QUARTERLY Communication Acetylation of methyl 5-amino-1H-[1,2,4]triazole-3-carboxylate ��

Acetylation with acetic anhydride of methyl 5-amino-1H-[1,2,4]triazole-3-carboxylate, one of the hetareneamino acids, was studied using HPLC, H NMR, FTIR and GC-MS. The compound has a significantly decreased susceptibility to acetylation compared to 5-amino-1H-[1,2,4]triazole itself. Two isomeric diacetylated products were found.

Hetareneamino acids have the potential to form hetarene oligopeptides, ones of the most promising small molecules controlling gene expression [1][2][3], and moreover to be the constituents of natural [4] and artificial [5] peptides useful for biological and non-biological purposes.They owe this to their properties such as a flat, rigid system capable of acting both as a hydrogen bond acceptor and donor, the electrostatic potential near nitrogen atoms, the ability to tautomerism and the amphoteric character.The readily available 5-amino-1H- [1,2,4]triazole-3-carboxylic acid (ATC) [6] is such an interesting amino acid.Its chemistry as well as the properties, including structural and spectral ones, of its derivatives are, however, little known.Only the methyl ester obtained through direct esteri-fication [6] and its 1-acetyl derivative synthesised totally via linear substrate cyclisation [7] are described.
Acylation of the hetareneamino acid exoamino group poses, however problem [e.g.8].The ability of ATC and its derivatives to undergo acylation, especially a selective one, which is of practical importance for the potential incorporation into a peptide chain, is unexplored.A model reaction of acylation is acetylation.Based on our previous work on the acetylation of 5-amino-1H- [1,2,4]triazole [9] we studied acetylation of methyl 5-amino-1H- [1,2,4]triazole-3-carboxylate, which is the simplest protected form of ATC allowing one to avoid undesired side reactions at its free carboxyl group.The reactions with Ac 2 O were investigated using HPLC, 1 H NMR spectroscopy, FTIR spectroscopy and GC-MS.The results were compared with those from the acetylation of 5-amino-1H- [1,2,4]triazole itself.The compounds isolated were characterized.

MATERIALS AND METHODS
The results of acetylation experiments were followed first of all using a Beckman "System Gold" chromatograph working at 210 nm as a rule and occasionally at 250 nm, with an Alltech Alltima C 18 , 5 mm, 150 ´4.6 mm reversed-phase column, 0.1% trifluoroacetic acid/acetonitrile (90:10, v/v) as a mobile phase and reference substances. 1 H NMR spectra were taken in a (CD 3 ) 2 SO solution on a Bruker Advance DRX 300 MHz spectrometer with tetramethylsilane internal standard.FTIR spectra were recorded on a Philips Analytical PU9800 FTIR spectrometer at a 2 cm -1 nominal resolution in KBr and dimethylsulfoxide.A GC HP 6890 chromatograph with an HP-5 column and an MS 5973 (EI) mass spectrometer as a detector were used.
The annular acetylation of 5-amino-1H-[1,2,4]triazole is very fast and gives two monoacetylated products, the kinetic one:  For picture clarity, units have been omitted.
The reaction of 5-amino-1H-[1,2,4]triazole with acetic anhydride in excess at room temperature gives the diacetyl derivative in quantitative yield [9].Under the same conditions, the investigated ester exoamino group does not react at all.Upon 10 days, merely minute quantities of the diacetyl compound methyl     1 and additional ones allow the following conclusions to be drawn.ATC(Ac)-OMe is the first product of acetylation.Of the two monoacetylated derivatives of methyl 5-amino-1H-[1,2,4]triazole-3carboxylate, Ac-ATC-OMe reacts faster with acetic anhydride and it is this compound that is the main substrate for the second acetylation.This is also shown by the results of acetylation of each of the monoacetylated substrate separately at room temperature.Ac-ATC-OMe can be assumed to be mostly formed from ATC(Ac)-OMe by intermolecular transacetylation, although direct acetylation of the exoamino group of ATC-OMe cannot be ruled out.Each experiment from Table 1 shows that Ac-ATC(Ac')-OMe is formed as the first diacetylated product and subsequently converted into its isomeric compound Ac-ATC(Ac)-OMe.The system aims at equilibrium with approximately equimolar quantities of both isomeric diacetyl derivatives, 45% each, and with 10% of Ac-ATC-OMe.This shows that the isomerisation can be considered as a process with the participation of the latter compound, but not as an intramolecular phenomenon.Both diacetylated compounds were characterised by 1 H NMR (Fig. 1) and FTIR spectroscopy (Fig. 2).An outstanding feature of methyl 1-acetyl-1H-[1,2,4]triazole-5-acetylamino-3-carboxylate is a very strong intramolecular hydrogen bond.Triacetylation that takes place in the case of 5-amino-1H [1,2,4]triazole [9] could not been observed for methyl 5-amino-1H-[1,2,4]triazole-3-carboxylate.  1  H NMR, FTIR and GC-MS

Figure 3 .
Figure 3. Hypothetical main pathways of the acetylation of ATC-OMe with neat Ac 2 O at boiling.