Simultaneous use of 1-hydroxybenzotriazole and copper(II) chloride as additives for racemization-free and efficient peptide synthesis by the carbodiimide method

In the carbodiimide mediated coupling of Z-Gly-l -Val-OH with H-l -Val-OMe in DMF, the simultaneous use of HOBt and copper(II) chloride as additives was found to give the desired peptide in a high yield without racemization. In the presence of HOBt, reducing the amount of copper(II) chloride produced a higher yield. Besides improving the coupling efficiency as compared with the case using copper(II) chloride alone as an additive, the present procedure offered another advantage for racemization suppression. Thus, even for the couplings where a low level of racemization was observed in the presence of copper(II) chloride, the simultaneous addition of HOBt and copper(II) chloride resulted in the elimination of racemization. The effectiveness of this new procedure using the two carbodiimide additives in the synthesis of biologically active peptides was assessed by the preparation of a protected Leuenkephalin. In the 4+1 segment condensation using HOBt and copper(II) chloride simultaneously as additives, no racemization was detected and the yield was high enough. The elimination of racemization and improvement of coupling efficiency produced by the present procedure can be attributable to a reduced tendency for the activated forms of the carboxyl component to form a 5(4H)-oxazolone by the action of HOBt, and to the prevention of racemization by copper(II) chloride of the small amount of the oxazolone formed which is not eliminated by the action of HOBt alone.     

Effect of tertiary amine on the carbodiimide-mediated peptide synthesis

The effect of tertiary amine (DIEA) on reaction rate and product purity of a carbodiimide/HOBt-mediated peptide synthesis was studied. It was found that very rapid activation can be achieved using carbodiimide/HOBt in non-polar solvents, such as DCM. Although the HOBt is poorly soluble in DCM, the activation proceeds within 2 min, probably forming the HOBt-ester. By such a preactivation followed by a coupling in the presence of DIEA the rate of coupling is comparable with other rapid methods using BOP or TBTU, and no racemization was found in a model coupling (< 0.1%). For comparison, syntheses of neurotensin by means of different coupling reagents (BOP, TBTU, OPfp-esters) and the DIEA-catalyzed coupling after carbodiimide/HOBt-activation under comparable conditions have shown that these procedures are of the same value in view of coupling efficiency and product purity.     

3-Dimethylphosphinothioyl-2(3H)-oxazolone (MPTO), a promising new reagent for racemization-free couplings

3-Dimethylphosphinothioyl-2(3H)-oxazolone (MPTO) was synthesized, and its ability to effect racemization-free couplings and cyclization of a peptide and its C-terminal epimer was examined. MPTO showed good reactivity in aprotic polar solvents such as N,N-dimethylformamide (DMF) and N-methylpyrrolidone. In reactivity MPTO resembles DPPA and dimethylphosphinothioyl azide (MPTA) previously developed by us, but it is much better than these reagents because the side reactions specific to the azide method could be avoided. In coupling of Z-Gly-Val-OH with H-Val-OMe in DMF at 0°C, no racemization was observed without use of racemization-suppressing additives. Slight racemization observed at room temperature could be completely suppressed by addition of HOBt but not by HOSu. The utility of MPTO was demonstrated by the synthesis of cyclo-(d -Trp-d -Glu(OBzl)-Ala-D-Val-Leu), an intermediate for an endothelin-binding inhibitor BE 18257A. In a comparative study using DPPA, MPTA and MPTO, no racemization was observed for MPTA or MPTO, while DPPA caused considerable racemization. When MPTO was used in the presence of HOBt rapid cyclization (3 h at RT) occurred to give the optically pure cyclic product.     

Anchoring of Fmoc-amino acids to hydroxymethyl resins

A method to anchor Fmoc-amino acids to p-alkoxybenzyl ester resins without using DMAP is described. Esterification takes place with 3–4 equiv. of Fmoc-amino acid plus DCC and a slightly lower excess of HOBt in good yields, without racemization or dipeptide formation. Addition of N-methylmorpholine to the reaction medium enhances the reaction yield but is accompanied by a small amount of racemization and dipeptide formation. Coupling via Fmoc-amino acid chlorides has also been evaluated.     

Racemization during peptide coupling with N,N'-bis(2-oxo-3-oxazolidinyl) phosphinic chloride (BOP-Cl)

The extent of racemization under various circumstances is reported for the coupling of the N-protected model dipeptide Z-Gly-Phe to Val-OMe, mediated by N,N'-bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOC-Cl). BOP-Cl can be used for peptide syntheses either in a one-pot reaction or with preactivation. Maximum yields are obtained with 1.2 equiv. BOP-Cl and 1.2–1.5 equiv. amino-nucleophile and 2.2 equiv. mediatorial base in tetrahydrofuran, but the amount of stereomutation is only tolerable with the use of suppressors (4–6%). BOP-Cl/ HOBt (88% yield, 0% racemization) and BOP-Cl/imidazole (96% yield, 1.6% racemization) are the best choices in apolar solvents and HOBt is to be preferred in DMF (89% yield, 2.4% racemization). Most other additives, HOSu, NP, Pfp, ZnCl₂, DMAP, etc., are not good suppressors.     

(1H‐Benzotriazol‐1‐yloxy)‐N,N‐dimethylmethaniminium hexachloroantimonate (BOMI), a novel coupling reagent for solution and solid‐phase peptide synthesis

Abstract: A HOBt‐based immonium‐type compound,(1H‐benzotriazol‐1‐yloxy)‐N,N‐dimethyl methaniminium hexachloro‐antimonate (BOMI), was synthesized and successfully applied to the synthesis of various oligopeptides with good yields. The estimation of racemization and the influence of several reaction parameters such as solvents, bases and temperature were studied by HPLC using a model reaction. It was found that the reactivity of BOMI was much higher than that of HOBt‐based phosphonium‐ and uronium‐type coupling reagents. Moreover, its racemization was lower than that of other HOBt‐derived coupling reagents. The effectiveness of BOMI was also demonstrated by the synthesis of Leu‐enkephalin both in solution and in the solid‐phase.     

Racemization studies in peptide synthesis through the separation of protected epimeric peptides by reversed-phase high performance liquid chromatography

Separation of protected epimeric peptides, Z-Gly-Xaa-Xbb-OMe (where Xaa and Xbb = chiral amino acid residues), by reversed-phase HPLC was utilized for studying racemization in peptide synthesis. Thus, the following factors which might affect the extent of racemization during the coupling by the carbodiimide method were investigated: the combination of amino acid residues to be coupled, coexisting tertiary amine salts, and the relative configuration of the amino acid residues. The following points were revealed: the combination of bulky residues at the coupling site results in extensive racemization in a polar solvent such as DMF, the amine hydrochlorides cause less racemization than the p-toluenesulfonates in DMF, and the influence of relative configuration differs depending on the solvent and the individuality of the amino components. Furthermore, the racemization-suppressing effect of some additives in the carbodiimide method was reevaluated by employing the same procedure.     

Studies on sensitivity to racemization of activated residues in couplings of N-benzyloxycarbonyldipeptides

A series of 24 peptides Z-Gly-Xaa(R)-OH where Xaa= 15 different residues and R= H, NH₂, tBu, Bzl, Trt, Mtr, and StBu were coupled with valine benzyl ester in dimethylformamide or dichloromethane at +5°. The accompanying racemization was determined by analysis of the epimeric products by normal phase high-performance liquid chromatography (HPLC) for Xaa(R) = Met, Cys(StBu) and Lys(Z) and by reversed-phase HPLC after removal of benzyl-based protecting groups for Xaa(R) = Ser(tBu), Thr(tBu) and Arg(Mtr). The coupling methods examined included mixed anhydride (MxAn) at -5°, and N,N′-dicyclohexylcarbodiimide (DCC), benzotriazol-1-yl-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU) in the presence of 1-hydroxybenzotriazole (HOBt). Very few couplings gave stereochemically pure products. The order of sensitivity to racemization of residues depended on the method of coupling and the solvent. It varied most when comparing MxAn to HOBt-assisted reactions; it varied moderately when comparing HOBt-assisted reactions. There was less variation in comparing BOP and HBTU reactions that are initiated by the same mechanism. Leu, Nle, Phe, Asn, Lys(Z) and Asp(OBzl) are identified as the residues least sensitive to racemization. DCC-HOBt generally led to less epimerization than the other methods.     

Effects of amounts of additives on peptide coupling mediated by a water-soluble carbodiimide in alcohols

Abstract: The optimal amounts of 1-hydroxybenzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt) and 1-hydroxy-7-azabenzotriazole (HOAt) for enhancement of peptide coupling mediated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride in alcoholic solvents were found to be less than equimolar against the carboxyl component or the carbodiimide. In comparison with the use of equimolar additives, the use of less equimolar ones was more effective in suppressing the competitive ester formation and in increasing the yield of desired peptides. EDC hydrochloride/around 0.1 equimolar HOAt or HOOBt were efficient reagents for peptide synthesis in the media.     

Practical protocols for stepwise solid‐phase synthesis of cysteine‐containing peptides

Abstract: This study details a series of conditions that may be applied to ensure ‘safe’ incorporation of cysteine with minimal racemization during automated or manual solid‐phase peptide synthesis. Earlier studies from our laboratories [Han et al. (1997) J. Org. Chem. 62 , 4307–4312] showed that several common coupling methods, including those exploiting in situ activating agents such as N‐[(dimethylamino)‐1H‐1,2,3‐triazolo[4,5‐b]pyridin‐1‐ylmethylene]‐N‐methylmethanaminium hexafluorophosphate N‐oxide (HATU), N‐[1H‐benzotriazol‐1‐yl)‐(dimethylamino)methylene]‐N‐methylmethanaminium hexafluorophosphate N‐oxide (HBTU), and (benzotriazol‐1‐yl‐N‐oxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) [all in the presence of N‐methylmorpholine (NMM) or N,N‐diisopropylethylamine (DIEA) as a tertiary amine base], give rise to unacceptable levels (i.e. 5–33%) of cysteine racemization. As demonstrated on the tripeptide model H‐Gly‐Cys‐Phe‐NH₂, and on the nonapeptide dihydrooxytocin, the following methods are recommended: O‐pentafluorophenyl (O‐Pfp) ester in DMF; O‐Pfp ester/1‐hydroxybenzotriazole (HOBt) in DMF; N,N′‐diisopropylcarbodiimide (DIPCDI)/HOBt in DMF; HBTU/HOBt/2,4,6‐trimethylpyridine (TMP) in DMF (preactivation time 3.5–7.0 min in all of these cases); and HBTU/HOBt/TMP in CH₂Cl₂/DMF (1:1) with no preactivation. In fact, several of the aforementioned methods are now used routinely in our laboratory during the automated synthesis of analogs of the 58‐residue protein bovine pancreatic trypsin inhibitor (BPTI). In addition, several highly hindered bases such as 2,6‐dimethylpyridine (lutidine), 2,3,5,6‐tetramethylpyridine (TEMP), octahydroacridine (OHA), and 2,6‐di‐tert‐butyl‐4‐(dimethylamino)pyridine (DB[DMAP]) may be used in place of the usual DIEA or NMM to minimize cysteine racemization even with the in situ coupling protocols.     

Effect of copper(II) chloride on suppression of racemization in peptide synthesis by the mixed anhydride and related methods

In segment couplings by the mixed anhydride method using isobutyloxycarbonyl chloride, the use of copper(II) chloride as an additive suppressed racemization completely in the same manner as in the carbodiimide method reported previously. This was confirmed by employing a number of couplings between Z-dipeptides and amino acid esters. The racemization-suppressing effect of other compounds were also evaluated by employing one of these model couplings to be at best only limitedly effective. Copper(II) chloride was effective also in the related method using EEDQ. Thus, in the couplings where a low level of racemization was observed without an additive, the addition of copper(II) chloride eliminated racemization even at ambient temperature where EEDQ is usually used. The effectiveness of copper(II) chloride was confirmed also in the BOP-C1 method. In the presence of HOBt racemization was reduced to a low but still detectable level, while it was suppressed completely by the addition of copper(II) chloride.     

Coupling in the absence of tertiary amines

In order to avoid base catalyzed side reactions during coupling, attempts were made to render superfluous the addition of tertiary amines to the reaction mixture. Weak acids were applied for the removal of acid labile protecting groups. Acetic acid and other carboxylic acids were considered unsuitable for this purpose because the resulting salts could be the cause of chain termination in the next coupling step. Pentachlorophenol and 2,4-dinitrophenol cleaved the Bpoc, Nps and Trt groups but more practical rates were reached with solutions of 1-hydroxybenzotriazole (HOBt) in trifluoroethanol, in acetic acid, or in a mixture of phenol and p-cresol. In addition to acidolysis, HOBt salts of amino components could also be obtained through hydrogenolysis of the Z group or thiolysis of the Nps group in the presence of HOBt, or by the displacement of acetic acid from acetate salts with HOBt. Acylation of HOBt salts of amino components with symmetrical or mixed anhydrides or with active esters did not require the addition of tertiary amine.     

实用高效的酰胺键形成试剂

HOAt、HOBt和6-Cl-HOBt是高效实用的酰胺键形成促进剂。它们能防止或大幅减少消旋反应，抑制副产物的产生。在制备、运输、应用及废料处理中要注意安全，防止爆炸。     

Studies on racemization associated with the use of benzotriazol-1-yl-tris (dimethylamino)phosphonium hexafluorophosphate (BOP)

The influence of the amount and nature of the necessary tertiary amine on racemization attending the BOP-mediated coupling of N-benzyloxycarbonylglycyl-dipeptides has been examined by determining the epimeric products by high-performance liquid chromatography. Racemization was encountered in all cases examined that included dichloromethane as solvent. The least racemization occurred when the base was diisopropylethylamine, the most when the base was N-methylmorpholine. Excess base promoted racemization. Racemization was significantly diminished but not eliminated when 1-hydroxybenzotriazole was added to the reaction mixtures. The above results pertain to the coupling of peptide segments; they do not pertain to the coupling of N-alkoxycarbonylamino acids.     

Synthesis of cyclic peptides on solid support Application to analogs of hemagglutinin of influenza virus

In order to mimic a well-known loop structure (site A) of the hemagglutinin of influenza virus, a series of cyclic peptides derived from thc region 139–147 were synthesized. The lactam analogs cyclised between the N-terminus Cys 139 and the β-carboxyl of aspartic acid 148 (small loop) or the ENHZ of lysine 148 via succinimidyl linker (large loop) were synthesized by the solid phase method. Cyclisation was directly performed on the solid support prior to final cleavage of the peptide. We describe two protection schemes which allow us to obtain different loop sizes derived from the same sequence. Eight of the analogs contained relatively large ring structures (up to 38 membered). For protection of the side chain of aspartic acid in combination with N-α-Fmoc protection, the cyclohexyl ester was more satisfactory than the benzyl ester with respect to imide formation. When the rate of cyclodimerisation, as a function of resin substitution, was compared to the rate of cyclic monomer formation, it was found that dimerisation was proportional to the charge of the resin. Furthermore, a comparison of the recently reported BOP reagent over the classical DIPC/HOBt method for the cyclisation reaction shows that in our case the reaction proceeded more rapidly by the BOP procedure although it gave a less pure crude product.     

Synthesis of a cyclic hexapeptide with sequence corresponding to murine tumor necrosis factor-(127–132) as a novel potential antitumor agent

A cyclic hexapeptide cyclo(Lys-Gly-Asp-Gln-Leu-Ser-) 10 was synthesized stepwise in solution by acylation of peptide ester trifluoroacetates directly with preactivated Boc-amino acids using the DCC/HOBt method; the final cyclization reaction was performed using the pentafluorophenyl ester method in solution (1–4). This peptide is a cyclic derivative of murine tumor necrosis factor-(127–132) and is designed as a potential antitumor agent. The cyclic peptide 10 displayed weak cytotoxic activity on three of the four human tumor cell lines tested.     

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.     

Fast and efficient peptide bond formation using bis‐[α,α‐bis(trifluoromethyl)‐benzyloxy]diphenylsulfur. Part I

Abstract: This study towards the development of sulfurane‐based coupling agents shows that bis‐[α,α‐bis(trifluoromethyl)‐benzyloxy]diphenylsulfur (BTBDS) can facilitate rapid amide bond formation between N^α‐urethane‐protected l ‐amino acids and l ‐phenylalanine ethyl ester in the absence of an external base. The corresponding dipeptide esters were obtained in excellent yields and with no detectable racemization, as judged by analysis of the formed dipeptides by chiral‐phase HPLC. In addition, BTBDS‐mediated condensation of benzoyl‐l ‐phenylalanine with l ‐phenylalanine ethyl ester was also investigated. The results indicate that sulfuranes can be useful for application in racemization‐sensitive systems, such as segment condensation.     

High-performance liquid chromatography of epimeric N-protected peptide acids and esters for assessing racemization

The separations by reversed phase high-performance liquid chromatography on a μBondapak-C₁₈ column of 53 epimeric N-substituted di-, tri- and tetrapeptide acids and esters have been attempted, with success in three quarters of the cases. Substituents include acetyl, benzoyl, benzyloxycarbonyl, tert.-butoxycarbonyl and 9-fluorenylmethoxycarbonyl. The series N-benzyloxycarbonylglycyl-Xxx-valine ethyl ester with Xxx = alanyl, valyl, leucyl and phenylalanyl, is recommended for use in studies on racemization. Results on racemization attending the coupling of an amino acid ester as compared with a di- and tripeptide ester vary with the coupling method.     

Racemization during aminolysis of activated esters of N-alkoxycarbonylamino acids by amino acid anions in partially aqueous solvents and a tactic to minimize it

Racemization during the aminolysis of activated esters of N-alkoxycarbonylamino acids by amino acid anions in aqueous dimethylformamide was examined by determining the epimeric products by high-performance liquid chromatography. Partial racemization occurred for a variety of esters, particularly when sodium hydrogen carbonate was used to generate the anion of d -valine. The racemization results from prolonged contact of unconsumed ester with the alkaline medium. Variation of the stoichiometry of reagents for reactions with N-benzyloxycarbonylphenylalanine (Z-Phe) 4-nitrophenyl ester revealed that racemization could be minimized by using Na₂CO₃ as base and a 50% excess of amino acid anion. An efficient synthesis of optically pure Z-l -Phe-D-Val-OH was achieved with a reaction time of 15 min.