Protected amino acid tetrafluorophenyl esters for peptide synthesis

New 2,3,5,6-tetrafluorophenyl active esters of protected amino acids useful for peptide synthesis were prepared in high yield. For certain amino acid derivatives such as Boc-Pro-OH, Boc-Ile-OH, and Boc-Val-OH, their tetrafluorophenyl esters have significantly higher melting points than the corresponding pentafluorophenyl esters. Kinetic studies were carried out to compare the racemization rate constants (with triethylamine) and coupling rate constants (with valine methyl ester) of the tetrafluorophenyl and pentafluorophenyl esters of protected histidine and tyrosine. Results of the second-order kinetics showed similarly large k_coupling/k_racemization ratios for both tetra- and pentafluorophenyl esters. In particular, the use of 2,3,5,6-tetrafluorophenyl or pentafluorophenyl ester prevents extensive racemization of the N-tert.-butyloxycarbonyl-N^im-benzyl-histidine.     

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.     

Synthesis of N-carboxyalkyl and N-aminoalkyl functionalized dipeptide building units for the assembly of backbone cyclic peptides

Abstract: To improve the assembly of backbone cyclic peptides, N-functionalized dipeptide building units were synthesized. The corresponding N-aminoalkyl or N-carboxyalkyl amino acids were formed by alkylation or reductive alkylation of amino acid benzyl or tert-butyl esters. In the case of N-aminoalkyl amino acid derivatives the aldehydes for reductive alkylation were obtained from N,O-dimethyl hydroxamates of N-protected amino acids by reduction with LiAlH₄. N-carboxymethyl amino acids were synthesized by alkylation using bromoacetic acid ester and the N-carboxyethyl amino acids via reductive alkylation using aldehydes derived from formyl Meldrums acid. Removal of the carboxy protecting group leads to free N-alkyl amino acids of very low solubility in organic solvents, allowing efficient purification by extraction of the crude product. These N-alkyl amino acids were converted to their tetramethylsilane-esters by silylation with N,O-bis-(trimethylsilyl)acetamide and could thus be used for the coupling with Fmoc-protected amino acid chlorides or fluorides. To avoid racemization the tert-butyl esters of N-alkyl amino acids were coupled with the Fmoc-amino acid halides in the presence of the weak base collidine. Both theN-aminoalkyl and N-carboxyalkyl functionalized dipeptide building units could be obtained in good yield and purity. For peptide assembly on the solid support, the allyl type protection of the branching moiety turned out to be most suitable. The Fmoc-protected N-functionalized dipeptide units can be used like any amino acid derivative under the standard conditions for Fmoc-solid phase synthesis.     

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.     

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.     

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.     

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.     

Racemization during aminolysis of mixed and symmetrical anhydrides of N-alkoxycarbonylamino acids by amino acid anions in aqueous dimethylformamide

Racemization attending the aminolysis of symmetrical (Sy) and mixed (Mx) anhydrides (An) of N-alkoxycarbonylamino acids by amino acid anions in aqueous dimethylformamide has been examined by reversed phase high-performance liquid chromatography analysis of the N-protected dipeptides produced. Racemization occurred in most cases when the anion was generated using 1 equiv. sodium hydrogen carbonate, in most cases for MxAn reactions when the base was sodium carbonate, and only in a few cases for SyAn reactions when the base was sodium carbonate. Less racemization was associated with MxAn reactions when the chloroformate of a secondary alcohol had been used for their generation. The change in chirality is explained on the basis of the formation and racemization of the 2-alkoxy-5(4H)-oxazolone, which is greater in the presence of the weaker base because of incomplete deprotonation of the amino acid zwitter-ion.     

An efficient and highly selective deprotection of N‐Fmoc‐α‐amino acid and lipophilic N‐Fmoc‐dipeptide methyl esters with aluminium trichloride and N,N‐dimethylaniline

Abstract: A novel procedure for the deprotection of the carboxyl group of amino acid methyl esters is presented. The process is carried out by the reagent system aluminium trichloride/N,N‐dimethylaniline that can successfully be applied to unblock the carboxyl moiety either of N‐Fmoc‐protected amino acid methyl esters and N‐Fmoc‐protected short dipeptide methyl esters. The chiralities of the optically pure amino acid or peptide precursors are maintained totally unchanged.     

The concept of superactive esters

According to the concept presented, esters forming an amide (peptide) bond by the mechanism S^#_nD_N or S^# * * The market price of synthetic peptides contains a very small component from the cost of the amino acids: obviously the main share consists of expenses related to their processing, which in the net result simply comprises dehydration leading to the formation of peptide bonds between amino acid residues. For oxytocin (Bachem H-6885) amino acids comprise less than 0.004% of the cost of the peptide.
_ND_N involving fast decay of the tetrahedral intermediate may behave as ‘superactive acylating reagents’. These should render coupling involving less reactive substrates, i.e. sterically hindered or ‘difficult coupling sequences’, much faster and more uniform than classic active esters. In extremely cases this advantage could be significant, and the calculated increase in time required for 99.9% coupling substrates 6 pK_a units less reactive than the standard ones reaches 2512000 τ_1/2 units for classic active esters, but only 631 τ_1/2 units for reaction involving ‘superactive esters’. The postulated change of mechanism is expected for esters bearing a leaving group which is able to undergo an additional, synchronous, energetically favored process accompanying its departure, as has been observed in the case of triazine esters. Some advantages of triazine superactive esters in the condensation of sterically hindered substrates are demonstrated.     

Synthesis and Biochemical Studies of Spirocyclic Amino Acid Esters,Part 3 Activity of 2-Azaspiro[4.5]decane-6-carboxylates as GABA-Uptake Inhibitors

Novel GABA analogous spirocyclic amino acid esters 7a—d and 8a—d were prepared and investigated for interaction with GABA-A and GABA-B receptors as well as the GABA uptake system. Starting from known bromoethyl lactones 1 or 2 and arylalkylamines spirocyclic hydroxyalkyl lactams 3a—d and 4a—d were obtained and reduced by LiAlH₄ to yield spirocyclic hydroxymethyl pyrrolidines 5a—d and 6a—d. Oxidation by Jones reagent followed by subsequent esterification gave the title compounds 7a—d and 8a—d which represent conformationally restricted analogues of GABA. Whereas the new spirocyclic amino acid esters 7a—d and 8a—d showed no activity at GABA receptors they proved to be active as GABA uptake inhibitors. An examination of the relationship between structure and GABA uptake inhibition revealed a strong dependence of activity upon the length of the alkyl chain in N-arylalkyl substituents and upon the ring size of underlying spirocyclic system.     

A novel reagent,dialkylphosphite, for peptide synthesis

The same dialkylphosphite reagent can be used for both N protection and C activation of amino acids. Two N-diisopropyloxyphosphinyl (Dipp) tripeptide esters were prepared, and nine N-Dipp-dipeptide acids were synthesized through the activated amide intermediate. The positive ion FAB-MS of N-Dipp-tripeptide showed novel cleavage patterns in that only the N-phosphoryl fragment ions gave intense peaks while the C-terminal series ions did not appear. This novel character might be useful for peptide sequence analysis. In addition, dialkyloxyphosphinyl group can be examined by ³¹P-NMR for peptide conformational analysis and inspection for the degree of racemization during the coupling reaction.     

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.     

Methylthiomethyl,a new carboxyl protecting group in peptide synthesis

N^α-protected amino acid methylthiomethyl esters (MTM) were obtained in good yields under mild conditions using the «Bu^tBr/Me₂SO» reagent. Selective removal of the N-protecting group was achieved in HCl/anhydrous ethyl ether and the MTM ester hydrochlorides were successfully used in the synthesis of dipeptides.     

Activation of carboxylic acids by pyrocarbonates

Activation of carboxylic acids was achieved via dialkyl pyrocarbonates (ROCO₂O, R= C₂H₅, i-C₃H₇, sec-C₄H₉, tert.-C₄H₉) in aprotic solvents in the presence tertiary amines. A convenient procedure for the preparation of carboxylic acid anhydrides from carboxylic acids and di-tert.-butyl pyrocarbonate in the presence of pyridine is reported. Analogously, di-isopropyl- or diethyl pyrocarbonate may be used in the presence of N-methylmorpholine (triethylamine). With pyridine, di-isopropyl- or diethyl pyrocarbonate carboxylic acids form isopropyl- or ethyl esters, respectively. A wide variety of esters were prepared in good yields in a one-pot procedure from carboxylic acids, including N-protected amino acids, and alcohols or from phenols by means of di-tert.-butyl pyrocarbonate in the presence of pyridine (Boc₂O-pyridine system). t-Butyl esters of carboxylic acids were obtained by the same procedure with 4-dimethylarninopyridine. In the absence of carboxylic acid, with 4-dimethylaminopyridine Boc₂O and alcohols generate alkyl tert.-butyl carbonates.     

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%.     

Kinetic study of peptide synthesis in the presence of imidazole

The interaction of N-protected amino acid active esters with imidazole has been found to be a reversible reaction. The rate constant values for a direct reaction of tert.-butyloxycarbonylglycine p-nitrophenyl ester with imidazole and for a reverse reaction of tert.-butyloxycarbonylglycine imidazolide with p-nitrophenol are reported. The effect of imidazole on the kinetics of active ester and imidazolide reactions with l -threonine methyl ester were studied. The influence of imidazole on the kinetics of O-acylation of N-benzyloxycarbonyl-l -serine amide was also investigated. The data obtained enable the optimum strategy to be chosen in the synthesis of peptides containing hydroxy-amino acid and histidine residues.     

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.     

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.     

Specificity and formation of unusual amino acids of an amide ligation strategy for unprotected peptides

An important step in the recently developed ligation strategy known as domain ligation strategy to link unprotected peptide segments without activation is the ring formation between the C-terminal ester aldehyde and the N-terminal amino acid bearing a 8-thiol or 8-hydroxide. A new method was developed to define the specificity of this reaction using a dye-labeled alanyl ester aldehyde to react with libraries of 400 dipeptides which contained all dipeptide combinations of the 20 genetically coded amino acids. Three different ester aldehydes of the dye-labeled alanine: α-formylmethyl (FM), β-formylethyl (FE), and β,β,β-dimethyl and formylethyl esters (DFE), were examined. The DFE ester was overly hindered and reacted with N-terminal Cys dipeptides (Cys-X). Interestingly, it also reacted slowly with the sequences of X-Gly where Gly was the second amino acid and the X-Gly amide bond participated in the ring formation. Although the FE ester reacted similarly as the FM ester in the ring formation, the subsequent O,N-acyl transfer was at least 30-fold slower than those of the FM-ester. The FM α-formyl methyl ester was the most suitable ester and was reactive with dipeptides of six N-terminal amino acids: Cys, Thr, Trp, Ser, His and Asn. The order and extent of their reactivity were highly dependent on pH, solvent and neighboring participation by the adjacent amino acid. In general, they could be divided into three categories. (1) N-Terminal Cys and Thr were the most reactive. Cys reacted very rapidly and completely within 0.5 h to form thiazolidine in both aqueous and high content of water-miscible organic solvents. Thr reacted to form oxazolidine slowly in aqueous buffer (t₁/2 > 300 h) but rapidly and completely within 20 h in organic-water solvents. (2) N-Terminal Trp, His and Ser were comparatively much less reactive than Cys or Thr. Trp reacted slowly and completely in aqueous buffer but significantly more slowly and incompletely in water-organic solvents. Both His and Ser reacted very slowly and incompletely in both solvent systems. (3) Finally, Asn reacted nearly insignificantly in both solvent systems. The significant rate enhancement by the water-miscible organic solvent on Thr was particularly important to allow the synthesis of disulfide-rich protein domains. Furthermore, the ring formation with N-terminal Trp, His and Asn provided a convenient route to prepare their bicyclic and unusual heterocyclic derivatives for structure-activity study.