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.     

Unexpected racemization of proline or hydroxy-proline phenacyl ester during coupling reactions with Boc-amino acids

When L-proline or O-benzyl-trans-4–hydroxy-L-proline phenacyl ester was coupled with Boc-amino acids in dimethylformamide using water-soluble carbodiimide (WSCI) in the presence of anhydrous 1-hydroxybenzotriazole (HOBt) as coupling reagents, extensive racemization was observed at the Cα of the proline or hydroxy-proline residue. The extent of racemization was measured by HPLC after the coupling with Boc-L-Leu-OH in the presence or absence of HOBt. The extent of racemization increased when HOBt was added to the reaction mixture, but greatly decreased when it was not, indicating that HOBt was needed for inducing racemization. Almost no racemization was observed when the coupling reaction was carried out by the mixed anhydride procedure in tetrahydrofuran or by the carbodiimide method in dichloromethane without using HOBt. In the case of coupling reactions with ordinary L-amino acid phenacyl esters, no racemization was observed. Examination of some model systems yielded sufficient evidence to prove that HOBt is an efficient catalyst for racemizing proline or hydroxy-proline phenacyl ester not only in the stage of cyclic intermediate formation but also in the opening of the ring structure. Thus, the racemization reaction was found to be closely related to the formation of the cyclic carbinol-amine derivative.     

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.     

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.     

Effect of copper(II) chloride on suppression of racemization in peptide synthesis by the carbodiimide method

Copper(II) chloride was found to be an extremely efficient racemization-suppressing additive in the DCC method as compared with the hitherto known ones, by employing the model coupling Z-Gly-l -Val-OH +h-l -Val-OMe in DMF. Although some other copper salts also had a profound effect, copper(II) chloride was the best from the viewpoint of both racemization suppression and coupling efficiency. The effectiveness of copper(II) chloride was further confirmed by employing the EDC-mediated couplings of Z-Gly-containing dipeptides with amino acid esters or dipeptide esters, and those of Z-l -Ala (or l -Val)-l -Val-OH with amino acid esters or dipeptide esters. In almost all the cases studied, no detectable amount (< 0.1 %) of epimer was observed by the HPLC analysis in the presence of copper(II) chloride. This was also the case even with an extremely stringent coupling system Z-l -Pro-l -Val-OH + H-l -Pro-OMe. With reference to the mechanism of racemization suppression, it was found that copper(II) chloride has a strong ability to suppress the racemization of the 5(4H)-oxazolone, which may be formed from an activated carboxyl component during the coupling.     

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.     

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.     

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.     

(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.     

Synthesis of Ac-Asp-Gly-Ser and Ac-Asp-Pro-Leu-Gly-NH2

The syntheses of Ac-Asp-Gly-Ser and Ac-Asp-Pro-Leu-Gly-NH₂ are described. The two peptides were prepared in solution by stepwise elongation using the DCC method with additives (HOSu or HOBt). The push-pull method was also used in the synthesis of Ac-Asp-Gly-Ser. A racemization test of the amino acids in this peptide has been performed.     

N,N'-bis(2-oxo-3-oxazolidinyl) phosphinic chloride (BOP-Cl); a superb reagent for coupling at and with iminoacid residues

Analytically pure dipeptides containing two iminoacids [Pro, MeAla, MePhe, MeVal and SPip (γ-thiapipecolinic acid)] were prepared with BOP-Cl [N,N]-bis(2-oxo-3-oxazolidinyl) phosphinic chloride] at normal temperature (0° — r.t.) in high yields (80–100%) and with excellent optical purity (below noise limit in ¹H n.m.r.). Maximum yields were obtained with Zervas' preactivation procedure, using, for example, an excess of BOP-Cl followed by the addition of (an equal excess of) the aminocomponent. Racemization suppressors (HOBt, imidazole, etc.) are of no use in these difficult couplings. Z-Me Val-Me Val-OBu^t (yield: 89%, without racemization (!)) and Z-SPip-SPip-OMe (yield: 96%, less than 5% stereomutation) were prepared for the first time, whereas all other coupling reagents have failed. Peptide acids with N-methylaminoacids as the COOH-terminal residues were coupled with excellent yields (70–90%), but unfortunately chiral integrity could not be preserved. Pronounced chiral induction was observed during the preparation of Z-Pro-MePhe-MeAla-OBu^t, and an influence of the penultimate protecting group was detected. In contrast, Z-MeVal-MeVal-MeVal-MeVal-OBu^t was formed with probably only 5% stereomutation (yield: 52%). Some side reactions with BOP-Cl are observed: e.g. symmetrical anhydride and DKP formation.     

Suppression of epimerization by cupric (II) salts in peptide synthesis using free amino acids as amino components

An epimerization-free system for coupling N-protected peptides with free amino acids was developed. A number of inorganic substances were tested as epimerization suppressant additives during the coupling by various methods (carbodiimide plus additives, uronium salts, Woodward’s reagent-K, isobutyl-chloroformate, etc.). Some of them (ZnCl₂, RbClO₄, LiCl, SnCl₄, AlCl₃, etc.) in combination with some coupling methods can guarantee coupling with minimal epimerization (D -epimer < 1%). But only a simultaneous use of 1-hydroxybenzotriazole and Cu²⁺ ions as additives in carbodiimide-mediated peptide couplings appeared to give a standard result (D -epimer < 0.1%). There was no epimerization even in the case when N-methyl amino acid (sarcosine) was used as an amino component, while in the absence of Cu²⁺ ions an unacceptable level of epimerization was observed (D -epimer, 22% for carbodiimide with the 1-hydroxybenzotriazole method). So far it has been considered that Cu²⁺ ions prevent obtaining peptides in high yields (< 90%) by various coupling methods. We have found that the use of 1-hydroxybenzotriazole, CuCl₂ and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide instead of dicyclohexylcarbodiimide provides a possible method for obtaining the desired peptides in 90–99% yields without epimerization. All these results were shown by employing several model peptide couplings with free amino acids as amino components dissolved in an effective solvent system which readily dissolved them.     

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。     

Organic copper complexes as a new class of proteasome inhibitors and apoptosis inducers in human cancer cells

Here we report that organic copper complexes can potently and selectively inhibit the chymotrypsin-like activity of the proteasome in vitro and in vivo. Several copper compounds, such as NCI-109268 and bis-8-hydroxyquinoline copper(II) [Cu(8-OHQ)(2)], can inhibit the chymotrypsin-like activity of purified 20S proteasome. In human leukemia cells, proteasome inhibition occurs within 15min after treatment, followed by apoptosis. Neither proteasome inhibition nor apoptosis occurs in non-transformed, immortalized human natural killer cells under the same treatment. Furthermore, proteasome inhibition and apoptosis induction were detected in prostate cancer cells treated with the ligand 8-OHQ alone following pre-treatment with copper(II) chloride. None of these events occurred in cells treated with copper(II) chloride alone, 8-OHQ alone (without growth in copper-enriched media), or nickel(II) chloride pre-treatment followed by 8-OHQ. Furthermore, we found that copper-mediated inhibition of purified 20S proteasome cannot be blocked by a reducing agent and that organic copper compounds do not generate hydrogen peroxide in the cells, suggesting that proteasome inhibition and apoptosis induction are not due to copper-mediated oxidative damage of proteins. Our results suggest that certain types of organic ligands could bind to tumor cellular copper, forming potent proteasome inhibitors and apoptosis inducers at copper concentrations found in tumor tissues.     

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.     

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.     

α-Amino acids derived from ornithine as building blocks for peptide synthesis

Recently great interest has arisen in the synthesis of combinatorial libraries, and this technology provides a significant partner to contemporary strategies in rational design and lead discovery. By simple combination of a given set of building blocks, high numbers of different molecules are produced simultaneously, increasing the possibility of discovery of a lead compound in a limited time. One direction of research in this field focuses on the synthesis of libraries composed of modified amino acids. Here, the synthesis and characteristics of some building blocks derived from ornithine are described. The synthesis is based on the acylation/sulfonation of the copper complex of ornithine by aroyl and arylsulfonyl chlorides exemplified by 2-thiophenecarbonyl chloride, p-toluenesulfonyl chloride and 8-quinolinesulfonyl chloride. To evaluate the potential use of these modified α-amino acids as component in an oligopeptide library, all three derivatives were incorporated in a hexapeptide with a random sequence using a standard coupling procedure (DIC/HOBt/DIEA). Depending upon the acidity of the amido hydrogen on the δ-nitrogen, competition between intramolecular cyclization and peptide bond formation was observed. The higher the acidity, the more pronounced is this side reaction. Coupling conditions for peptide formation were optimized so that the newly described amino acid based building blocks are suitable for incorporation into libraries consisting of unnatural amino acids. The outlined procedures open up a broad avenue of possibilities for creation of diversity into peptidic libraries.     

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.     

Racemization free coupling of peptide segments

The total synthesis of the insect neuropeptide derivative Z-Gly-Gly-Ser-Leu-Tyr-Ser-Phe-Gly-Leu-NH₂ has been carried out by a convergent solid phase strategy. For the coupling of the N-terminal pentapeptide to the C-terminal tetrapeptide, three different methods were assayed. Racemization of the acyl activated amino acid during the fragment condensation reaction was monitored by HPLC. Best results were obtained by enzymatic coupling in a low water containing media using adsorbed α-chymotrypsin. An optically pure product was obtained in 82% yield after 1 h of reaction. Chemical methods such as DIC/HOBt and BOP/HOBt NMM always rendered highly optically impure products containing 10-20% of the d -epimer.     

Influence of simultaneous supplementation of zinc and copper during chelation of lead in rats.

The influence of zinc and copper supplementation during chelation therapy to reduce zinc and copper imbalance and promote lead elimination from the body, was investigated in rats poisoned with lead. The simultaneous supplementation of zinc and copper increased urinary lead excretion by calcium disodium ethylenediamine tetraacetic acid (CaNa2EDTA) compared to treatment with CaNa2EDTA alone. Combination therapy was effective in potentiating the depletion of blood and renal lead by CaNa2EDTA and meso 2,3-dimercapto succinic acid (DMSA). Combination therapy was also more effective in reducing hepatic lead by CaNa2EDTA and blood lead by 2,3-dimercapto propane sulphonate (DMPS). Zinc and copper supplementation produced a more effective reversal of inhibited blood delta-aminolevulinic acid dehydratase (ALAD) activity, urinary delta-aminolevulinic acid excretion and depleted body zinc and copper status.