Amino acids DABITC

B 2 The new reagent 4-Ar,A7-dimethyIaminoazobenzene-4 -isothiocyanate (DABITC) is often used for the Edman method of protein sequence analysis Wnte a reaction showing this use of the reagent 3 Describe how the HPLC and CE instruments detected the FMOC-amino acids eluting from the column... 

Amino acids derivatized with isothiocyanates (PITC, NITC, DNITC, DABITC), with AQC. or with sulphonyl chlorides (DNS-Cl. DABS-CI)... 

These derivatives are obtained in basic medium by the reaction of DABITC with the primary amino group of N-terminal amino adds in peptides. The color difference between DABITC (or its degradation products) and DABTH-amino adds fadlitates identification on TLC. These derivatives are colored compounds and, because of their stability and sensitivity, are usually used for qualitative and quantitative analyses of amino compounds such as amino acids and amines. 

Dabsyl chloride is an alternative to DABITC as a derivatization reagent to be used for manual sequencing. Dabsyl chloride reacts with primary and secondary amino acids forming red-orange derivatives that are stable for months. The method offers excellent sensitivity, ease, and speed of preparation and high-resolution capabilities. However, it suffers from interferences with ammonia present in biological samples. Furthermore, it results in a relatively reduced column lifetime due to the utilization of excess of Dabsyl chloride [9]. 

The identification of N-terminal amino acids in peptides and proteins is of considerable practical importance because it constitutes an essential step in the process of sequential analysis of peptide structures. Many N-amino acid derivatives have been proposed for this purpose and the ones most commonly studied by TLC are 2,4-dinitrophenyl (DNP)-and 5-dimethylaminonaphthalene-l-sulfonyl (dansyl, Dns)-amino acids, and 3-phenyl-2-thiohydantoins (PTH-amino acids). Recently, 4-(dimethylamino)azobenzaie-4/-isothio-cyanate (DABITC) and phenyl-isothiocyanate (PITC) have also been investigated as derivatizing agents of amino acids. 

Separation and identification of derivatives of amino acids such as DNP-, PTH-, dansyl-, and DABITC-, is very important, particularly in the primary structure determination of peptides and proteins. Adequate description of the preparation of PTH- (76-79), dansyl (80-82), and DNP-amino acids (83-86) is available in the literature, and the methods of identification of N-terminal amino acids by TLC and other techniques have been reviewed by various workers (87-91). The present section describes briefly the preparation of such derivatives and TLC resolution data reported in recent years. 

C. Oimethylamino Azobenzeneisothiocyanate (DABITC) Derivatives of Amino Acids... 

Amino acids (0.5 mg) are dissolved in 100 )il of triethylamine-acetic acid buffer (50 ml water -f 50 ml acetone + 0,5 ml triethylamine -i- 5 ml of 0.2 M acetic acid, pH 10.65) and treated with DABITC solution (50 pi, 4 nmole/pl in acetone). The mixture is heated at 54°C for 1 h, dried under vacuum, and then redissolved in water-acetic acid (40 pi + 80 pi) saturated with HCI (alternatively, 100 pi of 50% TEA can be used instead of this aqueous acid mixture). The acid solution is heated at 54°C for 45 min and then dried again under vacuum. The dried DABTH-amino acid (about 200 nmole) is dissolved in a suitable volume of 90% ethanol and stored at -20°C for TLC analysis. The presence... 

The classical method is the Edman degradation reaction. It involves stepwise degradation of peptides with phenylisothiocyanate (cf. 1.2.4.2.3) or suitable derivatives, e. g. dimethylaminoa-zobenzene isothiocyanate (DABITC). The resultant phenylthiohydantoin is either identified directly or the amino acid is recovered. The stepwise reactions are performed in solution or on peptide bound to a carrier, i.e. to a sohd phase. Both approaches have been automated ( sequencer ). Carriers used include resins containing amino groups (e. g. amino polystyrene) or glass beads treated with amino alkylsiloxane ... 

Amino acids and their derivatives] resolution of amino acids and dieir derivatives on impregnated layers, 406-408 resolution of enantiomeric mixtures of amino acids and their derivatives, 411-419 separation of amino acid derivatives, 398-406 DABITC derivatives of amino acids, 403-406 dansyl amino acids, 402 dinitrophenyl-amino acids, 406 PTH amino acids, 399-402 separation of amino acids, 389-398 chromatographic techniques, 393-395 preparation of test materials, 389-392 TLC system for amino acids, 395-398 Aminoglycosides, 457-461 Amino-modified precoated silica layers, 114,117-119... 

DABITC) derivatives of amino acids, separation of, 403-406 Dimethylformamide (DMF), 17 Dimethylsulfoxide (DMSO), 25 Dinittophenyl (DNP)-amino acids quantitation of, 420 separation of, 406... 

Other tests for the detection of amino functionalities on solid supports include the TNBS (2,4,6-trinitrobenzenesulfonic acid, picrylsulfonic acid) [Hancock and Battersby Anal Biochem 71 260 ]976], the DABITC [Shah et al. Anal. Commun. 34 325 7997] and the NF31 [Madder et al. Eur J Org Chem 2787 7999] tests. 

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