Preparation of oligomer-free Nα-Fmoc and Nα-urethane amino acids

A variety of N^α-urethane blocked amino acids, in particular 9-fluorenylmethyloxycarbonyl (Fmoc) derivatives, have been synthesized by utilizing intermediate O,N-bis-trimethylsilyl-amino acids, formed in situ by treating an amino acid with trimethylsilylchloride and a base in an aprotic solvent. The intermediate is then reacted with an acylating agent. A general procedure is given which eliminates the oligomerization side reactions normally observed in Schotten-Baumann type methods. Protected amino acids obtained from this procedure are of high purity as judged by t.l.c, HPLC, ion exchange chromatography, and other physical parameters.     

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.     

Synthesis and modification of dibenzylglycine derivatives via the Suzuki‐Miyaura cross‐coupling reaction

Abstract: The objective of this paper is to describe in details of various available methods to prepare C^α,α‐dibenzylglycines (Db_zg) and then include our work involving the synthesis of side chain Db_zg derivatives. α,α‐Disubstituted amino acids (α,αAAs) are important members in the family of modified amino acids. Replacement of the α‐hydrogens of glycine 1 by alkyl groups leads to α,αAAs. The steric hindrance of the quaternary centre of Aib 2 combined with the helix‐forming capacity has attracted the attention of structural biologists and protein crystallographers. Db_zg 3 is a special structural variant of Aib. The presence of two benzyl groups at C^α‐position not only impart rigidity to the peptide backbone in which it is incorporated, but also acts as a useful vehicle for studying π–π interactions. Although several C^α,α‐disubstituted glycines such as C^α,α‐diethyl glycine (Deg), C^α,α‐dipropyl glycine (Dpg) etc. have been studied in detail, not much has been known about Db_zg because of limited availability of synthetic procedures. Various Db_zg derivatives 19a–f have been prepared using ethyl isocyanoacetate 14 as a glycine equivalent (eq.). A useful and simple methodology has been developed using the Suzuki‐Miyaura cross‐coupling reaction for the modification of Db_zg derivatives 17d , 19d , 22 . Using this ‘Building Block Approach’ (Accounts of Chemical Research 36 , 2003, 342) one can generate a variety of Db_zg derivatives 20a–f and 23a–e , which may find useful applications in combinatorial synthesis and QSAR studies.     

Synthesis of a cyclic retro analogue of somatostatin suitable for photoaffinity labelling

Cyclic somatostatin analogues containing the modified retro sequence of the amino acids Phe⁷ to Phe¹¹ of the natural compound have been found to exhibit high activity for cytoprotection of rat hepatocytes against cell poisons such as phallotoxins and galactosamine. Cyclo(-Phe(p-NH(1-¹⁴C)Ac)-Thr-Lys(CO(p-N₃)C₆H₄)-Trp-Phe-d -Pro-), a photoreactive and radioactive analogue of one of the most active cyclohexapeptides, was synthesized by a combination of solid phase technique and classical solution peptide synthesis. This peptide labels the same proteins in rat liver cell membrane that are modified by photolysable derivatives of bile acids, phalloidin and antamanide.     

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.     

Dipeptide derivative synthesis catalyzed by Pseudomonas aeruginosa elastase

Pseudomonas aeruginosa elastase was used to synthesize various N-protected dipeptide amides. The identity of the products was confirmed by FAB⁺-MS. After recrystallization, the yield of their synthesis was calculated, their purity was checked by RP-HPLC and their melting point was measured. With regard to the hydrolysis, it is well-established that the enzyme prefers hydrophobic amino acids in P′₁ position and it has a wide specificity for the P₁ position. This specificity was demonstrated to be quite unchanged when comparing the initial rates of peptide bond formation between different carboxyl donors (Z-aa) and nucleophiles (aa-NH₂). The elastase, but not the thermolysin, was notably able to incorporate tyrosine and tryptophan in P′₁ position. Furthermore, synthesis initial rates were at least 100 times faster with the elastase. To overcome the problematic condensation of some amino acids during chemical peptide synthesis, it has been previously suggested that enzymatic steps can combine with a chemical strategy. We demonstrated that the elastase readily synthesizes dipeptide derivatives containing various usual N-protecting groups. It was especially able to condense phenylalaninamide to Fmoc- and Boc-alanine. Increasing interest in peptides containing unnatural amino acids led us to try the elastase-catalyzed synthesis of Z-dipeptide amides including those amino acids in the P₁ position. A synthesis was demonstrated with αAbu, Nle, Nva and Phg.     

Efficient synthesis of metal binding peptides incorporating aminodiacetic acid based ligands

New N^x-Fmoc/Bu^t protected amino acids bearing half-EDTA side chains (CH₂)_nN(Ada-O-Bu¹)₂n= 1 (5), n= 2 ( 24 ), n= 3 ( 10 ), n= 4 ( 15 ) were prepared in satisfactory yields. These derivatives can be conveniently used in a solid-phase peptide synthesis as they are devoid of serious shortcomings of Boc/Bzl based syntheses of metallopeptides, such as preliminary peptide capping as well as undesired lactamization of 5 during the peptide synthesis.     

Synthesis and stability of 3-nitro-2-pyridinesulfenyl chloride (NpysCl)

3-Nitro-2-pyridinesulfenyl chloride (NpysCl) is the starting material for the synthesis of N-, O- and S-Npys-protected amino acids. Two efficient, novel synthetic routes to NpysCl are described. The stability of NpysCl was determined in a variety of solvents, with and without base, to determine the most suitable solvent and base for the synthesis of N-Npys amino acids. The syntheses of Npys-Ala and Boc-Lys(Npys) tert-butylammonium salt are also described.     

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.     

Replacement of Tyr-SO3H by a p-carboxymethyl-phenylalanine in a CCK8-derivative preserves its high affinity for CCK-B receptor

The sulfated tyrosine present in the sequence of CCK₈ Asp²⁶-Tyr(SO₃H)-Met-Gly-Trp-Met-Asp-PheNH₂, seems to play a critical role in the recognition of CCK-A binding sites. In this work, we have investigated whether the presence of an anionic charge on the tyrosine moiety is strictly necessary and whether the sulfate moiety interacts with a divalent cation in the receptor subsite. For this purpose, the novel amino acids (l,d ) Phe(p-CH₂CO₂H) and (l,d ) Phe(p-CH₂CONHOH), as well as their l -resolved forms were introduced into the sequence of Ac[X²⁷, Nle²⁸, Nle³¹]-CCK_27-33 by solid phase method. The biological activities of these new derivatives were compared to two almost equiactive analogues of CCK₈, Ac[Phe(p-CH2SO₃H)²⁷, Nle²⁸, Nle³¹]-CCK_27-33 and Boc[Nle²⁸, Nle³¹]-CCK₂₇-3₃ (BDNL) and to the nonsulfated analogue of the latter peptide (BDNL NS). All these new CCK-related analogues behave as agonists in stimulating pancreatic amylase release and display high affinity for brain binding sites (K₁～ 3-11 nm ) but the only peptides which retain affinity for CCK-A receptors (K₁～ 20 nm ) are those containing a p-caiboxymethyl phenylalanine. Thus, introduction of this amino acid under an esterified form on the side chain, into specific and potent CCK-B agonists could allow compounds endowed with good bioavailabilities to be obtained.     

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.     

From chiral bromo[13,14Cn]acetyl sultams to complex molecules singly/multiply labelled with isotopic carbon

The chiral bromo[^13,14C_n]acetyl sultams (+)‐ and (?)‐[^13,14C_n]BABS 1a , 1b have been demonstrated to be highly efficient, versatile and practical synthons to numerous enantiomerically pure singly and multiply labelled building blocks. The trichlorotitanium enolates derived from 1a , 1b undergo aldol addition reactions with aldehydes providing easily purified, crystalline syn‐2‐bromo‐3‐hydroxy [^13,14C_n]carboxylic acid derivatives with excellent diastereo‐selectivity. These can serve as starting materials for e.p. singly/multiply labelled α‐substituted β‐hydroxy acids, β‐substituted/branched α‐hydroxy acids and α‐unsubstituted β‐hydroxy acids. Furthermore, 1a , 1b can be easily converted to the (+)/(?)‐[^13,14C_n]DPMGBS 6 , (+)/(?)‐[^13,14C_n]ITCABS 8 and (+)/(?)‐[^13,14C_n]PABS 10 synthons, which significantly enlarges the spectrum of readiliy accessible intermediates. 6 Provides e.p. labelled α‐amino acids, 8 can be employed for the preparation of e.p. labelled α‐amino‐β‐hydroxy acids (threonine type). Synthon 10 reacts with aldehydes to chiral E‐configured enoyl sultams 11 which serve as starting materials for a broad variety of e.p. singly/multiply labelled α,β‐substituted/branched, acyclic and cyclic carboxylic acid derivatives. Finally, aldehydes, generated by reductive cleavage of the auxiliary from the primary α,β‐substituted acyl sultams, react with Ph₃P=COOR to give γ,δ‐substituted α,β,‐unsaturated esters, which in turn can be readily converted to highly functionalized e.p. labelled intermediates. This methodology has been extensively exploited for the synthesis of a broad spectrum of carbon‐14 labelled drug substances e.g. Taxol, Valsartan, Everolimus, Lipid X, NVP IMM125, SDZ ISQ844, SDZ PRI05 and the cyclosporin derivatives Valspodar, NVP IMM125, NVP NIM811. Copyright © 2002 John Wiley & Sons, Ltd.     

A sensitive HPLC-FLD method combined with multivariate analysis for the determination of amino acids in l-citrulline rich vegetables

The proposed analytical method reports the separation and quantification of 21 amino acids including l-citrulline from fresh vegetables and commercial juices using a C₈ column. Optimal separation conditions for amino acids analysis were obtained with 20 mM sodium acetate (solvent A) and water with organic modifier acetonitrile and methanol (solvent B; 18/50/32 V/V). The ideal pH and column temperature were found to be 5.40 and 35 °C, respectively. The LOD and LOQ values were obtained in the range of 0.02–0.19 ng/mL and 0.04–0.39 ng/mL for all amino acids respectively. Relative standard deviations (RSD) of intraday and interday analysis were found to be <2.7% and 7.9%, respectively. The recovery of amino acids were found be satisfactory for all the tested crops. The developed method was successfully used for the quantification of amino acids in six fresh vegetable juices including watermelon, cucumber, celery, calabaza squash, zucchini squash, yellow squash and commercial juices. Multivariate analysis was used to determine the significant differences in the amino acids profiles. l-citrulline content was highest in fresh watermelon juice (716.57 ± 24.80 μg/mL) and commercial watermelon lime juice (826.48 ± 34.48 μg/mL). The optimized analytical method is rapid, sensitive, accurate and reproducible for analysis of free amino acids including l-citrulline from different vegetable juices and other food products. To the best of our knowledge, this is the first report to separate OPA derivatives of amino acids using C₈ column from watermelon, cucumber, zucchini squash, yellow squash, calabaza squash, and celery in a HPLC-FLD system.     

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.     

Nsc and Fmoc Nα‐amino protection for solid‐phase peptide synthesis: a parallel study

Abstract The 2‐(4‐nitrophenylsulfonyl)ethoxycarbonyl (Nsc) group is an alternative to Fmoc for N^α‐protection in solid‐phase peptide synthesis. Nsc‐amino acids may be particularly suitable for automatic synthesizers, in which the amino acids are stored in solution, and the incorporation of residues prone to racemization such as Cys and His. Owing to the hydrophilicity of the Nsc group, these derivatives are useful for the preparation of protected peptides in convergent solid‐phase peptide synthesis strategies.     

Peptides containing dialkylglycines

A short route for the preparation of 2-trifluoromethyl-4,4-dialkyloxazolin-5-ones (Tdo's), useful reagents for the addition of dialkylglycine residues to the N-terminus of peptides, was examined. 2-Trifluoromethyl-oxazolin-5(4H)-one proved too unstable for a general substrate for alkylation, but 2-trifluoromethyl-4-alkyl-oxazolin-5(2H)-ones, readily available from protein amino acids, could be alkylated to Tdo's in the presence of mild base using active alkyl halides. 2-Trifluoromethyl-4,4-dibenzyl-oxazolin-5-one, prepared in this way as a stable crystalline solid, coupled well with protein amino acid esters or amides, confirming the utility of reagents of this type. Of a number of alkylations examined, only in the case of 2-trifluoromethyl-4-isobutyl-oxazolin-5(2H)-one using isobutenyl iodide was the isomeric 2-trifluoromethyl-2-isobutenyl-4-isobutyl-oxazolin-5-one observed at all; in this case, it predominated. Ammonolysis of Tdo's gave Tfa-dialkylglycine amides, N-deprotection of which using NaBH₄ unexpectedly gave 2-trifluoromethyl-4,4-dialkylimidazol-5-ones. The partition coefficients (P) of a series of N-acetyldialkylglycinamides were measured. Comparison with the values obtained for similar derivatives of the corresponding protein amino acids showed a close correlation between the increasing hydrophobicity of the second side chain of the dialkylglycine derivative and the log P values obtained.     

Comparative biological activities of potent analogues of α-melanotropin

Several α-melanotropin (α-MSH) analogues with para substituted aromatic and nonaromatic amino acids in the 7-position of the hormone were prepared and their melanotropic activities determined in the frog (Rana pipiens) and lizard (Anolis carolinensis) skin bioassays. D and L-Phe(p-NO₂), D- and L-Tyr, D- and L-Ala, and Gly were substituted in the 7-position. The use of substituted D or L-aromatic amino acids in the 7th position of the central Ac-[Nle⁴] -α-MSH_4–11 - NH₂ fragment resulted in a loss in potency relative to the corresponding phenylalanine-containing analogue. The loss in potency cannot be due entirely to steric hindrance at the melanophore receptor, since nonaromatic amino acids substituted in the 7th position of this octapeptide fragment also generally led to a loss in biological activity. We reported previously that replacement of phenylalanine-7 by its D enantiomer led to a marked increase in potency in each fragment analogue tested. Analogues containing other D amino acids in the 7th position also were more potent than their L amino acid-containing analogues with one exception: Ac-[Nle⁴, Ala⁷]-α-MSH_4–11-NH₂ was more potent than Ac-[Nle⁴, D-Ala⁷]-α-MSH_4–11-NH₂ in the frog skin bioassay. Replacement of phenylalanine-7 by glycine resulted in a large decrease in potency in both bioassays, illustrating the importance of the side chain group, in this position of α-MSH, to biological potency of the hormone.     

General procedure for preparing a reference mixture and determining the amount of dipeptide contaminant in N-alkoxycarbonylamino acids by high-performance liquid chromatography

The amount of N-alkoxycarbonyl-dipeptide contaminant in a sample of N-alkoxycarbonylamino acid is determined by reversed phase high-performance liquid chromatography on a μBondapak-C₁₈ column using aqueous methanol or acetonitrile as solvent and u.v.-absorbance monitoring. Reference mixtures of the N-substituted monomer and dipeptide are prepared by reaction of the symmetrical anhydride of the N-alkoxycarbonylamino acid with the sodium salt of the amino acid in aqueous dimethylformamide. The proportions of the two components in the reference mixture are established by ¹H-n.m.r. spectroscopy. N-Protected-dipeptide was detected (0.1–0.7%) in seven out of eight commercial benzyloxycarbonylamino acids examined.     

Solid-phase synthesis and conformational studies of helper T cell immunogenic peptides that carry carbohydrate haptens linked to serine

N ^α-Fmoc serine and its corresponding pentafluorophenyl ester were glycosylated with the 1,2-trans peracetates of the disaccharides galabiose and cellobiose. Complete stereoselectivity and 52-75% yields were obtained under boron trifluoride etherate promotion. Lower yields and loss of stereoselectivity were obtained when thioglycosides. trichloroacetimidates or glycosyl bromides were employed as glycosyl donors. The glycosylated building blocks were used in solid-phase synthesis of derivatives of a helper T cell immunogenic peptide consisting of amino acids 52-61 from hen-egg lysozyme. ¹H-NMR spectroscopy in DMSO-d₆ showed that the peptide moiety of the glycopeptides assumed random conformations which were not influenced by glycosylation at different positions.