Peter K. Bridson

Catalysis of accurate poly(C)-directed synthesis of 3′-5′-linked oligoguanylates by Zn2+

Abstract Zn 2+ is an efficient catalyst for the oligomerization of guanosine 5′-phosphorimidazolide on a polycytidylic acid template. Up to 75% of the input ImpG † is converted to oligomers with a mean chain length up to 10. Material longer than (pG) 30 can be detected. The oligomeric products are predominantly 3′-5′-linked. If poly(C) is incubated with ImpG and an equimolar quantity of the 5′-phosphorimidazolides of adenosine, uridine or cytidine, in the presence of Zn 2+ , ImpG is incorporated at least 200 times more efficiently than “incorrect” nucleotides.

Inactivation of aldophosphamide by human aldehyde dehydrogenase isozyme 3

Tumors resistant to chemotherapeutic oxazaphosphorines such as cyclophosphamide often overexpress aldehyde dehydrogenase (ALDH), some isozymes of which catalyze the oxidization of aldophosphamide, an intermediate of cyclophosphamide activation, with formation of inert carboxyphosphamide. Since resistance to oxazaphosphorines can be produced in mammalian cells by transfecting them with the gene for human ALDH isozyme 3 (hALDH3), it seems possible that patients receiving therapy for solid tumors with cyclophosphamide might be protected from myelosuppression by their prior transplantation with autologous bone marrow that has been transduced with a retroviral vector causing overexpression of hALDH3. We investigated whether retroviral introduction of hALDH3 into a human leukemia cell line confers resistance to oxazaphosphorines. This was examined in the polyclonal transduced population, that is, without selecting out high expression clones. hALDH3 activity was 0.016 IU/mg protein in the transduced cells (compared with 2x10(-5) IU/mg in untransduced cells), but there was no detectable resistance to aldophosphamide-generating compounds (mafosfamide or 4-hydroperoxycyclophosphamide). The lack of protection was due, in part, to low catalytic activity of hALDH3 towards aldophosphamide, since, with NAD as cofactor, the catalytic efficiency of homogeneous, recombinant hALDH3 for aldophosphamide oxidation was shown to be about seven times lower than that of recombinant hALDH1. The two polymorphic forms of hALDH3 had identical kinetics with either benzaldehyde or aldophosphamide as substrate. Results of initial velocity measurements were consistent with an ordered sequential mechanism for ALDH1 but not for hALDH3; a kinetic mechanism for the latter is proposed, and the corresponding rate equation is presented.

Autotaxin Structure Activity Relationships Revealed through Lysophosphatidylcholine Analogs

Autotaxin (ATX) catalyzes the hydrolysis of lysophosphatidylcholine (LPC) to form the bioactive lipid lysophosphatidic acid (LPA). LPA stimulates cell proliferation, cell survival, and cell migration and is involved in obesity, rheumatoid arthritis, neuropathic pain, atherosclerosis and various cancers, suggesting that ATX inhibitors have broad therapeutic potential. Product feedback inhibition of ATX by LPA has stimulated structure-activity studies focused on LPA analogs. However, LPA displays mixed mode inhibition, indicating that it can bind to both the enzyme and the enzyme-substrate complex. This suggests that LPA may not interact solely with the catalytic site. In this report we have prepared LPC analogs to help map out substrate structure-activity relationships. The structural variances include length and unsaturation of the fatty tail, choline and polar linker presence, acyl versus ether linkage of the hydrocarbon chain, and methylene and nitrogen replacement of the choline oxygen. All LPC analogs were assayed in competition with the synthetic substrate, FS-3, to show the preference ATX has for each alteration. Choline presence and methylene replacement of the choline oxygen were detrimental to ATX recognition. These findings provide insights into the structure of the enzyme in the vicinity of the catalytic site as well as suggesting that ATX produces rate enhancement, at least in part, by substrate destabilization.

PREPARATION OF XANTHINE-3-ACETIC ACID AND SOME DERIVATIVES

Abstract A general procedure for the preparation of 3-alkyl-xanthines from 7-benzylxanthine is described and used for the synthesis of xanthine-3-acetic acid.

Template-directed synthesis of oligoguanylic acids - Metal ion catalysis

The condensation of guanosine 5′-phosphorimidazolide on a poly(C) template is subject to regiospecific metal-ion catalysis. In the presence of the Pb2+ ion, predominantly [2′–5′]- linked oligomers up to the 40-mer are formed. The Zn2+ ion catalyzes the formation of predominantly [3′–5′]-linked oligomers up to the 35-mer. The Sn2+, Sb3+ and Bi3+ ions also catalyze the formation of [2′–5′]-linked products, but less efficiently than the Pb2+ ion.

Condensation of activated diguanylates on a poly(C) template

SummaryWe have studied the metal-ion catalysis of a number of reactions of the isomers of ImpGpG on a poly(C) template. In the absence of a catalytic metal ion, oligomers at least up to (pG)20 are obtained from the ImpGpG isomers in a l-methylimidazole buffer. The Pb2+ ion improves the yield of longer oligomers and changes substantially the distribution of linkage isomers. The Pb2+ ion greatly improves the yield of longer oligomers obtained from G and ImpGpG on a poly(C) template.The self-condensation of ImpGpG in a 2, 6-lutidine buffer is much less efficient than in a l-methylimidazole buffer. The Zn2+ greatly increases the yield of products from the [3′-5′]-linked dimer, but fails to catalyze the formation of long oligomers from the [2′-5′]-linked dimer. The bonds formed in the Zn2+-catalyzed selfcondensation of ImpG3pG on poly(C) are mainly [3′-5′]-linked.

Synthesis of deoxyazepinomycin

The syntheses of several new derivatives of imidazo[4,5-e] [1,4]diazepin-8-one are described

SYNTHESIS AND ADENOSINE RECEPTOR AFFINITY OF 7-BETA -D-RIBOFURANOSYLXANTHINE

7-beta-D-Ribofuranosylxanthine, a previously unreported isomer of xanthosine, was prepared in four steps from 7-benzylxanthine. The procedure, which involves the use of pivaloyloxymethyl groups to protect the xanthine ring, was also applied to preparation of some 1-N-alkyl derivatives of 7-ribosylxanthine. Adenosine receptor affinity for these compounds was determined. 7-beta-D-Ribofuranosylxanthine was found to have higher affinity and greater selectivity for the A1 receptor than previously reported xanthine nucleosides, and to be a partial agonist.

An NMR and computational study of diazepinediones

Abstract The structure and ring-inversion barriers of several imidazodiazepinediones and benzodiazepinediones are studied. The barrier for one of each type is experimentally determined by NMR and calculations are carried out with the AM1 semi-empirical method. For comparison, calculations are also carried out on several imidazodiazepines and benzodiazepines.

1-(5-Amino-1-β-D-Ribofuranosylimidazol-4-carbonyl)-3,5-dimethylpyrazole, a new Intermediate for the Preparation of Aicar Analogs

Abstract 1-(5-Amino-1-β-D-ribofuranosylimidazol-4-carbonyl)-3,5-di-methylpyrazole (2a), which is readily prepared from inosine, is shown to be a useful precursor for the preparation of 5-aminoimidazole nucleosides and nucleotides.