Trifluoroacetyl peptides

Several factors affect the volatility and stability of a peptide derivative, not least of these being the number and nature of the constituent amino acids. Heterocyclic and aromatic amino acids reduce volatility while those containing sulphur tend to decrease the thermal stability. Small naturally occurring peptides which are not derived from proteins often contain only aliphatic amino acids which lack functional groups in the side chains. Peptides of this type of up to about ten amino acids, after conversion to suitable derivatives, are amenable to analysis by mass spectrometry, e.g. [164]. A variety of derivatives has been reported and include N-trifluoroacetyl peptide esters [136,165], N-acetyl peptide esters [166-168], aromatic N-acyl peptide esters [169-172], and per-methylated N-acyl peptides [173]. The principal modes of the electron impact induced fragmentation of these peptide derivatives are well established and have been summarised in recent reviews [174,175]. Although the spectra of the permethylated derivatives [176] are perhaps the simplest and easiest to interpret and are now frequently used, the N-acyl peptide esters have been widely and successfully employed. 

At about the same time, in 1963, it was reported that simple iV-acetyl-peptides (Heyns and Griitzmacher [29]) or iV-trifluoroacetyl peptide esters (F. Weygand et al. [30]) are cleaved under electron impact at the peptide bonds, giving rise mainly to ions corresponding to N-terminal parts of the chain. If such fission occurs at each peptide bond, a series of ions are formed all having the same N-acyl group. This is equal to a stepwise degradation of the chain from its carboxyl end (Fig. 13). 

Since N-trifluoroacetyl peptide methyl esters are suitable for separation by gas chromatography, the procedure just described can also be applied to peptide mixtures obtained through partial methanolysis of larger peptides. This method permitted the structure elucidation of cyclic peptides such as antamanide (p. 213) and cyclolinopeptide (p. 216) by Prox and Weygand [31]. 

Tritylhydrazides s. Carboxylic acid tritylhydrazides, N-Trifluoroacetyl-peptide — Trityl salts as reactants 13, 914 Tropenium fluoroborates... 

The approach has been employed to determine the amino acid sequence in E, coli alanine t-RNA synthetase. After partial hydrolysis, the peptide mixtures were converted to their methyl esters, N-trifluoroacetylated, reduced with B2D6 and converted to their O-TMS derivatives (9). These compounds possess good GC properties and yield simple, yet informative fragmentations. A variety of derivatives have been explored for sensitive sequence analysis of small peptides and a new permethylation procedure has been described for trifluoroacetylated peptides at the 2 —10 nmol level. Mixtures of these derivatives, ideally of di- to hexa-peptides are readily analyzed by GC/MS 66). 

The original procedure for the trifluoroacetylation of amino acids used trifluoroacetic anhydride [Acetic acid, trifluoro-, anhydride].4 This reagent, although inexpensive and readily available, has certain disadvantages it is a highly reactive compound and thus has caused undesired reactions such as the cleavage of amide or peptide bonds,5 unsymmetrical anhydrides are formed between the newly formed A-trifluoroacetylamino acids and the by-product trifluoroacetic acid, and excess trifluoroacetic anhydride has caused racemization of asymmetric centers. 

Saturated 5(4//)-oxazolones are easily obtained from //-acylamino acids in the presence of a cyclization agent and have been used extensively in coupling reactions as synthetic equivalents of a-amino acids in the synthesis of peptides. In this context, tautomeric equilibrium can be a significant problem due to the racemization associated with the isomerization. For example, trifluoroacetylation of tryptophan in ether affords the 5(4//)-oxazolone 5 without racemization. However, upon dissolution in acetonitrile, 5 completely racemizes. Further, upon heating, an aqueous dioxane solution of 5 cleanly isomerizes to the isomeric 5(2//)-oxazolone 6 (Scheme 7.2). 

Alternatively, alkylation of 4-benzyl-2-(trifluoromethyl)-5(2//)-oxazolone 16 in the presence of mild base using active alkyl halides as electrophiles occurs at C-4 " Subsequent aminolysis of a 4,4-dialkyl-5(4f/)-oxazolone like 17 gave an Al-(trifluoroacetyl)-a,a-dialkylglycine amide 18 that was used to prepare important peptides incorporating an a,a-dibenzylglycine unit (Scheme 7.5). 

Racemization is not encountered when 4-unsubstituted-5(477)-oxazolones or 4,4-disubstituted-5(477)-oxazolones are used as reagents. Indeed, 4-unsubstituted-5(47/)-oxazolones function as glycine synthons in the synthesis of A-acylglycyl-a-amino acids. For example, aminolysis of 2-(trifluoromethyl)-5(47/)-oxazolone with a-methylphenylalanine affords A-(trifluoroacetyl)glycyl-a-methylphenylalanine. 4,4-Disubstituted-5(4//)-oxazolones, readily available by alkylation of the monosubstituted derivatives, are very useful intermediates in the synthesis of peptides that incorporate ot,ot-disubstituted amino acids. As an example, 4-(aryl-methyl)-2-phenyl-4-(trifluoromethyl)-5(4//)-oxazolones 260 are key intermediates... 

This reaction was first demonstrated using the desulfurization of /V,/V -bis(trifluoroacetyl)-L-cystine dimethyl ester which proceeded in 96% yield.121 However, mild alkaline hydrolysis to obtain the free acid resulted in significant product losses to yield only 64% of L-lanthionine (2R,6R-lanthionine). Alternative protecting strategies have been used in order to raise the versatility of the reaction for peptide synthesis, thus, A(Al -bis(benzyloxycarbonyl)-L-cystine diethyl ester could be converted into L-lanthionine in 86% yield. 

Chemical synthesis has provided an additional route to peptides containing halogenat-ed amino acids. Early 19F-NMR studies of proteins were performed on semi-synthetic polypeptides prepared by attachment of fluorinated probes to the polypeptide. For example, Heustis and Raftery modified ribonuclease by trifluoroacetylation of Lys residues 1 and 7. They then used 19F-NMR to study conformational changes brought about by the presence of inhibitors200. In his review, Gerig provides several other examples of this strategy187. 

Hoopes, E. A., Peltzer, E. T., and Bada, J. L., (1978). Determination of anino acid enantianeric ratios by gas liquid chromatography of the N-trifluoroacetyl-L-prolyl-peptide methyl esters. J. Chromatographic Sci.,... 

Peptide synthesis. The reagent is used to prepare N-trifluoroacetyl derivatives of amino acids and peptides. The substrate is warmed with 1.2-2 equivalents of the reagent in molten phenol at 120-150° for 2-20 min. Excess reagent and phenol are removed under vacuum or by solution in petroleum ether or carbon tetrachloride, from which the amino acid derivative crystallizes. The main advantage of the N-trifluoroacetyl protective group is that it can be removed under very mild conditions. 1,. Benoiton, H. N. Rydon, andJ. E. Willett, Chem. Ind., 1060(1960)... 

Trifluoroacetyl amino acids. Amino acids and peptides can be N-trifluoro-acetylated with this reagent in DMSO.1... 

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