Synthesis of Cytidine Nucleotides

CTP is synthesized from UTP by transfer of the amide nitrogen of glutamine to C-4 of the pyrimidine ring of UTP. This reaction requires ATP as an energy source. 

The deoxycytidine phosphates result from reduction of CDP to dCDP by a mechanism analogous to that described for the purine nucleotides. Then dCDP is converted to dCTP by nucleoside diphosphate kinase. 

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A series of other barbiturates (phenobarbital, barbital, thiopental, pentobarbital at 1 mmol l i concentration inhibit the orotate uptake system without affecting the incorporation of uracil into cellular pyrimidines [287]. While barbituric acid and hexobarbital are less active, phenylethylhydan-toin, chlorpromazine and phenethyl alcohol are extremely active. Phenobarbital also depresses the utilization of orotic acid for the synthesis of cytidine nucleotides in the liver [288]. a-Hexachlorocyclohexane, an inhibitor of the phenobarbital type, was even more effective in depressing de novo cytidine nucleotide synthesis from orotic acid [289]. 

Seifert, J. and Vacha, J. Decreased utilization of orotic acid for the synthesis of cytidine nucleotides in rat hver after administration of a-hexychlorocyclohexane. Toxicology, 7, 155-161 (1977)... 

The further conversion of UMP to cytidine nucleotides has not been demonstrated at the mononucleotide level. Apparently, uridine triphosphate was aminated by ammonia and ATP in E. coU to form cytidine triphosphate (CTP) no other nitrogen donors were able to perform the amination step (382, 383) (Fig. 22). Since the presence of glutamine was necessary for optimal synthesis of cytidine nucleotides from UMP in a homogenate of a hepatoma (384), it is possible that a different amination reaction exists in mammalian tissues. 

In this situation, two different polypeptide chains interact to form a new specific complex, whose biologic activity can be modified by ligands that bind to the precursor subunits. This type of regulation has been extensively studied for the aspartate transcarbamylase of E. coli (Gerhart, 1970). This enzyme catalyzed the initial step in the synthesis of cytidine nucleotides it is allosterically inhibited by CTP and shows positive cooperativity for substrate. It may be dissociated by mercurials into catalytic subunits, which are insensitive to CTP and which exhibit hyperbolic kinetics for substrate, and into regulatory subunits which bind CTP. 

A classic description of the role of cytidine nucleotides in phospholipid synthesis. 

UMP is the parent compound in the synthesis of cytidine and deoxycytidine phosphates and thymidine nucleotides (which are deoxyribonucleotides). 

It is an enzyme used in the early stages of cytidine nucleotide synthesis. 

Wolf et al. reported the synthesis of cytidine-5 -monophosphate-sialic acid derivatives (70). The synthesis involves the reaction of cyclo-Sal-nucleotides with different phosphate nucleophiles. Cyc/o-Sal-nucleotides were also used to prepare monophosphate-linked sugar nucleotides in high yields. These compounds were used for enzymatic studies (bacterial polysialyltransferase) and the enzyme showed marked differences in utilising these compounds. 

While mammahan cells reutilize few free pyrimidines, salvage reactions convert the ribonucleosides uridine and cytidine and the deoxyribonucleosides thymidine and deoxycytidine to their respective nucleotides. ATP-dependent phosphoryltransferases (kinases) catalyze the phosphorylation of the nucleoside diphosphates 2 "-de-oxycytidine, 2 -deoxyguanosine, and 2 -deoxyadenosine to their corresponding nucleoside triphosphates. In addition, orotate phosphoribosyltransferase (reaction 5, Figure 34-7), an enzyme of pyrimidine nucleotide synthesis, salvages orotic acid by converting it to orotidine monophosphate (OMP). 

The intermediate phosphite employed in this synthesis was prepared by condensation of duly protected sialic acid with the nucleosidyl phospho-roamidite in the presence of N-PhIMT. Oxidation by TBHP and deprotection according to standard procedures gave the cytidine-S -monophospho-iST-ace-tylneuraminic acid. This synthetic route is claimed to have advantages over procedures published earlier [26]. The same group demonstrated the importance of 3 A and 4 A molecular sieves as moisture scavengers in the reaction of nucleoside phosphoroamidite with a nucleotide. This approach should be likely to find application in the synthesis of biophosphates outside nucleotide chemistry. 

The structures of these cytidine nucleotides were confirmed by synthesis. Condensation of 1,2-0-isopropylidene-L-glyceritol 3-phosphate with cytidine 5-phosphate in the presence of dicyclohexylcarbodiimide, followed by careful acid hydrolysis of the isopropylidene group, yielded cytidine glyceritol pyrophosphate identical with the natural product. 

Cidofovir (Figure 24.4) is an antiviral cytidine nucleotide analog with inhibitory activity against HCMV and other herpes viruses. Cidofovir is first converted to an active diphosphate form by cellular enzymes. Antiviral effects of cidofovir are due to inhibition of viral DNA polymerase by the diphosphate metabolite (Neyts and De Clercq, 1994 Plosker and Noble, 1999 Scholar and Pratt, 2000). The diphosphate probably interacts with DNA polymerase either as an alternate substrate (incorporation at the 3 end or within the interior of the DNA chain) or as a competitive inhibitor (with respect to the normal substrate dCTP). Cidofovir inhibits HCMV DNA synthesis at intracellular concentrations 1000-fold lower than are required to inhibit cellular DNA synthesis (Neyts and De Clercq, 1994). For HSV-1 and HSV-2 corresponding concentrations are at least 50-fold lower. 

The utilization of ammonia resulting from the combination of carbamyl phosphate with aspartic acid, the initial reaction for the synthesis of the pyrimidine nucleotides, continues only as long as there is a requirement for them (Fig. 3). Regulation of this biosynthetic pathway is probably by way of feedback inhibition of aspartate transcarbamylase. The rat liver enzyme is inhibited by uridine, cytidine or thymidine or such derivatives as CMP, UTP, or TMP, all intermediates or products of this pathway (B8). This is not the only enzyme of the pathway which may be subject to feedback regulation. Dihydroorotase from rat liver is also inhibited by some pyrimidines and purines (B9). 

Purine [adenosine] nucleoside antimetabolite active triphosphate form can prevent elongation of DNA chain also inhibits ribonucleotide reductase, depleting intracellular deoxynucleotides and impairing DNA synthesis Antipyrimidine antimetabolite inhibits DNA polymerase with inhibition of DNA strand elongation and replication activated in tumor cells to triphosphate form competes with conversion of cytidine to deoxycytidine nucleotides, further blocking polymerization of DNA leads to production of short DNA strands cell-cycle specific (S phase) acts only on proliferating cells... 

Adenylic acids o and b were each converted by trifluoroacetic anhydride into the same 2 3 -cyclic phosphate (13, R = adenin-9-yl) by Brown, Magrath, and Todd. These authors also applied this method to the synthesis of 2 3 -cyclic phosphates (13) of cytidine, uridine, and guanosine. Hydrolysis of these cyclic phosphates gave the corresponding a and b nucleotide mixtures. Markham and Smith found that, during hydrolysis of ribonucleic acid with pancreatic ribonuclease, the 2 3 -cyclic phosphates of the pyrimidine nucleosides are formed as intermediates leading to the ribonucleoside phosphates b. The also showed that 2 3 -cyclic phosphates (13) are formed by very mild, alkaline hydrolysis of ribonucleic acid. The discoveries of these a and b isomers of mononucleotides from... 

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