Unique self-anhydride formation in the degradation of cytidine-5'-monophosphosialic acid (CMP-Neu5Ac) and cytidine-5'-diphosphosialic acid (CDP-Neu5Ac) and its application in CMP-sialic acid analogue synthesis.

Sialyloligosaccharides are synthesised by various glycosyltransferases and sugar nucleotides. All of these nucleotides are diphosphate compounds except for cytidine-5'-monophosphosialic acid (CMP-Neu5Ac). To obtain an insight into why cytidine-5'-diphosphosialic acid (CDP-Neu5Ac) has not been used for the sialyltransferase reaction and why it is not found in biological organisms, the compound was synthesised. This synthesis provided the interesting finding that the carboxylic acid moiety of the sialic acid attacks the attached phosphate group. This interaction yields an activated anhydride between carboxylic acid and the phosphate group and leads to hydrolysis of the pyrophosphate linkage. The mechanism was demonstrated by stable isotope-labelling experiments. This finding suggested that CMP-Neu5Ac might also form the corresponding anhydride structure between carboxylic acid and phosphate, and this seems to be the reason why CMP-Neu5Ac is acid labile in relation to other sugar nucleotides. To confirm the role of the carboxylic acid, CMP-Neu5Ac derivatives in which the carboxylic acid moiety in the sialic acid was substituted with amide or ester groups were synthesised. These analogues clearly exhibited resistance to acid hydrolysis. This result indicated that the carboxylic acid of Neu5Ac is associated with its stability in solution. This finding also enabled the development of a novel chemical synthetic method for CMP-Neu5Ac and CMP-sialic acid derivatives.

Enhanced subunit interactions with gemcitabine-5'-diphosphate inhibit ribonucleotide reductases.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The class I RNRs are composed of two subunits, alpha and beta, with proposed quaternary structures of alpha2beta2, alpha6beta2, or alpha6beta6, depending on the organism. The alpha subunits bind the nucleoside diphosphate substrates and the dNTP/ATP allosteric effectors that govern specificity and turnover. The beta2 subunit houses the diferric Y* (1 radical per beta2) cofactor that is required to initiate nucleotide reduction. 2',2'-difluoro-2'-deoxycytidine (F2C) is presently used clinically in a variety of cancer treatments and the 5'-diphosphorylated F2C (F2CDP) is a potent inhibitor of RNRs. The studies with [1'-(3)H]-F2CDP and [5-(3)H]-F2CDP have established that F2CDP is a substoichiometric mechanism based inhibitor (0.5 eq F2CDP/alpha) of both the Escherichia coli and the human RNRs in the presence of reductant. Inactivation is caused by covalent labeling of RNR by the sugar of F2CDP (0.5 eq/alpha) and is accompanied by release of 0.5 eq cytosine/alpha. Inactivation also results in loss of 40% of beta2 activity. Studies using size exclusion chromatography reveal that in the E. coli RNR, an alpha2beta2 tight complex is generated subsequent to enzyme inactivation by F2CDP, whereas in the human RNR, an alpha6beta6 tight complex is generated. Isolation of these complexes establishes that the weak interactions of the subunits in the absence of nucleotides are substantially increased in the presence of F2CDP and ATP. This information and the proposed asymmetry between the interactions of alphanbetan provide an explanation for complete inactivation of RNR with substoichiometric amounts of F2CDP.

Synthesis of 2'-deuterio and 3'-deuterio cytidine 5'-diphosphate.

2'-2H- and 3'-2H-CDP were synthesized from 5'-MMT-3'-O-TBDMS and 2',5'-O-diTBDMS cytidine derivatives, respectively, by oxidation followed by acidic removal of 5'-protection, reduction with [NaBD(OAc)3] and finally displacement of a tosyl group by pyrophosphate.

Alpha-phosphono lactone analogues of cytidine and cytosine arabinoside diphosphates: synthesis via ring closing metathesis.

alpha-Phosphono lactone derivatives of the nucleosides cytidine and cytosine arabinoside have been prepared from the corresponding nucleoside aldehydes. The stereochemical outcome of allylation reactions with these aldehydes was found to be dependent upon both the choice of protecting groups for the 2'- and 3'-hydroxyl groups and, to some extent, the nature of the Lewis acid catalyst employed. Ultimately, conditions were found that favored either the 5'R or 5'S diastereomer from different cytidine aldehydes, and gave some stereoselectivity in additions to an aldehyde derivative of ara-C. The resulting homoallylic alcohols were used as substrates in attempted Knovenagel and Horner-Wadsworth-Emmons condensations, but elimination was found to predominate over lactone formation under the conditions employed. The desired alpha-phosphono lactones could be prepared through a reaction sequence that included ring-closing metathesis on acrylate esters of the homoallylic alcohols, followed by reduction of the resulting alpha,beta-unsaturated lactones and carbon-phosphorus bond formation on enolates generated from the saturated lactones.

The novel fluorescent CDP-analogue (Pbeta)MABA-CDP is a specific probe for the NMP binding site of UMP/CMP kinase.

Direct thermodynamic and kinetic investigations of the binding of nucleotides to the nucleoside monophosphate (NMP) site of NMP kinases have not been possible so far because a spectroscopic probe was not available. By coupling a fluorescent N-methylanthraniloyl- (mant) group to the beta-phosphate of CDP via a butyl linker, a CDP analogue [(Pbeta)MABA-CDP] was obtained that still binds specifically to the NMP site of UmpKdicty, because the base and the ribose moieties, which are involved in specific interactions, are not modified. This allows the direct determination of binding constants for its substrates in competition experiments.

Synthesis and antiproliferative activity of cytidine-5'-alkylphosphonophosphates and structurally related compounds.

The chemical synthesis of cytidine-5'-alkyl- and cytidine-5'-alkyl (acyl)deoxyglycerophosphonophosphates is reported. The compounds obtained represent a novel class of cytostatically active agents based on phospholipids, which inhibit the growth of various tumor cell lines in vitro. They are phosphono analogs of the cytidine-5'-diphosphate-diacylglycerol (CDP-DAG) possessing a structurally modified lipid moiety and a phospholipase C-resistant P-C bond. The antiproliferative efficacy of the cytidine-5'-alkylphosphonophosphates strongly depends on the alkyl chain length. The cytidine-5'-hexadecylphosphonophosphate was found to be the most effective compound tested in this study. Its cytostatic effect was distinctly higher than that of the alkyl(acyl) deoxyglycero derivatives and of the corresponding diphosphates. The structure of the new compounds were confirmed by fast atom bombardment mass spectrometry (FAB). The FAB fragmentation pattern is discussed.

Synthesis of the coenzymes adenosine diphosphate glucose, guanosine diphosphate glucose, and cytidine diphosphoethanolamine under primitive Earth conditions.

The nonenzymatic synthesis of the coenzymes adenosine diphosphate glucose (ADPG), guanosine diphosphate glucose (GDPG), and cytidine diphosphoethanolamine (CDP-ethanolamine) has been carried out under conditions considered to have been prevalent on the early Earth. The production of these compounds was performed by allowing simple precursor molecules to react under aqueous solutions, at moderate temperatures and short periods of time, with mediation by cyanamide or urea. These two condensing agents are considered to have been present in significant amounts on the primitive Earth and have been previously used in the nonenzymatic synthesis of several other important biochemical compounds. In our experiments, ADPG was obtained by heating glucose-1-phosphate (G1P) and ATP in the presence of cyanamide for 24 h at 70 degrees C. The reaction of G1P and GTP under the same conditions yielded GDPG. The cyanamide-mediated production of CDP-ethanolamine was carried out by reacting a mixture of ethanolamine phosphate and CTP for 24 h at 70 degrees C. The separation and identification of the reaction products was carried out by paper chromatography, thin-layer chromatography, high performance thin-layer chromatography, high performance liquid chromatography, both normal and reverse-phase, UV spectroscopy, enzymatic assays, and acid hydrolysis. Due to the mild conditions employed, and to the relative ease of these reactions, these studies offer a simple attractive system for the nonenzymatic synthesis of phosphorylated high-energy metabolic intermediates under conditions considered to have been prevalent on the ancient Earth.