Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Synthesis of peptides
Patent
1994-09-23
1998-05-26
Tsang, Cecilia J.
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Synthesis of peptides
530402, 514389, C07K 1107, C07D23372
Patent
active
057566674
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention consists in a method of preparing amino acid thiohydantoins either in isolation or as the C-terminal residue of a peptide. The method of the present invention is particularly useful in the preparation of proline thiohydantoins. The present invention also relates to an improved method of C-terminal peptide sequencing involving the preparation and cleavage of amino acid thiohydantoins.
BACKGROUND OF THE INVENTION
The search for a chemical method for step wise removal of amino acids from the C-terminal end of proteins began early in this century when Schlack and Kumpf (Hoppe-Seyler's Z. Physiol. Chem. 154: 125, 1926) applied the method of Johnson and Nicolet (J. Am. Chem. Soc. 33: 1973, 1911), for conversion of acylamino acids to acylthiohydantoins, to a small peptide, and then cleaved the thiohydantoins from the molecule with 1M sodium hydroxide. While alternative chemistries have been proposed over the years scientists have periodically attempted to utilize or modify the Schlack and Kumpf degradation procedure for C-terminal sequence analysis. None of the procedures proposed have, however, led to an accepted routine determination.
It has been recognised that the major hurdle to clear before full rewards can be obtained from application of the thiohydantoin procedure is the difficulty in producing prolyl residues.
Currently workers in the field are using different reagents to form thiohydantoins of amino acids. None of them have claimed to be successful with these reagents for proline. Earlier workers, Kubo et al. (Chem. Pharm. Bull. 19: 210, 1971), claim that acetyl proline and proline in two peptides reacted well at 30.degree. with a two step reaction consisting of acetyl chloride-trifluoroacetic acid treatment, then addition of thiocyanic acid dissolved in dioxane. Unfortunately, this procedure is not a good practical solution, partly because of difficulties in preparing and storing the thiocyanic acid and it has not been elaborated by others in the field.
Earlier papers by the present inventors have reported the successful application of a thiohydantoin procedure to both manual and automatic C-terminal sequence analysis of peptides and proteins. With the exception of proline, all amino acids that might have been expected to give difficulties were shown to be degraded normally in peptides (Inglis et al In: Methods in Protein Sequence Analysis, Ed. Wittman-Liebold, 1989; Inglis et al In: Modern Methods in Protein and Nucleic Acid Research, Ed. Tschesche, 1990) These findings have been largely corroborated by others (Bailey et al J. Prot. Sci. 1:68, 1992) using different reaction conditions, despite some initial problems with amino acids such as aspartic acid, asparagine and glutamic acid (Bailey and Shively Biochemistry 29:3145, 1990). Apparently dithiothreitol (DTT) in the cleavage reagent caused adverse effects. The analysis of proline is now the major obstacle to a general routine method employing ammonium thiocyanate as the coupling reagent.
Stark (Biochemistry 7:1796, 1968) believed that the thiohydantoin procedure would not work for proline and aspartic acid. While that has now been disproved for the latter (Inglis et al In: Methods in Protein Sequence Analysis, Ed. Wittman-Liebold, 1989), it is clear from recent work that the former is quite unreactive under some circumstances and, indeed, the premise has again been advanced that the chemical pathway is such as to preclude the reaction of thiocyanate reagents with proline (Bailey et al J. Prot. Sci. 1:68, 1992). On the other hand, Kubo et al (Chem. Pharm. Bull. 19:210, 1971) obtained a product from acetylproline on which they did elemental, UV, IR and TLC analyses, all of which indicated that it was a pure sample of proline thiohydantoin. The present inventors initial findings with HSCN and acetylproline (Inglis et al In: Methods in Protein Sequence Analysis, Ed. Wittman-Liebold, 1989) were completely consistent with their results in that the present inventors also obtained a major product with an abs
REFERENCES:
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Adams Peter Laurence
Inglis Adam
Tseng Albert Peng Sheng
Garvan Institute of Medical Research
Gupta Anish
Tsang Cecilia J.
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