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S-triazolo[3,4-A]phthalazines : implications for C-terminal peptide sequencing

University of British Columbia

The syntheses of s-triazolo[3,4-a]phthalazines (15, 3-R-TAP) by reaction of hydralazine (12, 1-hydrazinophthalazine) with N-protected amino acids and dipeptides under homogeneous (solution) and heterogeneous ("solid-phase") conditions are reported. Transition metal complexes containing the TAP ligand were prepared and their spectral properties investigated. The use of metal-ions and a cation-exchange resin (H+ form) were considered for the mild hydrolysis of side-chain amide bonds in TAP derivatives. The objective of these studies was to determine the feasibility of reacting the carboxyl groups in amino acids with hydralazine to afford the TAP derivatives as a method for peptide sequencing from the C-terminal residue. Hydralazine reacts with carboxylic acids to form an amide intermediate which undergoes ring closure with elimination of water to form the s-triazolo[3,4-a]phthalazine derivative. To promote the initial binding of hydralazine to the acid, coupling reagents were used to activate the carboxylate group towards nucleophilic attack. N-Ethyl-5-phenylisoxazolium-3'-sulfonate (17, NEPIS), 1-ethoxy -carbonyl-2-ethoxy-l,2-dihydroquinoline (27, EEDQ), various phosphorus compounds, carbodiimides, and chloroformates were carboxyl activating agents used to synthesize TAP derivatives. In solution studies, the carbodiimides (EDC, 16 and DCC, Si), NEPIS (17), and a combination of triphenylphosphite with imidazole are the most successful procedures for TAP synthesis In solid-phase studies, the best procedures for activating immobilized amino acids, are with isobuty'l chloroformate, NEPIS (l?), and DCC (51). Transition metal complexes were synthesized with the general formula: [M(3-H-TAP) (H20)6_n](C104)m (n = 4, m = 2, M = Co, Ni, Cu; n = 2, m = 2, M = Ni; n = 6, m = 3, M = Co). The infrared and visible spectra of these complexes are reported. [Co(.trien)C3-(N-Ac-gly)-TAP)J(C104)2 was also prepared and under acidic conditions, no hydrolysis of the side-chain amide bond was observed. There was also no significant hydrolysis of the side-chain with free ?+ 2+ 3-(N-Ac-gly)-TAP in the presence of Co and Cu under acidic conditions, or when it was eluted through a cation-exchange (H+ form) column. The decomposition of hydralazine in non-aqueous media was investigated and a major product of the decomposition was identified as diphthalazinylhydrazine {82). The implication of our studies is that the modification of amino acids with hydralazine is not yet a viable method for C-terminal peptide sequencing. Improvements are required for improving the yields of the coupled product, and the lack of a mild and selective method for hydrolyzing the C-terminal peptide bond limits the method at present to determination of the C-terminal residue only.

Text

2010-02-21

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http://hdl.handle.net/2429/20641

Chemistry

University of British Columbia

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