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 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.     

Effect of tertiary amines and amine salts on racemization in peptide synthesis

An effect of different tertiary amines, anions and cations on yield, product purity and reaction rate in DCC-mediated Ac-Phe-Leu-OMe synthesis was studied. Degree of racemization and side-product yields down to 0.05% were readily determined by HPLC. The increasing effect of tertiary amino salts on degree of racemization was determined more precisely than previously and, more interestingly, their suppression of N-acylurea formation was discovered. Unexpectedly, we found that the suppressing effect of the racemization of l-hydroxybenztriazole was completely blocked by tribenzylamine. Furthermore, kinetic investigation showed that degree of racemization was not directly linked with 5(4H)-oxazolone formation and aminolysis. The much greater racemization observed than calculated from the rate of formation and aminolysis of 5(4H)-oxazolone led us to the conclusion that an activated form of Ac-Phe other than 5(4H)-oxazolone could be co-responsible for racemization in the investigated reaction. An efficient method for preparation of N-(acetyl-DL-phenylalanyl)-dicyclohexylurea is described.     

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.     

Repetitive BOP coupling (REBOP) in solid phase peptide synthesis

The coupling reagent (benzotriazol-1-yloxy)tris-(dimethylamino)phosphonium (BOP) hexafluorophosphate was tested in the synthesis of luliberin (LH-RH) with inexpensive classically protected Boc-amino acids, in slight excess, and benzhydryl amino resin, without any other additive. The good solubility of this reagent and its by-products is of particular interest for automated peptide synthesis. [D-H-S¹]LH-RH was also synthesized and compared with LH-RH by proton nuclear magnetic resonance spectroscopy. As shown by the biological tests and the high performance liquid chromatography study, unprotected pyroClu and Boc-His can be used without any significant racemization but Boc-His(Boc) was found to be preferable since it gave no detectable racemization and no by-products. The difficult isolation of the minority D-derivative from the crude preparation of LH-RH was resolved by a recycling procedure in reversed phase HPLC.     

Studies on asymmetric induction associated with the coupling of N-acylamino acids and N-benzyloxycarbonyldipeptides

The asymmetric induction occurring during aminolysis by an amino acid benzyl ester of the 5(4H)-oxazolones obtained from N-acyl-dl -valine for acyl = formyl, acetyl, benzoyl, trifluoroacetyl and A′-benzyloxycarbonyl-Xaa where Xaa = Gly, Ala and Leu in dichloromethane and dimethylformamide at + 5° and – 5° was determined by analysis of the epimeric products by high-performance liquid chromatography after removal of protecting groups by hydrogenolysis. The influence of the side-chain of the activated residue on induction was assessed by examining aminolysis of the 5(4H)-oxazolones from N-benzyloxycar-bonyl giycyl-Xaa-OH for Xaa = Ala, Leu, Val, and Phe. The contribution of induction to the epimeric content of products obtained from couplings mediated by N,N′-dicyclohexylcarbodiimide in the presence and absence of 1-hydroxybenzotriazole, and by the mixed-anhydride method, were calculated. The induction was affected at varying levels by the nature of the N-acyl group, the side-chain of Xaa, the nature of the aminolyzing nucleophile, the nature of the solvent, and the temperature, with diastereomeric excesses reaching – 32 and +53. The influence of the side-chain of Xaa on the induction was different in the two solvents. For the N-acyl series, the epimeric content of products did not always correctly reflect the relative tendencies of the derivatives to racemize. The order for epimeric content of the products also depended on the method of coupling.     

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.     

Urethane-protected N-carboxyanhydrides (UNCAs) as unique reactants for the study of intrinsic racemization tendencies in peptide synthesis

We have established that urethane-protected N-carboxyanhydrides (UNCAs) are uniquely suited for the study of intrinsic racemization tendencies in peptide synthesis. The UNCA allows epimerization only by the direct enolization pathway (proton abstraction from the α-carbon) and does not decompose upon epimerization. A protocol employing the quantitative separation and analysis of enantiomeric N-protected amino acid derivatives by chiral HPLC has been developed to measure the intrinsic rate of racemization of UNCAs under widely varying reaction conditions. The influence of the tertiary amine structure, UNCA side chain structure, and solvent were studied. The same protocol was employed to study the intrinsic rate of racemization of N-protected activated amino acid intermediates generated via ‘onium-type’ activating reagents. We have shown that the trends influencing the intrinsic rate of racemization of UNCAs are maintained under the conditions of in situ activations, and are consistent with the trends found in classical studies in the literature. The results are relevant to peptide synthesis both in solution and on solid phase. The intrinsic rate of racemization for any type of activation with any tertiary amine can be measured by this protocol. © Munksgaard 1997.     

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.     

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.     

Comparison of the radiorecovery activity of copper(II)2(3,5-diisopropylsalicylate)4 and copper(II) (chloride)2

Copper(II)₂(3,5-diisopropylsalicylate)₄ [Cu(II)₂(3,5-DIPS)₄] and copper(II)(chloride)₂ [Cu(II)Cl₂ were used to treat γ-irradiated female C57BL/6 mice after irradiation at levels LD50/30 to compare their efficacy in facilitating recovery from radiation-induced systemic inflammatory disease accompanied by loss of body mass and in increasing survival of irradiated mice. Doses of 5, 10 or 20 μmol Cu(II)Cl₂ or 5, 10 or 20 μmol [Cu(II)₂(3,5-DIPS)₄]/kg were administered subcutaneously 3 h after LD_50/30 irradiation and body mass and survival determined throughout the 30-day post-irradiation period compared with controls. Treatment with Cu(II)₂(3,5-DIPS)₄ or Cu(II)Cl₂ facilitated recovery of radiation-induced systemic inflammatory disease, recovery of body mass, and increased survival. Treatment with 5, 10 or 20 (μmol [Cu(II)₂(3,5-DIPS)₄]/kg produced a 44%, 67% or 44% increase in survival, respectively, compared with the vehicle-treated control group. Treatment with 5,10 or 20 μmol Cu(II)Cl₂/kg produced a 7%, 21% or 29% increase in survival, respectively, compared with the vehicle-treated control group. The recovery of radiation-induced loss in body mass and an increase in survival document that both Cu(II)₂(3,5-DIPS)₄ and Cu(II)Cl₂ are effective radiorecovery agents. In addition, Cu(II)₂(3,5-DIPS)₄ is a more effective radiorecovery agent than Cu(II)Cl₂.     

Use of palladium(II) chloride as colour-forming reagent in determination of pralidoxime chloride in water and tablets

Pralidoxime chloride (PAM-2Cl) has been determined spectrophotometrically in Britton—Robinson buffer solution at pH = 6.45; the method is based on measurement of the absorbance of the Pd(II)-pralidoxime complex at 327 nm. Studies of the composition of the complex by Job's continuous variation method, the molar ratio method and Bent—French's method yielded a Pd(II):pralidoxime ratio of 1:1. The conditional stability constant (K′) of the complex at the optimum pH of 6.45 and an ionic strength (μ) of 0.3 M was found to be 10^5.2. The molar absorptivity was 1.05 × 10⁴ 1 mol⁻¹ cm⁻¹. Beer's law was obeyed at concentrations up to 60 μM. The detection limit was 0.55 μg ml⁻¹. The relative standard deviation (N = 10) was 0.28–1.03%. The method was accurate and sensitive for the analysis of PAM-2Cl in water and tablets.     

BOP reagent for the coupling of pGlu and Boc-His(Tos) in solid phase peptide synthesis

The model peptide TRH was successfully synthesized using benzotriazol-l-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent). The coupling reactions were carried out in N,N-dimethylformamide or N-methylpyrrolidone. These solvents allowed the incorporation of the N-terminal pyroglutamic acid residue into the peptide chain, without using the derivative bearing the N-benzyloxycarbonyl group, which acts as a solubility promoter. A comparative racemization study showed that Boc-His(Tos) can be coupled by means of BOP reagent with less racemization than with DCC when the amount of diisopropylethylamine (DIEA) is kept minimal (same ratio of equivalents as for Boc-His(Tos), i.e. 3 equiv.). However, with the use of a larger amount of DIEA in the coupling mixture (9 equiv.), approximately 3% of epimer was found in the crude product. Our study showed that even under low DIEA conditions, the rate of coupling of the residues with BOP remained comparable to that observed with DCC.     

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.     

Use of the excluded protecting group (EPG) method for peptide synthesis

Abstract: The excluded protecting group (EPG) method has been used for the solution synthesis of several peptides including Merrifield's Model Tetrapeptide, linear antamanide and an analogue of magainin‐1, [Ala¹⁹, Asn²²]magainin‐1. In the approach reported, the C‐terminal amino acid is esterified to the 2‐position of cholestane as the [2s,3s]iodohydrin ester and the penultimate amino acid added to the aminoacyl‐steroid as the Fmoc‐pentafluorophenyl‐ester. The Fmoc group is removed with Et₂NH/DMF (～15% v/v) and, after evaporation to ～10 mL, the solution chromatographed on Sephadex LH‐20 in DMF. The dipeptidyl‐steroid elutes as the free amine well separated from other reaction mixture components. Fractions containing the dipeptide, as determined by counting and TLC, are pooled and reacted with the next Fmoc‐amino acid‐pentafluorophenyl ester in the sequence. Repetition of the deprotection/purification/reaction cycle yields the fully protected peptide.On completion of the synthesis, the cholestane iodohydrin ester is selectively removed by treatment with Zn°/AcOH to yield the peptide with intact α‐amino and side chain protecting groups. Global deprotection is achieved with HF. All intermediates from the syntheses reported were characterized. The magainin analogue was shown to have full biologic activity. The Fmoc iodohydrin esters of 16 of the 20 proteogenic amino acids have been prepared and characterized for use as the C‐terminal amino acids in other EPG syntheses.     

N-Fmoc-protected(alpha-dipeptidoyl)benzotriazoles for efficient solid-phase peptide synthesis by segment condensation

N-Fmoc-protected(alpha-aminoacyl)benzotriazoles 1a-d readily afford chiral N-Fmoc-protected-alpha-dipeptides 2a-f (77-89%). Compounds 2a-f are further converted into N-Fmoc-protected(alpha-dipeptidoyl)benzotriazoles 3a-f (71% average yield). Under mild microwave irradiation, 3a-f are used in solid-phase peptide segment condensation syntheses to give tri-, tetra-, penta-, hexa-, and heptapeptides (20-68%).     

Effect of calcium (II) and magnesium (II) ions on the 18–23 γ-carboxyglutamic acid containing cyclic peptide loop of bovine prothrombin An AMBER molecular mechanics study

The effect of calcium (II) and magnesium (II) ions on the conformation of the 18–23 cyclic peptide loop of bovine prothrombin are investigated by the molecular mechanics program AMBER (Assisted Model Building with Energy Refinement). The work is an extension of an earlier paper (Eastman et al, Int. J. Peptide Protein Res. 27, 1986, 530–553) that employed the program ECEPP (Empirical Conformational Energy Program for Peptides). In the absence of either metal ion, or in the presence of either one Ca(II) or one Mg(II) ion, the lowest-energy forms found by AMBER have the Gla21-Pro22 peptide bond in a trans conformation. In the presence of two Ca(II) or Mg(II) ions, the loop form of lowest energy is decidedly cis. The coordination about the Ca(II) and Mg(II) ions is different in both the single and double metal cases. In addition, the peptide chains that emerge from the loop are oriented parallel to each other in the lowest-energy complex with two Ca(II) ions, but are not parallel in the lowest-energy complex with two Mg(II) ions.     

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.     

1 H n.m.r. long-range coupling in 2,4-disubstituted-5(4H)-oxazolones and 4-alkyl-5(2H)-oxazolones generated therefrom by the action of triethylamine

Long-range couplings were observed between H-4 and 2-CCH_a of 2,4-disubstituted-5(4H)-oxazolones, and H-4 and H-2 of 4-alkyl-5(4H)-oxazolones. In the presence of triethylamine, H-4 of the latter migrates to C-2 accompanied by a shift of the double bond to give 4-alkyl-5(2H)-oxazolones which show ⁵J coupling between H₂-2 and 4-CCH_a protons.