fluorenyl-methoxycarbonylamino acid reactive esters or coupling by preactivation with 1-hydroxybenzotriazole and Castro's reagent. In two separate experiments the synthesis was carried out with or without protection of the side-chain hydroxyl group of threonine as the tert-butyl ether. Comparison of the crude peptides after deprotection and detachment from the synthesis resin suggests that side-chain protection of threonine is unnecessary under the synthetic conditions employed.     

Solid-phase synthesis of peptide haptens containing a cysteine—sulfur mustard adduct

Solid-phase synthesis of peptide haptens containing 2-[2-(S-cysteinyl)ethylthio]ethanol has been achieved by solution-phase synthesis of a properly protected S-alkylated cysteine derivative and subsequent solid-phase incorporation. © Munksgaard 1995.     

From peptide libraries to optimized nonpeptide ligands in the search for S‐farnesyltransferase inhibitors

Abstract: A complete 331 776‐member library of tetrapeptides made of 24 amino acid building blocks was synthesized robotically on solid phase and subjected to a deconvolution based on the inhibitory potency of the sublibraries in a HPLC assay of the S‐farnesyltransferase activity in vitro. One of the non‐natural peptide and noncysteine‐containing leads Nip‐Trp‐Phe‐His (Nip = p‐nitrophenyl‐l ‐alanine) was optimized chemically to give a proteolytically stable pseudopeptide with a 200‐fold potency compared with the original lead. The final compound was converted to the C‐terminal ethyl ester: p‐F‐C₆H₄‐CO(CH₂)₂‐CO‐Bta‐d ‐Pheψ[CH2NH]His‐OEt (Bta = benzothienyl‐l ‐alanine) and shown to behave as a prodrug which was hydrolyzed back to the C‐terminal acid following cell penetration. The method confirmed that several structurally original leads can be discovered in large libraries when deconvolution relies upon a highly specific assay and that these leads can be optimized by chemical modification to impart the final compound the desired pharmacological and pharmacokinetic properties.     

Solid-phase synthesis of O-mannosylated peptides: two strategies compared

A comparison of the O-glycosylation of resin-bound assembled peptides with the incorporation of glycosylated amino acids using established chemistry is presented. Fmoc/tert-butyl-based protecting groups were used for the peptidic moieties in conjunction with acetyl sugar protection. Koenigs-Knorr glycosylations were carried out using protected bromomannose derivatives, the acceptor being threonine or serine, either in solution or within a resin-bound peptide. The characterisation of microgram quantities of glycopeptides by the use of glycosidases in combination with mass spectrometry is also described.     

Solid-phase synthesis of a nonadecapeptide coded for by the v-myb oncogene

A nonadecapeptide comprising a predicted B-cell determinant from the v-myb oncoprotein was synthesized by Merrifield's solid-phase method. Hydrogen chloride in dichloromethane was used for protective t-butyloxycarbonyl group removal; the deprotection was monitored using a new qualitative deprotection test. The nonadecapeptide coupled to a carrier elicited a high titre of protein-reactive antipeptide antibodies.     

Multiple continuous-flow solid-phase peptide synthesis Synthesis of an HIV antigenic peptide and its omission analogues

Multiple continuous-flow solid-phase peptide synthesis was performed on a standard polystyrene-based resin under low-pressure conditions using a simple manually operated synthesizer. Stable-flow resin-packed columns were prepared in small polypropylene flow reactors, adjustable for volume. The concurrent synthesis of 10 peptides was carried out in flow reactors concatenated together; solvents and reactants were passed through this set of columns using moderate overpressure. One decapeptide, H-Val-Tyr-Tyr-Arg-Asp-Ser-Arg-Asn-Pro-Leu-NH₂, containing an antigenic determinant of the p31 protein product of the pol gene of the human immunodeficiency virus, and its nine omission analogues were synthesized.     

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.     

General method for rapid synthesis of multicomponent peptide mixtures

A method is suggested for the synthesis of multicomponent peptide mixtures. The method is a solid phase synthesis modified in order to give a closely equimolar mixture of peptides with predetermined sequences. The main point of modification is that before every coupling cycle the resin is divided into equal parts and each portion is coupled with a different amino acid. Then the portions are mixed and before the next coupling cycle the resin is again distributed into equal portions. The method is illustrated by the synthesis of a mixture of 27 tetrapeptides and that of 180 pentapeptides.     

Solid-phase peptide synthesis and biological activity of bovine thymopoietin II (bTP–11)

Bovine thymopoietin (bTP), a 49 amino acid polypeptide, was synthesized using Merrifield's solid-phase peptide synthesis methodology. The polypeptide was purified using anion-exchange chromatography and reversed-phase HPLC and characterized by mass spectrometry and amino acid analysis of the full-length peptide and of products derived from digestion with Staphylococcus aureus V8 protease. The biological activity of the synthesized product was tested in several assay systems. Synthetic bTP was found to induce the expression of Thy 1.2 antigen on T-lymphocytes from athymic mice, in agreement with previous studies on the biological activity of endogenous bTP. Biological activity at skeletal muscle and neuronal nicotinic acetylcholine receptor sites, as reported by others for bTP, could not be confirmed in our studies. The absence of biological activity at nicotinic receptor sites may be related to the results of a recent report demonstrating the presence of a cobratoxin-like molecule in preparations of natural bTP. These data indicate that synthetic peptides have an important role for the evaluation of the specificity of the biological activity of polypeptides.     

Pepsin as a catalyst for peptide synthesis: formation of peptide bonds not typical for pepsin substrate specificity

Abstract: Porcine pepsin in water solutions containing 15–28% of dimethylformamide at pH 5 and 20–37°C catalysed the formation of peptide bonds between Z-Ala-Ala-Phe-OH and various amino acid or peptide derivatives. Substrate binding subsite S′₁ of pepsin demonstrated broad specificity in these reactions but revealed a certain preference for hydrophobic amino acid residues, including non-proteinous homophenylalanine, p-nitrophenylalanine, S-methylcysteine, as well as for those that contained, in addition to the hydrophobic elements, a group capable of donating a hydrogen bond, e.g. o-nitrotyrosine. This observation increases the range of peptides that might be prepared by pepsin-catalysed synthesis.     

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.     

Pepsin as a catalyst of peptide synthesis Enzyme co-precipitation with emerging peptide products

Pepsin successfully catalyzed the synthesis of several peptide derivatives from N-protected di- or tripeptides and amino acid or peptide esters or p-nitroanilides in dimethylformamide-water solutions at pH 4.6. An optimal substrates:pepsin ratio depended on the structure of starting peptides, especially their fit to the substrate binding sites of the enzyme. For hexapeptide Z-Ala-Ala-Phe-Leu-Ala-Ala-OCH₃ formation, an equilibrium yield was attained at 1:3. 10⁵ enzyme-substrates ratio that indicated high efficiency of pepsin in synthesis reactions. In the course of the equilibrium peptide synthesis, pepsin gradually disappeared from the liquid phase due to its entrapment within a gel, formed by the hexapeptide product, while retaining its activity. The inclusion into the precipitate was not specific for pepsin, so far as inert proteins, lysozyme, ribonuclease A and carbonic anhydrase, when added to the reaction mixture, became also co-precipitated with the hexapeptide formed. It appears that co-precipitation of pepsin, an important factor limiting the enzyme efficiency, might be operative as well for other proteinases used to catalyze peptide synthesis.     

Solid-phase synthesis of a peptide derivative of thymosin alpha1 and initial studies on its (99m)Tc-radiolabelling

A derivative (1) of the immunopotentiating 28-peptide thymosin alpha1 has been especially designed, so that it can be (99m)Tc-radiolabelled, and synthesized following the Fmoc solid-phase peptide synthesis approach. Derivative 1 contains the N-terminal fragment Talpha1[1-14] as a bioactive segment, at the C-terminus of which a (99m)Tc-chelating moiety consisting of N(alpha),N(alpha)-dimethylglycine, serine and cysteine is linked through the N(epsilon)-amino group of a 'bifunctional' lysine residue; the latter is indirectly anchored on the solid-phase peptide synthesis resin through 6-aminocaproic acid (dmGSCK{N(epsilon)-Talpha1[1-14]}Aca). Synthetic derivative 1 was obtained at high overall yield (approximately 35%) and purity (>95%) and shown to be efficiently radiolabelled with (99m)Tc, thus resulting in the first, to our knowledge, so far reported (99m)Tc-radiolabelled derivative of thymosin alpha1, which may be eventually used as a specific molecular tool for the in vitro/in vivo study of the mode of action of the parent bioactive peptide.     

Use of a new sonically cleavable and acido-labile handle for solid-phase peptide amide synthesis

A new handle usable for solid-phase peptide amide synthesis was designed. New releasing conditions of the peptide using sonication allowed much shorter reaction times at lower TFA concentrations.     

Design and synthesis of a novel peptide for selective detection of cancer cells

Using a minimalist approach, an 11‐residue peptide (Peptide 1 ) tagged with rhodamine fluorophore was designed and synthesized for selective detection of cancer cells. Peptide 1 contains RGD and NGR motifs to bind, respectively, integrins and aminopeptidase CD13, which are over expressed in cancer cells. Surface tension measurements revealed that peptide 1 possess surface‐active property owing to the overall hydrophobicity and cationic nature of the peptide. Peptide 1 displays cancer cell‐selective binding at ≤5.0 µM concentrations, while peptide 2 (randomized sequence of 1 ) shows non‐selective binding to normal and cancer cells. Fluorescence microscopy and FACS analysis demonstrated the intracellular localization of peptide 1 in three different cancer cell lines, confirming the role of RGD and NGR motifs. Cytotoxicity assay exhibited the viability of normal and cancer cells up to 100 µM concentrations of peptide 1 . Steady‐state fluorescence measurements disclosed the preferential interactions of the peptide 1 with anionic POPC/POPG bilayers rather than with zwitterionic POPC lipid bilayers. Circular dichroism studies showed minimal changes in the secondary structure of peptide 1 upon binding with the anionic lipid bilayers. Peptide 1 is largely unordered, non‐toxic, and useful for identification of cancer cells. Peptide 1 provides a template for designing drug‐loaded peptides for targeted delivery into cancer cells.     

Use of tetrabutylammonium salts of amino acids in peptide synthesis

Tetrabutylammonium (TBA) salts of amino acids and peptides have increased solubility, as compared with that of alkali metals salts, in organic solvents. We have compared the reaction rates for tripeptide formation in methylene chloride from Boc-Gly-Phe activated with various phenols, N-oxysuccinimide and azide, and TBA-salt of tryptophan, as well as Trp-OCH₃. H-Trp-O^? TBA⁺ as an amino component significantly accelerates the rate of reaction. Although a significant degree of racemization has been found, the use of TBA-salt of amino acids and peptides is justified in many cases due to high conversion rates.     

Automated synthesis and use of N-chloroacetyl-modified peptides for the preparation of synthetic peptide polymers and peptide-protein immunogens

A method to incorporate N-chloroacetyl moieties at the amino termini of synthetic peptides using a standard program with an automated peptide synthesizer has been developed. The N-chloroacetyl-modified peptides react well with sulfhydryl containing proteins such as 4-mercaptobutyrimide-modified bovine serum albumin to form stable protein-peptide conjugates. By incorporating cysteine into the synthetic peptide, autopolymerization or cyclization of the synthetic peptide occurs by reaction of the free sulfhydryl with the chloroacetyl group. N-Chloroacetyl-derivatized peptides may be useful as reagents for potential peptide immunogens and vaccines.     

Disulfide linkage to polyacrylic resin for automated Fmoc peptide synthesis. Immunochemical applications of peptide resins and mercaptoamide peptides

The use of disulfide bonds for peptide–resin linkage in solid-phase peptide synthesis was investigated using polyacrylic polymers (Expansin^TM) and automated Fmoc methodology. The disulfide moiety was bound to the support either by coupling a protected bifunctional handle or by an original stepwise procedure. Among the three different disulfide handles that were investigated, only the aminoethyldithio-2-isobutyric acid (AEDI) handle was stable enough to achieve peptide synthesis. A series of peptides of up to 10–20 amino acids were prepared in this manner, in good yield and purity. Rapid and quantitative peptide release was obtained by reduction with equimolecular amounts of dithiothreitol at pH 9 or tris(2-carboxymethyl) phosphine at pH 4.5. This allowed direct and rapid coupling of the released cysteamide peptides to an activated protein carrier and the use of free or resin-bound forms of the antigen in immunoassays.