The Board of Governors for Higher Education, State of Rhode Island and

The Board of Governors for Higher Education

Ching-Shih Chen

Da-Ming Gou

Yeuk-Chuen Liu

Woan-Ru Shieh

Antibodies against Ins(1,4,5)P.sub.3 were raised by immunizing rabbits with two types of IP.sub.3 -BSA conjugates which were synthesized by covalently coupling Ins(1,4,5)P.sub.3 to the carrier protein via alkyl linkages. The anti-Ins(1,4,5)P.sub.3 antibodies were detected by an ELISA where Ins(1,4,5)P.sub.3 was covalently attached to a microplate well surface. Both antiserum preparations showed specific binding with Ins(1,4,5)P.sub.3. The specificity of these antibodies was enhanced by affinity purification for the antiserum through Ins(1,4,5)P.sub.3 -agarose chromatography. The affinity-purified antibodies have IC.sub.50 values of 12 nM and 73O nM for Ins(1,4,5)P.sub.3 and Ins(1,3,4,5)P.sub.4, respectively. These antibodies showed many properties similar to those of biologically relevant receptors for Ins(1,4,5)P.sub.3.

Antibodies specific for d-myo-inositol 1,4,5-trisphosphate and the

(2R,3S)- and (2S,3R)-enantiomers of trans-.beta.-phenylglycidic esters, prepared by lipase-mediated enantioselective transesterification, are used for the synthesis of the taxol C-13 side chain with good yield.

Chemoenzymatic synthesis of the taxol C-13 side chain N-benzolyl-

Antibodies against Ins(1,4,5)P.sub.3 were raised by immunizing rabbits with two types of IP.sub.3 -BSA conjugates which were synthesized by covalently coupling Ins(1,4,5)P.sub.3 to the carrier protein via alkyl linkages. The anti-Ins(1,4,5)P.sub.3 antibodies were detected by an ELISA where Ins(1,4,5)P.sub.3 was covalently attached to a microplate well surface. Both antiserum preparations showed specific binding with Ins(1,4,5)P.sub.3. The specificity of these antibodies was enhanced by affinity purification for the antiserum through Ins(1,4,5)P.sub.3 -agarose chromatography. The affinity-purified antibodies have IC.sub.50 values of 12 nM and 73OnM for Ins(1,4,5)P.sub.3 and Ins(1,3,4,5)P.sub.4, respectively. These antibodies showed many properties similar to those of biologically relevant receptors for Ins(1,4,5)P.sub.3.

Antibodies specific for D-myo-inositol 1, 4, 5-trisphosphate and the

Two types of structural analogues of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P.sub.4 ] are prepared by a chemo-enzymatic route. These 6-O-substituted analogues retain the biological activity of Ins(1,4,5)P.sub.3 and are able to elicit Ca.sup.2+ release from porcine brain microsomes. Moreover, these analogues allow the preparation of Ins(1,4,5)P.sub.3 -based immunogens and affinity matrix which are successfully applied to the preparation and purification of antibodies against Ins(1,4,5)P.sub.3. These antibodies display discriminative affinity toward Ins(1,4,5)P.sub.3, and provide a useful tool to study intracellular Ca.sup.2+ mobilization.

D-myo-inositol 1,4,5-trisphosphate analogues

A computer modeling approach is taken to delineate the structural requirements of substrate molecules for biocatalytic turnover and enantioselective discrimination. This leads to an efficient enzymatic preparation of optically active propranolol. The acyl-sustituents on the alcohol and the secondary amine of propranolol play crucial roles in controlling the catalysis and enantiospecificity, respectively, of the enzymatic hydrolysis.

Enzymatic preparation of optically active propanolol and

Multigram quantities of Ins(1,4,5) P.sub.3, Ins(1,3,4) P.sub.3, and Ins(1,3,4,5)P.sub.4 are prepared in their enantiomerically pure forms from the two enantiomers of 1,2:5,6-di-O-cyclohexylidene myo-inositol. Also, a facile enzymatic preparation is also described of these chiral precursors through enantiospecific deacylation of the corresponding racemic esters is disclosed.

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Wakefield, RI, United States of America

Ching-Shih Chen