TMS derivatives, of amino acids

The silylation of amino acids with BSTFA was studied in detail by Gehrke and coworkers [254—256]. BSTFA—acetonitrile (1 1) was applied first and fourteen amino acids were silylated at 135°C for 15 min. Glu, Arg, Lys, Trp, His and Cys, however, require up to 4 h, in order for measurable peaks to be obtained in the chromatogram. Despite such a long reaction, Gly and Glu gave two peaks and also it was difficult to separate the tris-TMS derivative of Gly from the derivatives of lie and Pro. The influence of polar and non-polar solvents was demonstrated later and was decisive mainly with respect to uniformity of the products. Only the bis-TMS derivative was produced in hexane, methylene chloride, chloroform and 1,2-dichloroethane bis- and tris-derivatives were produced in six more polar solvents. On the other hand, Arg did not provide any peak in the less polar solvents that were used and only one peak in the six more polar solvents. The best and most reproducible results were obtained when silylating seventeen amino acids with BSTFA—acetonitrile (1 1) at 150°C for 15 min 2.5 h at 150°C were necessary for the reproducible derivatization of Gly, Arg, and Glu. These reaction conditions were recommended for the analysis of all twenty amino acids. The TMS derivatives of amino acids were found to be stable on storing them in a sealed vial at room temperature for 8 days, with no decomposition. 

Particular attention should be devoted to the selection of the column packing for the separation of TMS derivatives of amino acids as they are, as already mentioned, very sensitive towards moisture and can decompose on supports that have not been deactivated sufficiently. Silicone stationary phases of the SE-30, OV-1, OV-17 and DC-550 type and supports such as Gas-Chrom Q, Chromosorb W HP and Diatoport S have mostly been applied. One of the best GC separations of TMS derivatives of amino acids was obtained by Gehrke and Leimer [256] on a 6 m X 2 mm I.D. column packed with 10%... 

TMS derivatives of amino acids were also combined with other procedures and some difficulties were thus avoided. N-TMS-methyl and -ethyl esters of most protein amino acids were prepared by the action of TMSDEA on alkyl esters of amino acids and were chromatographed on methylsilicone stationary phases [246], Their retention times were found to be 15—20% lower than those of the corresponding TMS derivatives. Despite having an additional step in comparison with direct silylation, the procedure was applied by Hardy and Kerrin [259] to the GC analysis of twenty protein amino acids, including Hypro and CysH. Amino acids were esterified with a 3 N HC1 solution in n-butanol at 150°C for 15 min with subsequent silylation with BSTFA for 90 min at the same temperature. Acetonitrile and methylene chloride were used as solvents for the silylation. In the former solvent double derivatives of Gly and Lys (bis- and tris-) were produced, whereas in the latter the less silylated form only was produced. As Arg, in contrast to direct silylation, also leads to one peak in this instance, methylene chloride is recommended as the silylation solvent. The separation of all twenty amino acids was achieved on a simple column with 2% of OV-7 on GLC-110 textured glass beads (100—120 mesh). 

The known disadvantage of TMS derivatives of amino acids is the easy postreaction hydrolysis of N-Si bonds in the formed derivatives, which leads to uncertainty regarding the formed products. At the same time, silylation of carboxylic acids is the most popular method for their derivatization (see Acids Derivatization for GC Analysis, p. 3). Hence, it seems reasonable to combine the silylation of CO2H groups in amino acids with the formation of other derivatives of amino groups, e.g., amides. This method of derivatization was realized only in 2007. " The first step is the formation of 0-TMS derivatives under mild conditions (with MSTFA as reagent), followed by trifluoroacetylation of amino groups by MBTFA (Fig. 5). 

Procedures A and B illustrate the two current methods for preparation of N-9-phenylfluoren-9-yl derivatives of amino acids and amino acid esters. Free carboxylate (as in alanine in Step A) or free hydroxyl (e.g., serine7) functions can be blocked for the duration of the reaction as trimethylsilyl (TMS) esters or ethers, respectively, by treatment with chlorotrimethylsilane and triethylamine. The TMS group(s) are then removed by methanolysls from carboxylic acids (as in Step A) and mild acidic hydrolysis from hydroxyl groups, both being accomplished during product isolation. In addition to 2, the N-9-phenylfluoren-9-yl derivatives of serine,7 glutamic add y-methyl ester,8 and aspartic acid 3-methyl ester3 9 have been prepared in this manner. 

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,... 

Cimbura and Kofoed (50),mentioned earlier, used GLC to separate amphetamine and methamphetamine after acetylation with acetic anhydride in methanol. Derivatives were extracted using diethyl ether and chromatographed op columns of either 3% OV-17, OV-1, or SE-30. Column temperature was 160°C. They also reported the chromatographic determination of acetylated morphine on 3% SE-30, OV-1, or OV-17 at temperatures of 220°C. Cruickshank et al.(21) separated 21 amino acids as their trifluoroacetylated methyl esters. The column was 5% neopentyl glycol succinate on Gas Chrom P. Column temperatures were both isothermal and programmed 65°C for 20 min at 1.5°C/min then 2°C/min until 42.5 min then 4°C/min until 60 min then isothermal until about 75 min (see Figure 12.2). Chang et al. (19), used BSA/pyridine to form the TMS derivatives of levodopa, methyldopa, tyrosine. 

Pocklington [257] applied the method to the determination of nanomole amounts of amino acids in sea water. Dry marine salt was extracted with acidified ethanol. Interfering components having been removed, the extract was purified on an ion-exchange resin. TMS derivatives were prepared by the action of pure BSTFA (10-fold excess) at 78°C for 1 h. The analysis was performed on 3% of OV-1 or OV-17 with temperature programming. Suitable conditions were found for the detection of down to 10-11 mol of all protein amino acids (except Arg and His) plus several non-protein amino acids. 

Fig. 5.21. Gas chromatogram of TMS derivatives of five iodinated amino acids and tyrosine. Peaks 1 = Tyr 2 = MIT 3 = DIT 4 = T2 5 = T3 6 = T4. Conditions borosilicate-glass column, 1 m X 3.5 mm I.D., 0.5% SE-30 on Chromosorb G (60-80 mesh, AW, DMCS-treated) nitrogen flow-rate, 40 ml/min temperature programme, 4.6°C/min from 70°C. (Reproduced from Anal. Biochem.,...

Silyl esters are stable to nonaqueous reaction conditions, but are often too labile to mild acid or base or even neutral aqueous media to survive many simple manipulations. Thus, they have not found wide application in peptide synthesis. Due to easy formation and cleavage they may play an important role as intermediates in the synthesis of amino acid derivatives and for temporary carboxy protection in the preparation of small peptide fragments. The TMS group has been used for the solubilization of H-Arg-OH for the synthesis of Z-Arg(Z2)-OHP l and in the synthesis of Al -Nps- and Al -Tfa-protected amino acids.P Amino acid trimethylsilyl esters as well as the related A1 -TMS derivatives react rapidly with acylating agents and are used for the preparation of peptides with amino acid active esters, e.g. A-hydroxysuccinimide-, 4-nitrophenyl-, or 2,4,5-trichlorophenyl esters, or mixed anhydrides. 

This paper (S4) compares the two silylating reagents bis (trimethyl-silyl) acetamide (BSA), first described by Klebe et ad. (K6), and bis-(trimethylsilyl) trifluoroacetamide (BSTFA), for the preparation of volatile trimethylsilyl (TMS) derivatives of 12 sulfur-containing amino acids. BSTFA was recommended as the reagent of choice for taurine, cysteic acid, homocystine, djenkolic acid, ethionine, methionine sulfone, L-2-thiolhistidine, cysteine, and cystine. For S-methyl-L-cysteine, methionine sulfoxide, and methionine, BSA was used as silylating reagent. 

Using bis (trimethylsilyl) acetamide (BSA), volatile trimethylsilyl (TMS) derivatives of the active components 3,5,3, 5 -tetraiodothyronine (thyroxin, TJ and 3,5,3 -triiodothyronine (T3) have been prepared as well as TMS derivatives of the nonphysiologically active components 3,3, 5 -triiodothyronine (T3 ), 3,5-diiodothyronine (T2), and 3,5-diiodo-tyrosine (DIT). Separation and quantitative estimation of these iodinated amino acids is achieved by gas-liquid chromatography. The method is... 

Lithiation at the C2 position of the imidazole ring by means of n-BuLi was followed by a treatment with tosyl azide which furnished the corresponding 2-azido imidazole in a yield of 89%. This azido group was reduced to an amino group with palladium on carbon under hydrogen atmosphere (in a yield of 95%). Finally, the 2-aminoimidazole was treated with a TMS-activated derivative of parabanic acid, a reaction that afforded the target molecule naa-midine G in a yield of 78%. This eight-step synthesis provided naamidine G in a total yield of (95 x 97 x 85 x 98 x 81 x 89 x 95 x 78) 41%. 

The primary limitation associated with GC/MS is the need for derivatization. Derivatization introduces additional complexity to the system and is not 100% efficient. Inefficient reactions result in the presence of multiple derivatized forms of the same compound. For example, we can detect three different derivatization products of the amino acid asparagine (mw = 132) in M. truncatula roots (Fig.3.4). These include asparagine, N,0-TMS (mw = 276), asparagine, N,N,0-TMS (mw = 348), and asparagine, N,N,N,0-TMS (mw = 420). Inefficiency of the derivation reactions also limits the lower concentration range of analytes that can be profiled. Finally, derivatization is not capable of achieving volatility for all compounds, such as many of the flavonoid glycosides. If derivatization is successful and the analyte is... 

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