Racemization mechanism of serine dipeptide active ester derivatives*

The racemization of Z-Gly-Ser(Bzl)-OPcp was studied using the isotope effect to distinguish between enolization and 5(4H)-oxazolone mechanism. Labeled Z-Gly-L(α²H)-Ser(Bzl)-OPcp was racemized with NEt₃ in THF to give k_r^2H = 570; similarly the unlabeled Z-Gly-L-Ser(Bzl)-OPcp yielded a racemization rate constant of k_r^H = 720. The k_r^H/k_r^2H ratio of 1.3 indicates that the serine dipeptide active ester derivatives racemize mainly through 5(4H)-oxazolone and to a lesser extent through the enolization mechanism, provided the 5(4H)-oxazolone racemizes much faster than it couples. 5(4H)-Oxazolone was prepared from Z-Gly-L-Ser(Bzl)-OH with DCC. Its first order racemization rate constant in THF is independent of its concentration, indicating that the racemization is not an intermolecular autoracemization. The unimolecular, that is intramolecular self-catalyzed, racemization is not possible based on molecular models. Therefore, it must be a solvent catalyzed bimolecular reaction. Its racemization was instantaneous with NEt₃ while its coupling with H-Val-OMe was found to be 4 times 10^-2M^-1 s^-1, that is k_r± k_c. The k_c/k_r values, which indicate the extent of racemization during coupling, are compared for benzyloxycarbonylamino and benzyloxycarbonylglycylamino acid active esters; the most vulnerable amino acids are His, Cys and Ser. The k_c/k_r values also indicate that for practical synthetic purposes the pentafluorophenyl esters should be preferred over other active esters to minimize racemization.     

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.     

Serine and d-serine position-1 substituted angiotensin II analogues Synthesis using new 2,3,5,6-tetrafluorophenyl active esters and biological activities

[Ser¹], ( 32 ), [d -Ser¹]- ( 29 ), [Ser¹, Leu⁸]- ( 31 ), and [d -Ser¹, Leu⁸] angiotensin II ( 30 ) were synthesized by a repetitive method in solution using new protected amino acid 2,3,5,6-tetrafluorophenyl active esters. 32 and 29 were agonists, and 31 and 30 were specific antagonists to angiotensin II (AII) receptors determined by the rabbit aortic strip (RAS) and rat blood pressure (RBP) assays. It was found that the hydroxymethyl side chains of serine and D-serine in position-l has an important influence on the agonistic activity of the analogues. The pressor activities of 32 and 29 were 129 and 314%, respectively, as potent as AII. On the other hand, 31 and 30 were effective in antagonizing the AII-induced contraction of RAS and rise in RBP.     

p-Chlorotetrafluorophenyl esters of N-protected amino acids

p-Chlorotetrafluorophenyl (Tfc) esters of protected amino acids and peptides are more reactive than are the well known pentafluorophenyl (Pfp) esters. Two reagents, p-chlorotetrafluorophenyltrifluoroacetate (Tfc-OTfa) and di-(p-chlorotetrafluorophenyl)carbonate (di-Tfc-carbonate), can be used for their syntheses, thereby avoiding use of the allergic dicyclohexylcarbodiimide. This is especially important for bulk preparations. Many Fmoc- and Boc-amino acid-OTfc esters have been synthesized and characterized. The hexadecameric tanden1 repeat H-(AlaAlaLysPro)₄-OH was synthesized using di-Tfc-carbonate for the preparation of Tfc-esters.     

Isosteric conversion of protein carboxyl groups into carboxamide groups. II. Application to lysozyme

For isosteric conversion of carboxyl groups of proteins into amide groups, ammonolysis of protein esters under mild conditions was attempted. Ammonolysis of methyl esters of lysozyme and bovine serum albumin proved to be incomplete. Highly reactive N-ethylsalicylamide esters of guanylated lysozyme were therefore prepared by subjecting the protein to reaction with N-ethylbenz-isoxazolium ion at pH 4.2, 0°. Per molecule, 5–7 ester groups were introduced, with concomitant decrease of activity of 80–90%. Only 0.3 tyrosine was modified. On hydrolysis at pH 9.2 the activity was completely restored. At pH 7.9 three classes of ester groups could be distinguished: one group of high rate of hydrolysis (k₁ = 1.5 min^-1), three groups of intermediate rate (k₂ = 0.13 min^-1) and two groups of low rate (k³ = 0.018 min^-1). The intermediate rate approximated the rate of hydrolysis of the model compound benzoylglycine N-ethyl-salicylamide ester (k = 0.15 min^-1). Ammonolysis at pH 9.2 in 2.0 M ammonia/ ammonium acetate provided complete conversion of the ester groups into amide groups without restoration of activity, confirming the essentiality of certain carboxyl groups. In particular, rearrangement of the ester groups into relatively stable imide groups by O–N acyl migration was found to be completely absent. When native lysozyme was esterified with N-ethylbenzisoxazolium ion the activity did not completely return on hydrolysis.     

Preparation and properties of Nα-trityl amino acid 1-hydroxybenzotriazole esters

N ^α-Trityl amino acid 1-hydroxybenzotriazole active esters, in contrast to HOBt esters of other protected amino acids, exhibit high hydrolytic stability and can be isolated in excellent yields and purity. The derivatives occur in three isomeric forms (an ester=I, two amides=II and III). All active ester forms react at high concentrations within 2 h with various alkyl amino acid esters to produce the corresponding protected dipeptides in high yields. Amorphous active esters on prolonged storage at — 20° rearrange partially towards form III without any sign of decomposition.     

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.     

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.     

4-Sulfobenzyl ester – an anionic protecting group for peptide synthesis

The preparation of the 4-sulfobenzyl esters of 18 amino acid derivatives is described. This carboxyl protecting group was introduced according to Hubbuch et al. (1980). The caesium or dicyclohexylammonium salts of N-terminal protected amino acids were reacted with 4-(bromomethyl)benzenesulfonate (1). After N-terminal deblocking, the amino acid-4-sulfobenzyl esters were isolated as zwitterions. The protecting group was removable by catalytic hydrogenation and by saponification. The 4-sulfobenzyl esters could be easily converted to amides and hydrazides. They were stable to 2 M hydrogen bromide in acetic acid as well as to a 10-fold excess of trifluoromethane sulfonic acid in trifluoro-acetic acid. The behaviours of ⁺H₂-Gly-Phe-Leu-OBzl-SO^-₃ and the corresponding methyl, benzyl and 4-nitrobenzyl esters were compared under various conditions.     

Esterification of 9-Fluorenylmethoxycarbonyl-glycosylated serine and cysteine derivatives with an hydroxymethyl resin

Esterification of glycosylated serine and cysteine derivatives with a 4-alkoxybenzyl alcohol (Wang) resin is described. The classical methods of ester bond formation (symmetrical anhydride, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate [TBTU]. /4-dimethylaminopyridine [DMAP]. with or without 1-hydroxybenzotriazole [HOBT]., pentafluorophenyl [Pfp]. esters) gave high percentages of racemization of the glycosylated serine or cysteine residues. To reduce the d -amino acid content, we found that the best results were obtained with the highly efficient MSNT reagent (2,4,6-mesitylenesulfonyl-3-nitro-1,2,4-triazolide), which gave a high yield of substitution of the resin and the lowest percentage of racemization. A difference in behavior was observed between the two amino acids. The glycosylated cysteine derivative always gave lower racemization than the analogous glycosylated serine.     

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.     

Kinetics of peptide synthesis studied by fluorescence of fluorophenyl esters

The kinetics of the reaction of Boc-alanine-trifluorophenyl, Boc-alanine-tetrafluorophenyl, Boc-alanine-pentafluoropbenyl, and Boc-alanine-p-chlorotetrafluorophenyl esters (BocAlaOTrf, BocAlaOTfp, BocAlaOPfp, and BocAlaTfc, respectively) with leucine amide and with valine methyl ester have been measured using changes in fluorophenyl chromophore emission at 375 nm. The kinetic data cannot be well fit with a simple second-order reaction scheme. Measurements of the reaction kinetics at different concentrations of the reagents showed that the expression for the reaction rate is in which k is the reaction rate constant, C_N is the concentration of either LeuNH₂ or ValOCH₃, and C_AE is the concentration of the fluorophenyl ester. This reaction equation indicates a complex, probably chain-like, reaction mechanism. The order of reactivity for these active esters with ValOCH₃ is BocAlaOTfc > BocAlaOPfp > BocAlaOTfp > BocAlaTrf. The apparent rate constant, k, for the reaction with LeuNH₂ is higher than that for the reaction with ValOCH₃.     

4-Sulfobenzyl ester – its use in peptide synthesis

Amino acid 4-sulfobenzyl esters were employed for the synthesis of peptides in solution. They significantly increase the hydrophilicity of protected intermediates as shown by analytical reversed-phase high performance liquid chromatography and counter-current distribution. General methods for working up are described which permit simple and standardized isolations of products. The use of several coupling methods was demonstrated in the synthesis of Z-Val-Gly-OBzl-SO₃NH₄. Ion-exchange chromatography was introduced as a selective purification procedure for protected peptide 4-sulfobenzyl esters. A step-wise synthesis of [Leu⁵]-enkephalin was performed, starting from leucine 4-sulfobenzyl ester. Removal of N-terminal protecting groups produced zwitterionic peptide 4-sulfobenzyl esters. These were readily purified by crystallization from slightly acidic media; no further purification was necessary. The biologically fully active pentapeptide Tyr-Gly-Gly-Phe-Leu was obtained in an overall yield of 30.2%.     

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.     

ACTIVE ESTERS IN THE FORMATION OF ESTER BONDS BETWEEN AMINO ACIDS AND POLYMERIC SUPPORTS

The imidazole catalyzed transesterification of active esters was used for the formation of the ester bond between the carboxyl group of protected amino acids and the hydroxyl group of a polymeric support applied in solid phase peptide synthesis. Anchoring of the C-terminal residue to the hydroxymethyl polymer proceeded smoothly and provided a high degree of incorporation. No racemization was observed in the imidazole-catalyzed alcoholysis. The procedure could be carried out with various active esters such as esters of o- and p-nitrophenol, 2, 4, 5–trichlorophenol, pentachlorophenol and N-hydroxysuccinimide.     

The in Vitro Enzymic Labilities of Chemically Distinct Phosphomonoester Prodrugs

The kinetics of decomposition of phosphomonoesters of hydroxy-methyl-5,5-diphenylhydantoin (1), estrone (2), 17-testosterone (3), 1-phenylvinyl alcohol (4), and 17-testosterone (5) were studied in rat whole blood at 25 and/or 37°C. As the acidity of the leaving hydroxyl group of the phosphomonoester increased, there was a tendency for the rate of hydrolysis to increase, except for the anomalous behavior of 4, which was consistent with its relative rate of hydrolysis in aqueous solutions (1). In addition, the kinetics of hydrolysis of 1–5 and p-nitrophenyl phosphate (p-NPP) were studied in the presence of isolated alkaline phosphatases from a variety of sources. The initial rate of production of 17- and 17-testosterone from their respective phosphate esters (5 and 3), in the presence of human placental alkaline phosphatase, revealed that 3 was hydrolyzed 5.3-fold more rapidly than 5. This difference in reactivity might have been the result of differences in the stereochemical and/or steric nature of the two isomers. For p-NPP, 1, 2, and 4, the k _cat and k _cat / K _m values determined in the presence of the various alkaline phosphatases showed little variation, whereas for 3, the catalytic constants, k _cat and k _cat / K _m, were found to be dramatically less than those found for p-NPP, 1, 2, and 4. This suggested that the reaction steps, involving the noncovalent binding of the phosphomonoester to the enzyme and/or the nucleophilic displacement of the leaving alcohol of the phosphomonoester by the reactive amino acid residue of the enzyme, might have been less favorable in the case of 3, where the carbon atom of the ester linkage was secondary and was associated with a rigid ring system.     

Acetylated glucopyranosyl esters of enkephalins

Acetylated d -glucopyranosyl esters of enkephalins were prepared by two different fragment condensation procedures involving direct participation of imidazole in the ester linkage formation. By both methods anomeric mixtures of d -glucosyl esters were obtained and resolved by column chromatography. Depending on coupling conditions, racemization of either the C-terminal or the penultimate amino acid residue of the enkephalin molecule occurred. The glucoconjugates with inverted stereochemistry were quantitated and separated from the main product by reversed-phase high-performance liquid chromatography. The opioid agonist potencies of the synthesized glucopyranosyl esters of enkephalins on electrically stimulated guinea pig ileum and mouse vas deferens preparations were determined in comparison with [Leu⁵]enkephalin.     

Solid-phase synthesis of phosphopeptides

We report the solid-phase synthesis of peptides containing O-phosphoserine. Coupling was with commercially available Fmoc-amino acid pentafluorophenyl esters, with base used at each cycle to cleave Fmoc. Phosphorylation of those serine residues left unprotected on the peptide-resin was achieved with dibenzylphosphochloridate, and finally trifluoroacetic acid was used to remove side-chain protecting groups (including the benzyl groups used for the phosphate), and to cleave the peptide from the resin in the same step. This synthetic strategy enables the preparation of peptides with individual, selectively phosphorylated residues. Alternative approaches to introduce protected phosphate and continue with coupling of further amino acids were less advantageous due to the lability of the phosphate group to base and to steric hindrance.     

Synthesis of glycosylated tuftsins and tuftsin-containing IgG fragment undecapeptide

Syntheses are described of two new tuftsin derivatives containing a 2-acetamido-2-deoxy-D-galac-topyranosyl unit α- or β-glycosidically linked to the threonine's hydroxy side chain function and of the glycosylated undecapeptide corresponding to the tuftsin region of the heavy chain of IgG (amino acid sequence 289–299). The glycosylated tuftsins were synthesized by the solution procedure. Fmoc-[Gal NAc(Ac)₃α]Thr-OH and Fmoc-[GalNAc(Ac)₃β]Thr-OH were allowed to react with H-Lys(Z)-Pro-Arg(NO₂)-OBzl by the mixed anhydride procedure and the resulting glycosylated tetrapeptides were fully deblocked by catalytic hydrogenation followed by treatment with potassium cyanide, purified by ion exchange chromatography and characterized by analytical HPLC, elemental and amino acid analyses, optical rotation, and proton NMR spectroscopy. Synthesis of the glycosylated undecapeptide was achieved by the continuous flow solid phase procedure on 4-hydroxymethylphenoxyacetyl-norleucyl derivatized Kieselguhr-supported resin. Fmoc-amino acid symmetrical anhydrides or pentafluorophenyl esters, in the presence of N-hydroxybenzotriazole, were used as the acylating agents. To mimic the native sequence of the tuftsin region at the Fc-domain of immunoglobulin G a 2-acetam～do-2-deoxy-β-D-glucopyranosyl unit was N-glycosidically linked to the amide side chain of Asn 297. The glycosylated asparagine residue was introduced as N²-fluorenylmethyloxycarbonyl-N⁴-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopy-ranosyl)-asparagine pentafluorophenyl ester. After cleavage from the resin the glycopeptide was deprotect-ed, purified by ion exchange chromatography, and characterized by analytical HPLC, amino acid analysis, high voltage electrophoresis, and proton NMR. The conformational features of the glyco-undecapeptide were determined by circular dichroism measurements both in water and in 98% trifluoroethanol. Results of biological assays will be published elsewhere.     

Ac10c: a medium‐ring,cycloaliphatic Cα,α‐disubstituted glycine. Incorporation into model peptides and preferred conformation

Abstract: Two complete series of N‐protected oligopeptide esters to the pentamer level from 1‐amino‐cyclodecane‐1‐carboxylic acid (Ac₁₀c), an α‐amino acid conformationally constrained through a medium‐ring C^α_i ? C^α_i cyclization, and either the l ‐Ala or Aib residue, along with the N‐protected Ac₁₀c monomer and homo‐dimer alkylamides, were synthesized using solution methods and fully characterized. The preferred conformation of these model peptides was assessed in deuterochloroform solution using FT‐IR absorption and ¹H NMR techniques. Furthermore, the molecular structures of two derivatives (Z‐Ac₁₀c‐OH and Fmoc‐Ac₁₀c‐OH) and two peptides (the dipeptide ester Z‐Ac₁₀c‐l ‐Phe‐OMe and the tripeptide ester Z‐Aib‐Ac₁₀c‐Aib‐OtBu) were determined in the crystal state using X‐ray diffraction. The experimental results support the view that β‐bends and 3₁₀‐helices are preferentially adopted by peptides rich in Ac₁₀c, the third largest cycloaliphatic C^α,α‐disubstituted glycine known. This investigation allowed us to complete a detailed conformational analysis of the whole 1‐amino‐cycloalkane‐1‐carboxylic acid (Ac_nc, with n = 3–12) series, which represents the prerequisite for our recent proposal of the ‘Ac_nc scan’ concept.