Synthesis of the C-terminal half of thymosin α1 by the polymeric reagent method

In this report we further show the utility and efficiency of polymer-bound 1-hydroxybenzotriazole (PHBT) as an almost ideal support for the polymeric reagent method of peptide synthesis. This was demonstrated by the synthesis of thymosin α₁ (15–28), in which two suitably blocked segments, Boc-Asp (OtBu)-Leu-Lys (2Cz)-Glu (OBzl)-Lys (2Cz)-Lys (2Cz)-OH ( 3 ) and Boc-Glu (OBzl)-Val-Val-Glu (OBzl)-Glu (OBzl)-Ala-Glu (OBzl)-Asn-OBzl ( 2 ), were prepared entirely by utilizing PHBT activation for each coupling step. After appropriate deblocking of 2 , segments 2 and 3 were coupled by the DCC-HOBT method, followed by complete deblocking and ion-exchange chromatographic purification, affording the C-terminal half of thymosin α₁, H-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH ( 1 ).     

Enhancement of peptide coupling reactions by 4-dimethylaminopyridine

4-Dimethylaminopyridine (DMAP) was found to be useful in the enhancement of peptide coupling reactions mediated by dicyclohexylcarbodiimide or symmetrical anhydrides. In an automated synthesis of the model heptapeptide Boc-Ala-Cle-Ile-Val-Pro-Arg(Tos)-Gly-OCH₂-Resin (Cle, cycloleucine), the efficiencies of various coupling methods such as dicyclohexylcarbodiimide, dicyclohexylcarbodiimide plus 1-hydroxybenzotriazole, and symmetrical anhydride were compared with that of dicyclohexylcarbodiimide plus 4-dimethylaminopyridine. Based on the amino acid composition of the peptide-resin samples and high pressure liquid chromatographic analyses of the protected heptapeptide amide obtained from the ammonolytic cleavage of the peptideresin samples, it was concluded that only dicyclohexylcarbodiimide plus 4-dimethylaminopyridine gave the desired near quantitative couplings in those cycles involving the sterically hindered amino acid residues. Observations were also made that 4-dimethylaminopyridine was a useful additive in a modified symmetrical anhydride method of coupling. In the synthesis of the model tetrapeptide Leu-Ala-Gly-Val on a Pam resin, the anhydride couplings were accelerated by DMAP and the product was equivalent in homogeneity to that obtained by the best previous methods. In addition, no racemization was detectable by a sensitive chromatographic method. There also was no detectable racemization found in a DCC-DMAP coupling of Boc-Ile-OH with H-Val-OCH₂-resin. However, significant racemization was observed during the coupling of Boc-Phe-OH with H-Glu(OBzl)-OCH₂-resin. DMAP is recommended as an additive for coupling hindered amino acids, particularly C^α-substituted residues, where little or no racemization is expected.     

固相法合成心肌兴奋肽及其类似物

用Boc-和Tos-基团分别保护氨基和侧链胍基,以1%交联度聚苯乙烯二苯甲氨基树脂为载体,用DCC固相法合成肽,HF断裂肽树脂键和去除侧链保护基团,粗产物经高效液相层析纯化,合成了心肌兴奋肽Phe-Met-Arg-Phe-NH₂及其类似物Phe-Pro-Arg-Phe-NH₂,并观察了此二种肽对大鼠血压和心率的影响。     

Synthesis of oligophosphoseryl sequences occurring in casein

The protected oligophosphoseryl peptides from bovine caseins, Z-Xxx-{Ser[PO(OPh)₂]}₃-Glu(OBzl)-OBzl for Xxx = Ile, Val, Gly, Leu and Ph = phenyl, were synthesized in high yields by stepwise lengthening using Boc-Ser[PO(OPh)₂]-OH as acylating carboxyl component and N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride as coupling reagent. The hydrogenolytic deprotection (PtO₂) was carried out with the valine derivative and with the tetrapeptide Ser[PO(OPh)₂]₃-Glu(OBzl)-OBzl. Phosphorylation of oligoseryl peptides failed to give the expected products. Large scale phosphorylation of protected serine was carried out in the presence of triethylamine using absolute ether as a solvent. 2,2,2-Trichloroethyl group (Tc) was shown to be a useful phosphorus protecting moiety in phosphopeptide synthesis: Boc-Ser[PO(OTc)₂]-OBzl, Z-Ser[PO(OTc)₂]-OBzl and Boc-Glu(OBzl)-Ser[PO(OTc)₂]-OBzl were synthesized in high yields using bis-(2,2,2-trichloroethyl) phosphochloridate.     

Application of 2-chlorotrityl resin in solid phase synthesis of (Leu15)-gastrin I and unsulfated cholecystokinin octapeptide

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/dichloro-methane. 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.     

Synthesis of O-glycosylated tuftsins by utilizing threonine derivatives containing an unprotected monosaccharide moiety

Synthesis is described of four tuftsin derivatives containing a D-ghcopyranosyl or a D-galactopyranosyl unit covalently linked to the hydroxy side chain function of the threonine residue through either an α or βO-glycosidic linkage. Fmoc-threonine derivatives containing the suitable unprotected sugar were used for incorporating the O-glycosylated amino acid residue. Z-Thr[α-Glc(OBzl)₄]-OBzl and Z-Thr[α-Gal(OBzl)₄]-OBzl were prepared from the tetra-O-benzylated sugar and Z-Thr-OBzl by the trichloroacetimidate method in the presence of trimethylsilyl trifluoromethane sulfonate. The α glycosylated threonine derivatives were converted into Fmoc-Thr(α-G1c)-OH and Fmoc-Thr(α-Gal)-OH by catalytic hydrogenation followed by acylation with Fmoc-OSu. β-Glucosylation and β-galactosylation of threonine were carried out by reacting the proper per-O-acetylated sugar with Z-Thr-OBzl and boron trifluoride ethyl etherate in dichloromethane. Catalytic hydrogenation of the β-O-glycosylated threonine derivatives followed by acylation with Fmoc-OSu and deacetylation with methanolic hydrazine yielded Fmoc-Thr(β-Glc)-OH and Fmoc-Thr(β-Gal)-OH, respectively. The O-glycosylated threonine derivatives were then reacted with H-Lys(Z)-Pro-Arg(NO₂)-OBzl in the presence of DCC and HOBt and the resulting glycosylated tuftsin derivatives were fully deblocked by catalytic hydrogenation, purified by HPLC, and characterized by optical rotation, amino acid analysis, and ¹H NMR. The β-galactosylated tuftsin was also prepared by the continuous flow solid phase procedure.     

A cleavage method which minimizes side reactions following Fmoc solid phase peptide synthesis

The success of solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl (Fmoc) amino acids is often limited by deleterious side reactions which occur during TFA peptide-resin cleavage and side-chain deprotection. The majority of these side reactions modify susceptible residues, such as Trp, Tyr, Met, and Cys, with TFA-liberated side-chain protecting groups and linkers. The purpose of this study was to assess the relative effectiveness of various scavengers in suppressing these side reactions. We found that the cleavage mixture 82.5% TFA: 5% phenol: 5% H₂O : 5% thioanisole : 2.5% EDT (Reagent K) was maximally efficient in inhibiting a great variety of side reactions. Synthesis and cleavage of 10 peptides, each containing 20-50 residues, demonstrated the complementarity of Fmoc chemistry with Reagent K for efficient synthesis of complex peptides.     

Amino acids and peptides. XVIII. Dipeptide formation during the synthesis of Z-Asp(OBzl)-OH

During the benzyloxycarbonylation of H-Asp(OBzl)-OH by the Schotten-Bauman reaction with benzyloxycarbonyl chloride in the presence of NaHCO₃ or Na₂CO₃, besides Z-Asp(OBzl)-OH, Z-Asp(OBzl)-Asp(OBzl)-OH was formed as side product, although the extent of the dipeptide formation differed depending on the base used (10% and 20% respectively). It was found that melting point, rotation value and Rf values upon thin-layer chromatography of Z-Asp(OBzl)-Asp(OBzl)-OH were quite similar to those of Z-Asp(OBzl)-OH.     

Preparation of protected peptide amides using the Fmoc chemical protocol

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 isopropanoll, 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.     

Asparagine coupling in Fmoc solid phase peptide synthesis

To investigate side reactions during the activation of side chain unprotected asparagine in Fmoc-solid phase peptide synthesis the peptide Met-Lys-Asn-Val-Pro-Glu-Pro-Ser was synthesized using different coupling conditions for introduction of the asparagine residue. Asparagine was activated by DCC/HOBt, BOP (Castro's reagent) or introduced as the pentafluorophenyl ester. The resulting peptide products were analyzed by HPLC, mass spectrometry and Edman degradation. In the crude products varying amounts of β-cyano alanine were found, which had been formed by dehydration of the side chain amide during carboxyl activation of Fmoc-asparagine. A homogeneous peptide was obtained by using either side chain protected asparagine derivatives with BOP mediated activation or by coupling of Fmoc-Asn-OPfp. Fmoc-Asn(Mbh)-OH and Fmoc-Asn(Tmob)-OH were coupled rapidly and without side reactions with BOP. For the side chain protected derivatives the coupling was as fast as that of other Fmoc-amino acid derivatives, whereas couplings of Fmoc-Asn-OH proceed more slowly. However, during acidolytic cleavage both protection groups, Mbh and Tmob, generate carbonium ions which readily alkylate tryptophan residues in a peptide. Tryptophan modification was examined using the model peptide Asn-Trp-Asn-Val-Pro-Glu-Pro-Ser. Alkylation could be reduced by addition of scavengers to the TFA during cleavage and side chain deprotection. A homogeneous peptide containing both, asparagine and tryptophan, was obtained only by coupling of Fmoc-Asn-OPfp.     

Solid phase synthesis of the protected 43–55 tridecapeptide of the heavy chain of myeloma immunoglobin M603, employing cyclohexyl ester protection for glutamic acid

A protected tridecapeptide of the sequence Boc-Lys(2ClZ)-Arg(Tos)-Leu-Glu(OcHex)-Trp(For)-Ile-Ala-Ala-Ser(Bzl)-Arg(Tos)-Asn-Lys(2ClZ)-Gly-OH, representing residues 43–55 of the variable region of the heavy chain of mouse myeloma protein M603, was synthesized. It was assembled by a stepwise solid phase method designed to give a fully protected peptide in high yield and purity with minimal side reactions. Thus, the peptide chain was attached as an α-methyl phenacyl ester to a 2-bromopropionyl-resin. After the synthesis the protected peptide fragment was obtained in 89% yield by photolytic cleavage from the resin. The peptide was purified by multiple precipitation and column chromatography. It was shown to be homogeneous by reverse phase high pressure liquid chromatography, and it had the correct amino acid composition and sequence. In the course of this work it was shown that tert.-butyloxycarbonyl-amino acids caused the formation of significant amounts of pyrrolidone carboxylic acid residues during the coupling reaction when a γ-benzyl glutamyl residue was NH₂-terminal. Other weak-acid additives also caused this chain terminating side reaction. The cyclization was markedly suppressed by protection of the glutamyl side chain as a cyclohexyl ester. With this protecting group, no evidence of pyrrolidone carboxylic acid formation could be detected in the tridecapeptide 43–55.     

Peptide synthesis using novel S-sulfocysteine derivatives

New cysteine S-sulfonate derivatives, Boc-Cys(SO₃Na)-ONa 2 and Fmoc-Cys(SO₃Na)-ONa 3 , were prepared and their utility for peptide synthesis examined. The Fmoc derivative 3 was used in the solid-phase peptide synthesis of Arg⁸-vasopressin 9 via the Bunte salt 7 . Satisfactory S-sulfonate stability was observed when p-cresol scavenged the cleavage from the resin. The intermediate 7 was purified by ion-exchange chromatography prior to S-sulfonate cleavage with tributylphosphine. © Munksgaard 1995.     

Lability of N-alkylated peptides towards TFA cleavage

Trifluoroacetic acid (TFA) is a common reagent in both solid-phase and solution peptide synthesis. It is used for the deprotection and/or cleavage of the synthesized peptide from the resin. The use of TFA under these standardized conditions is thought to be sufficiently mild, thereby preventing degradation of the desired product. However, peptides of the general structure R¹-(N-alkyl X₁)-X₂-R² are hydrolyzed by standard TFA solid-phase peptide synthesis (SPPS) cleavage/deprotection conditions providing fragments R¹-(N-alkyl X₁)-OH and H-X₂-R². The fragmentation is observed during a TFA cleavage both from the resin and in solution. The hydrolysis is proposed to proceed via an oxazolone-like intermediate in which equilibration of the chiral center of the N-alkylated residue occurs. This mechanism is supported by H/D exchange as observed by MS and NMR in conjunction with HPLC. © Munksgaard 1996.     

固相法合成心肌兴奋肽及其类似物

用Boc-和Tos-基团分别保护氨基和侧链胍基,以1%交联度聚苯乙烯二苯甲氨基树脂为载体,用DCC固相法合成肽,HF断裂肽树脂键和去除侧链保护基团,粗产物经高效液相层析纯化,合成了心肌兴奋肽Phe-Met-Arg-Phe-NH_2及其类似物Phe-Pro-Arg-Phe-NH_2,并观察了此二种肽对大鼠血压和心率的影响。     

SYNTHESIS OF α- AND β-MELANOCYTE STIMULATING HORMONES*

The solid phase synthesis of α-melanocyte stimulating hormone (α-MSH) using the benzhydrylamine resin and a number of recently described side chain protecting groups is given. The carboxamide terminal peptide was obtained directly by treatment of the peptide resin with liquid hydrogen fluoride at 0 in the presence of carbonium ion scavengers. The solid phase synthesis of the C-terminal carboxylate hormone, porcine β-melanocyte stimulating hormone (β_p-MSH), using a 1% cross-linked Merrifield resin and the same side chain protecting groups as in the α-MSH synthesis also is presented. Purification of both peptides was carried out by conventional chromatographic techniques. Both hormones were fully active and β_p-MSH was slightly more potent than previously reported in the literature.     

SYNTHESIS OF PROTECTED SECRETIN16–27 ON A MERRIFIELD RESIN

The partially protected dodecapeptide to secretin, H - Ser(Bzl) - Ala - Arg(Tos) - Leu - Gln - Arg(Tos) - Leu - Leu - Gln - Gly - Leu - Val - NH₂ (protected secretin_16–27) was prepared using a standard Merrifield resin and solid phase synthesis methods. For comparative purposes the unprotected peptide also was prepared on a benzhydrylamine resin. Contrary to previous reports, the valine C-terminal peptide can be cleaved from the resin and the amide obtained in high yield. A variety of conditions were examined to accomplish the cleavage of the peptide from the resin in its carboxamide terminal form. The best conditions found were transesterification followed by ammonolysis in a mixed solvent system. A thin-layer chromatography system which clearly separates the methyl ester and carboxamide terminal secretin_16–27 was developed.     

Synthesis of phospho-urodilatin by combination of global phosphorylation with the segment coupling approach

The chemical synthesis of biologically active phosphorylated urodilatin (CDD/ANP-95-126) was achieved by using a strategy of coupling protected peptide segments in solution. Three protected peptide segments corresponding to urodilatin (1-14) with side chain-unprotected Ser¹⁰, (15-24) and (25-32) were prepared manually using Fmoc chemistry on an aminopropyl polystyrene resin with the super acid-labile HMPB linker. For the coupling of segments, the carboxy group of the C-terminal segment (25-32) was converted into the tert-butyl ester by treatment with TBTA. The protected peptide segments were coupled in the presence of EDC/HOOBt or TBTU/HOBt to yield fully protected urodilatin with a free hydroxy function at Ser¹⁰. Introduction of the phosphate was performed with Et₂NP(OtBu)₂ and tetrazole followed by oxidation of the phosphite. Alternatively, a prephosphorylated protected segment (1-14) was used in the segment condensation. Our investigations indicate that both pathways, phosphorylation of protected urodilatin in solution and use of a prephosphorylated building block, are suitable methods to obtain a large phosphopeptide of high purity without formation of H-phosphonates or other by-products. © Munksgaard 1996.     

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

The preparation and application of a new linker for the synthesis of peptide amides using a modified Fmoc-method is described. The new anchor group was developed based on our experience with 4,4′-dimeth-oxybenzhydryl (Mbh)-protecting group for amides. Lability towards acid treatment was increased dramatically and results in an easy cleavage procedure for the preparation of peptide amides. The synthesis of N-9-fluorenylmethoxycarbonyl-[(5-carboxylatoethyl-2.4-dimethoxyphenyl)-4′-methoxyphenyl]-methyla-mine is reported in detail. This linker was coupled to a commercially available aminomethyl polystyrene resin. Peptide synthesis proceeded smoothly using HOOBt esters of Fmoc-amino acids. Release of the peptide amide and final cleavage of the side chain protecting groups was accomplished by treatment with trifluoroacetic acid-dichloromethane mixtures in the presence of scavengers. The synthesis of peptide amides such as LHRH and C-terminal hexapeptide of secretin are given as examples.     

MODIFICATIONS OF SOLID PHASE PEPTIDE SYNTHESIS TO OBTAIN HOMOGENEOUS OLIGOPROLINES IN HIGH YIELD

When oligoprolines containing a [³H] proline in the second residue from the carboxyl terminus are synthesized by the standard solid phase method using stepwise addition of prolines there appears, in addition to the oligoprolines, a steady increase of ninhydrin-negative, but radioactive, impurities. These impurities were found to be N-trifluoroacetyl oligoprolines, and a substantial amount of diketopiperazine was present at the diproline stage. The trifluoroacetyl blockage of the N-termini was almost complete by the time 14 residues were added. To eliminate these side reactions, a block of Boc[³H]Pro₂ was coupled to prolyl-resin in order to avoid the diprolyl-resin stage, and 25% CF₃CO₂H in CHCl₃ was replaced by 4N HCl in dioxane as the deprotecting reagent. These changes eliminated the formation of diketopiperazine and trifluoroacetyl impurities, but still a steady increase of ninhydrin-negative, but radioactive, impurity was seen, reaching 22% after three couplings. These side reactions were traced to the neutralization step, and in order to avoid them it became necessary to eliminate the neutralization step, and replace it by incremental additions of Et₃N during the coupling step. As a result of these modifications, we now obtain homogeneous length oligoprolines upon cleavage from the resin, with no further purification, when analyzed by reversed-phase liquid chromatography.     

Further studies into the Boc/solid-phase synthesis of Ser(P)- and Thr(P)-containing peptides

The Ser(P)-containing peptide corresponding to phospholamban 11-19, Ac-Ala-Ile-Arg-Are-Ala-Ser(P)-Thr-Ile-Glu-NH₂, was prepared by the use of Boc-Ser(PO₃Ph₂)-OH in Boc/solid-phase peptide synthesis followed by HF cleavage of the peptide from the polystyrene resin and subsequent platinum-mediated hydrogenolytic cleavage of the phenyl phosphate groups. A study of the HF deprotection step showed that extensive dephosphorylation of the Ser(PO₃Ph₂)-residue occurred using three commonly used HF conditions and gave rise to large quantities of the Ser-containing peptide. The subsequent study of model peptide systems under standard HF conditions established firstly that the extent of dephosphorylation was dependent on the HF-contact time, and secondly that the Ser(PO₃Ph₂) residue underwent dephosphorylation at a slightly higher rate than the Thr(PO₃Ph₂) residue. © Munksgaard 1994.