N-Butyl esters, of amino acids

MethElute, Pierce Chemical Co.), or methanol/NA -dicyclohexylcarbodi-imide (Felder et ai, 1973) are to be recommended as a substitute for diazomethane. The dime thy Iformamide dialkyl acetals are particularly applicable to the preparation of methyl, ethyl, propyl and n-butyl esters of fatty acids. They have not, however, been studied extensively with a wide range of acids with multiple functional groups and care should be taken to examine their reactions with the pure acids of interest before application to biological extracts. With amino acids they form A -dimethylaminomethylene alkyl esters (Thenot and Horning, 1972), for example. 

The advantage of trimethylsilyl (TMS) derivatives lies in the simplicity of the derivatization procedure, which is carried out by the addition of N,0-bis(trimethylsilyl)trifluoroacetamide (BSTFA) in acetonitrile and heating for approximately 2 h at 150 °C under anhydrous conditions in a sealed tube. However, there may be problems owing to the formation of multiple derivatives of each amino acid. Another technique involves the formation of n-butyl esters of the amino acids and their subsequent trimethylsilylation by a similar procedure. The n-butyl esters are formed by heating the amino acids for 15 min in n-butanol and HC1 and these are then converted to the A-TMS-n-butyl ester derivatives. A-acyl amino acid alkyl esters are commonly used. Acetylation of the butyl, methyl or propyl esters of amino acids,... 

The 1-adamantyl (1-Ada) esters exhibit increased acid stability if compared to the tert-butyl esters.These amino acid esters are prepared by reaction of the amino acid tosylates with adamantan-l-ol and dimethyl sulfite in boiling toluene. Removal of the adamantyl ester is effected by neat TFA within 1 hour or TFAyCH2Cl2 (1-2) within 15 hours. N-Terminal Boc groups are selectively cleaved with 4 M HCl in anhydrous dioxane (rt, 25 noin) in the presence of the 1-adamantyl ester. 

Conversions of the amino acids to methyl-a-hydroxy esters (L8, Wl) and methyl a-chloro esters (M5) have been described, and Bier and Teitelbaum (B13) have chromatographed the volatile amines produced by decarboxylation of amino acids. More recently, attention has been focused on the use of the AT-acylated esters of amino acids, e.g., n-amyl JV-aoetyl (J7), n-butyl iV-trifluoroacetyl (Z3), w-propyl iV-acetyl (G6), n-amyl A -trifluoroacetyl (B14), and methyl AT-trifluoroacetyl (C13, HI). Silyl derivatives have also been prepared (R5). 

Methyl esters undergo trans-esterification with the quaternary ammonium salts at high temperature and the reaction has been used with some effect for the preparation of, for example, n-butyl esters by heating the methyl ester with tetra-n-butylammo-nium chloride at 140°C [31]. Optimum yields (>75%) are obtained in HMPA or in the absence of a solvent. A two-step (one-pot) trans-esterification under phase-transfer catalysed conditions in which the carboxylate anion generated by initially hydrolysis of the ester is alkylated has been reported for Schiff s bases of a-amino acids [32] and for A-alkoxycarbonylmethyl [1-lactams [33]. Direct trans-esterification of methyl and ethyl esters with alcohols under basic catalytic conditions occurs in good yield in the presence of Aliquat [34, 35]. 

Lanikin, W. M., and C. W, Gehrke Quantitative gaschromatography of amino acids. Preparation of butyl N-trifluoracetyl esters. Analytic. Chem. 37, 383-389 (1965). 

TABLE 9.4 RELATIVE WEIGHT RESPONSES (RWR)1 OF AMINO ACID N-TFA n-BUTYL ESTERS ON 10% APIEZON M2 (41)... 

N-trifluoroacetyl amino acids have been reported as stable at -10°C over a 2-week period (53) and the TFA n-butyl esters are stable for about 72 hr when stored at 0 C in a nitrogen atmosphere (32). Reasonable stability of derivatives is necessary for repetitive analyses when many samples are prepared and stored prior to chromatography. Instability of silyl derivatives to hydrolysis has been a major problem which has led to decreased activity in research on these materials. The efficient treatment of amino acids with N,0-bis(trimethylsilyl)acetamide (51) and subsequent GLC of the derivatives is now used only occasionally as a result of instability of the derivatives. 

The method of N-chlorination of amino acid esters 40 and AT-acylamino acid esters with tert-butyl hypochlorite and subsequent elimination of hydrogen chloride is of general use in the preparation of DHA derivatives 41/42 122-1241 (Scheme 15). 

Gas chromatography is used to analyze volatile derivatives of amino acids. Phenylthiohydantoins (products of Edman degradation) may be analyzed directly by GC but are better resolved if converted to their trimethylsilyl derivatives with N, O-bis(trimethylsilyl) acetamide. Free amino acids are generally converted to their 7V-trifluoroacetyl- -butyl esters or trimethylsilyl derivatives before GC analysis. For best results, all gas chromatography of amino acid derivatives should be done with a glass column and injection port, as contact with metals causes extensive decomposition of the derivatives. 

Gas chromatography (GC) for amino acid analysis is the alternative to HPLC that has found the greatest acceptance. It requires the preseparation derivatization of the amino acids to render them volatile. For this purpose, amino acids are frequently converted into acylated esters. N-Trifluoroacetyl-n-butyl esters and /V-heptafluorobutyrylisobutyl esters are most commonly employed. There have been comparative studies (3,4) that document similar (if not equivalent) analytical results for GC and the classic ion-exchange chromatographic method applied to a variety of food samples. Comparison (5) of GC to the reversed-phase HPLC determination of amino acids (phenylisothiocyanate derivatized) also shows excellent agreement. 

Alkylation of amino acids.1 This base is preferable to LDA for dideprotonation of di-f-butyl N-formylaspartate (1) to give 2. The dianion is alkylated by CH3I, CH2 CHCH2Br, and C6H5CH2Br to give a mixture of / - and a-alkylated aspartate derivatives (3 and 4) in a ratio of ca. 7 2. As expected, the /3-alkylated a-amino acid esters 3 are optically pure surprisingly, the products 4 are also optically active and, after crystallization, can be obtained in optical yields of 60%. 

Asymmetric amino-acid synthesis.1 The (S)-l-f-butyl 4-methyl N-benzyloxy-carbonylaspartate (1), prepared in 80% yield from the 4-methyl ester of aspartic acid, undergoes diastereoselective alkylation at the -carbon (LDA or lithium hexa-... 

As far as quantitative chemical derivatization GC analysis is concerned, it is necessary to mention especially the work of Gehrke and his collaborators, who specified the fundamental concepts of quantitative GC analysis combined with the chemical derivatization of sample compounds and applied them to the accurate determination of the twenty natural protein amino acids and other non-protein amino acids as their N-TFA-n-butyl esters [5 ], the urinary excretion level of methylated nucleic acid bases as their TMS derivatives [6], TMS nucleosides [7] and other investigations. Further examples include a computer program for processing the quantitative GC data obtained for seventeen triglyceride fatty acids after their transesterification by 2 NKOH in n-butanol [8], a study of the kinetics of the transesterification reactions of dimethyl terephthalate with ethylene glycol [9] and the GC-MS determination of chlorophenols in spent bleach liquors after isolation of the chlorophenols by a multi-step extraction, purification of the final extract by HPLC and derivatization with diazoethane [10]. 

The reaction of 2 equivalents of tert-butyl dicarbonate with A -Z-protected amino acids in tBuOH and catalytic DMAP (0.1 equiv) proceeds smoothly over 30 minutes to give the corresponding esters in high yields (see Section 3.9.3.2).P Preparation of tert-butyl esters of N-protected anoino acids via tert-butyl mixed carbonic anhydride generated in situ by reaction with tert-butyl fiuorocarbonate has been reported by Loffet et al.t l Finally, N -Z-protected amino acids are esterified in good yields with tert-butyl bromide.P ... 

Trityl (triphenylmethyl, Trt) esters are extremely labile toward acidic conditions and are selectively cleaved in the presence of ferf-butyl esters by treatment with AcOH at room temperature for a few minutes.The hydrochloride salts of amino acid trityl esters are hydrolyzed in aqueous and methanolic solutions. Amino acid trityl esters are obtained in satisfactory yields by treatment of the corresponding N -protected amino acid cesium salts with trityl chloride.t l... 

N-ProtecHon of amino acids. The reagent reacts readily with amino acids and esters (aqueous dioxane, MgO) to give f-butyloxycarbonylamino acids, which are not otherwise available, since /-butyl chloroformate, (CHslsCOCOCl, is too un-... 

M26. Mussini, E., and Marcucci, F., Separation of amino acid N-butyl esters by means of thin-layer chromatography. J. Chromalogr. 17, 576-577 (1965). 

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