The synthesis and resolution of N-(benzyloxycarhony1)-y,y-di-tert-butyl-d,ly-carboxyglutamic acid (5a) is described. Resolution using quinine allowed separation of the D enantiomer from the racemic mixture in 15% yield from N-(benzyloxycarbonyl)-0-tosyl-d,l-serine methyl ester (la). Liberation of the L enantiomer from the remaining oily quinine salt followed by purification of the L-tyrosine hydrazide salt of 5a provided an overall yield of 13°C of the L enantiomer from la. The synthesis of N-(benzyloxycarbonyl)-y,y-di-tert-butvl-D-y-carhoxyglutamyl-,y,y-di-tcrt-butyl-D-y-carhoxyglutamic acid (19) is described.
At acidic pH the -proton of the -carboxyglutamic acid (Gla) side chain undergoes rapid exchange. We have utilized the reactivity of the resulting enol form to develop a method for the chemical modification of peptide-bound Gla residues. Reaction of Gla peptides with a morpholine-formaldehyde mixture at pH 4.5 yields the Mannich base adduct. Fragmentation of the Mannich base occurs rapidly in 50% aqueous DMF to yield carbon dioxide, morpholine, and the corresponding -methyleneglutamyl residue.
X=P(0)0R1 + R2OH -* CH3C0CH(CH3)0P(0)(0R1)(0R2) (2) Alcohol R^H reacts much faster with X-P(0)0Ar than with the product X=P(0)0R1, and, therefore, the symmetrical phosphates, CH3C0CH(CH3)0P(0)(0R1)2, are not formed in any appreciable extent. Moreover, the phenols with electron -withdrawing substituents are much less reactive than alcohols toward both X=P(0)0Ar and X=P(0)0R1, and hence the corresponding aryl phosphates are not produced.The effective catalysis of reaction 2 by the phenol salts [e.g., a factor of ~140 in the reaction of ¿-C4H9OH with X=P(0)0-c-C5H9 by p-N2C6H4-0(C2H5)3NH+ in 0.2 M CDCI3 at 25°] could involve 5-and 6-coordinate phosphorus intermediates4 4 and 5; the latter, 5, is analogous to compounds isolated from the reaction of stable pentaoxyphosphoranes, phenols and tertiary amines.4,5These results may have a bearing on the mechanism of action of enzymes that catalyze the reactions of phosphates and pyrophosphates, since the presence of tyrosine, lysine, arginine, and histidine residues could facilitate the addition of nucleophiles to 4-coordinate phosphorus by analogous mechanisms.
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