Nucleoside monophosphates conversion

In the preceding sections the conversion of purines and purine nucleosides to purine nucleoside monophosphates has been discussed. The monophosphates of adenosine and guanosine must be converted to their di- and triphosphates for polymerization to RNA, for reduction to 2 -deoxyribonucleoside diphosphates, and for the many other reactions in which they take part. Adenosine triphosphate is produced by oxidative phosphorylation and by transfer of phosphate from 1,3-diphosphoglycerate and phosphopyruvate to adenosine diphosphate. A series of transphosphorylations distributes phosphate from adenosine triphosphate to all of the other nucleotides. Two classes of enzymes, termed nucleoside mono-phosphokinases and nucleoside diphosphokinases, catalyse the formation of the nucleoside di- and triphosphates by the transfer of the terminal phosphoryl group from adenosine triphosphate. Muscle adenylate kinase (myokinase)... 

The synthesis of purine nucleotides (1) starts from IMP. The base it contains, hypoxanthine, is converted in two steps each into adenine or guanine. The nucleoside monophosphates AMP and CMP that are formed are then phos-phorylated by nucleoside phosphate kinases to yield the diphosphates ADP and GDP, and these are finally phosphorylated into the triphosphates ATP and CTP. The nucleoside triphosphates serve as components for RNA, or function as coenzymes (see p. 106). Conversion of the ribonucleotides into deoxyribo-nucleotides occurs at the level of the diphosphates and is catalyzed by nucleoside diphosphate reductase (B). 

F. Conversion of nucleoside monophosphates to nucleoside diphosphates and triphosphates... 

Conversion of Nucleoside Monophosphates to Triphosphates Goes through Diphosphates Inhibitors of Nucleotide Synthesis Catabolism of Nucleotides... 

All biosynthetic pathways are under regulatory control by key allosteric enzymes that are influenced by the end products of the pathways. For example, the first step in the pathway for purine biosynthesis is inhibited in a concerted fashion by nucleotides of either adenine or guanine. In addition, the nucleoside monophosphate of each of these bases inhibits its own formation from inosine monophosphate (IMP). On the other hand, adenine nucleotides stimulate the conversion of IMP into GMP, and GTP is needed for AMP formation. 

The conversion of IMP to AMP and GMP is shown in Figure 10.8. Note that GTP is required for the biosynthesis of AMP and ATP for GMP biosynthesis, and high-ATP levels channel the conversion of IMP to GMP. In addition, IMP dehydrogenase is inhibited by GMP. We therefore have additional loci for controlling cellular concentrations of purine nucleotides by regulating the fate of IMP. The conversion of nucleoside monophosphates to di- and triphosphates is discussed later. 

Synthetic non-nucleoside RT inhibitor (NNRTI) in clinical use Synthetic nucleoside reverse transcriptase inhibitors (NRTIs) in clinical use metabolic conversion to the nucleoside triphosphate (NTP) (via the nucleoside monophosphate (NMP) and diphosphate (NDP)) gives DNA chain termination because of absence of 3 -hydroxyl (Note PMEA yields the phosphonate diphosphate ... 

How is the other major pyrimidine ribonucleotide, cytidine, formed It is synthesized from the uracil base of UMP, but UMP is converted into UTP before the synthesis can take place. Recall that the diphosphates and triphosphates are the active forms of nucleotides in biosynthesis and energy conversions. Nucleoside monophosphates are converted into nucleoside triphosphates in stages. First, nucleoside monophosphates are converted into diphosphates by specific nucleoside monophosphate kinases that utilize ATP as the phosphoryl-group donor (Section 9.4). For example, UMP is phosphorylated to UDP by UMP kinase. 

The cascade begins with stoichiometric amounts of phosphoenolpyruvate (PEP), 8-allyl-A-acetyl lactosamine 120, NeuAc 1, and catalytic quantities of ATP and CMP. Initially, CMP is converted to CDP by nucleoside monophosphate kinase (NMK) in the presence of ATP. The CDP produced reacts with PEP under pyruvate kinase (PK) catalysis to form CTP. Next, CMP-NeuAc synthetase catalyzes the in situ formation of the sialyl donor from NeuAc and CTP. The pyrophosphate byproduct is decomposed to inorganic phosphate by inorganic pyrophosphatase (PPase). Subsequently, the a-2,6-sialyltransferase accomplishes the sialyation of the lactosamine acceptor 120 and produces the ttansferase inhibitor CMP as a by-product. The CMP concentrations are kept low by conversion to CDP, and in so doing the problem of product inhibition is minimized. The cycle afforded 21% of the sialylated ttisac-charide 121, which is remarkable considering the complexity of the system and number of synthetic steps that can be avoided. 

Subsequent phosphorylation reactions produce purine nucleoside diphosphates (ADP and GDP) and triphosphates (ATP and GTP). The purine nucleoside monophosphates, diphosphates, and triphosphates are all feedback inhibitors of the first stages of their own biosynthesis. Also, AMP, ADP, and ATP inhibit the conversion of IMP to adenine nucleotides, and GMP, GDP, and GTP inhibit the conversion of IMP to xanthylate and to guanine nucleotides (Figure 23.22). 

Nucleoside analogues are widely used as antiviral agents in the treatment in AIDS and AIDS-related complex. The only clinical agent approved in the United States for the treatment of AIDS is 3 -azido-3 -deoxythymidine (AZT) [54,55]. The molecular mechanism of action for this nucleoside includes conversion into its corresponding 5 -monophosphate by the action of cellular nucleoside kinase, followed by stepwise phosphorylation catalyzed by cellular nucleoside kinase to the corresponding 5 -triphosphate. These inhibit proviral DNA synthesis [55-57], catalyzed by HIV reverse transcriptase (RT), and incorporation to the 3 end of the growing DNA chain [55,58]. 

The addition of a third phosphate to any of the nucleoside pyrophosphates, such as UDP, is catalyzed by an enzyme found in yeast and several animal tissues. Since the reaction catalyzed by this enzyme appears to be nonspecific with respect to the base of the nucleotides, it has been named nucleoside diphosphate kinase (nudiki). The phosphate donor in this reaction may be any of the nucleoside triphosphates. As in the case of the nucleoside monophosphate kinases, there are no significant differences in the free energies of hydrolysis of the various nucleoside triphosphates, so all of the reactions are freely reversible with equilibrium constants near 1. Since the phosphorylation of nucleoside diphosphates is reversible, it is necessary that each of the corresponding triphosphates serve as phosphate donor. The phosphorylation of the monophosphates is similarly reversible, but in this case one of the sites on the enz3one appears to react only with adenine, and involves the conversion of ATP to ADP. The complementary reaction involves XMP XDP. Thus the nonadenine nucleotides are never equivalent to ATP, and the reaction is limited to the phosphorylation of a specific nucleoside monophosphate by ATP. 

The regeneration system for CMP-NeuAc is more complicated than that for NDP-sugars (Scheme 7) [24]. An additional phosphorylation step must be incorporated, because CMP, a nucleoside monophosphate, is released after reaction with the sialyltransferase. For recycling purposes, nucleoside monophosphate kinase (NMK EC 2.7.4.4) or myokinase (MK EC 2.7.4.3) is added for the conversion of CMP to CDP. In this reaction, the phosphoryl donor is ATP. Subsequently, both CDP and ADP must be re-phosphorylated to CTP and ATP, respectively. Thus, for regeneration of CMP-NeuAc, an additional kinase and two equivalents of PEP are required. The condensation of NeuAc with CTP is catalyzed by CMP-NeuAc synthetase (EC 2.7.7.43). This system was used for the large-scale synthesis of 6 -sialyl-LacNAc(6 -SLN) from LacNAc catalyzed by a2,6-SiaT (EC 2.7.7.43) in 97% yield. 

Fig ire 2 shows the possible interconversions between purine bases, nucleosides and nucleoside monophosphates. Prom genetic and enzymatic studies it has been shown that the conversion of the free bases(adenine, hypoxanthine,xanthine and guanine) to the monophosphate level is carried out by at least three different enzymes (the phosphoribosyltransferases)(1,5). This is in contrast to htunan tissues, which have one specific adenine phosphoribosyltransferase and one transferase with activity towards hypoxanthine,xanthine and guanine. 

Various synthetic routes to novel nucleoside diphosphate derivatives have been described. Tonn and Meier describe a solid-phase protocol for the synthesis of cjc/oSal-protected nucleoside monophosphates, which can then be used for the synthesis of diphosphates and triphosphates, and conjugates of both, by treatment with the appropriate phosphate or pyrophosphate derivative, including methylene pyrophosphate derivatives. " A synthesis of lamivudine (3TC) diphosphate has been reported which involves oxidation of lamivudine /f-phosphonate, conversion of the phosphate to its imidazolate followed by reaction with phosphate. A few syntheses of glycosyl conjugates of nucleoside diphosphates, e.g. (17), have been reported for their study with glycosyltransferases, which play a key role in a variety of cellular processes. ... 

Acyclovir (acycloguanosine. Fig. 5.221) is a novel type of nucleoside analogue which becomes achvated only in herpes-infected host cells by a herpes-specific enzyme, thymidine kinase. This enzyme inihates conversion of acyclovir initially to a monophosphate and then to the antiviral triphosphate which inhibits viral DNA polymerase. The host cell polymerase is not inhibited to the same extent, and the antiviral triphosphate is not produced in uninfected cells. Ganciclovir (Fig. 5.22J) is up to 100... 

Pyrimidine 5 -nucleotidase (P5N) is a unique enzyme that was recognized from studies of families with relatively common hemolytic disorders. The enzyme catalyzes the hydrolytic dephosphorylation of pyrimidine 5 -nucleotides but not purine nucleotides. The role of this enzyme is to eliminate RNA and DNA degradation products from the cytosol during erythroid maturation by conversion of nucleotide monophosphates to diffusible nucleosides. P5N is inhibited by lead, and its activity is considered to be a good indicator of lead exposure (PI). 

Aciclovir is a member of a group of nucleoside derivatives termed acyclonucleosides, in that there is an incomplete sugar ring. The structural relationship to 2 -deoxyguanosine should be very clear. Aciclovir is converted into its monophosphate by the viral enzyme thymidine kinase - some viruses also possess enzymes that facilitate their replication in the host cell. The viral enzyme turns out to be much more effective than that of the host cell, and conversion is, therefore, mainly in infected cells. The monophosphate is subsequently converted into the triphosphate hy the host cell enzymes. Aciclovir triphosphate inhibits viral DNA polymerase, much more so than it does the host enzyme, and so terminates DNA replication. 

Trifluridine (Viroptic) is a fluorinated pyrimidine nucleoside that has in vitro activity against HSV-1 and HSV-2, vaccinia, and to a lesser extent, some adenoviruses. Activation of trifluridine requires its conversion to the 5 monophosphate form by cellular enzymes. Trifluridine monophosphate inhibits the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) by thymidylate synthetase. In addition, it competes with deoxythymidine triphosphate (dTTP) for incorporation by both viral and cellular DNA polymerases. Trifluridine-resistant mutants have been found to have alterations in thymidylate synthetase specificity. 

Didanosine is a synthetic purine nucleoside analog that inhibits the activity of reverse transcriptase in HIV-1, HIV-2, other retroviruses and zidovudine-resistant strains. A nucleobase carrier helps transport it into the cell where it needs to be phosphorylated by 5 -nucleoiidase and inosine 5 -monophosphate phosphotransferase to didanosine S -monophosphate. Adenylosuccinate synthetase and adenylosuccinate lyase then convert didanosine 5 -monophosphate to dideoxyadenosine S -monophosphate, followed by its conversion to diphosphate by adenylate kinase and phosphoribosyl pyrophosphate synthetase, which is then phosphorylated by creatine kinase and phosphoribosyl pyrophosphate synthetase to dideoxyadenosine S -triphosphate, the active reverse transcriptase inhibitor. Dideoxyadenosine triphosphate inhibits the activity of HIV reverse transcriptase by competing with the natural substrate, deoxyadenosine triphosphate, and its incorporation into viral DNA causes termination of viral DNA chain elongation. It is 10-100-fold less potent than zidovudine in its antiviral activity, but is more active than zidovudine in nondividing and quiescent cells. At clinically relevant doses, it is not toxic to hematopoietic precursor cells or lymphocytes, and the resistance to the drug results from site-directed mutagenesis at codons 65 and 74 of viral reverse transcriptase. 

Phosphoromorpholidate intermediates, initially developed by Khorana and Moffatt,19 have been widely used for the construction of nucleoside diphosphates. The construction of the diphosphate linkage typically involves exposure of a carbohydrate-derived phosphate nucleophile to phosphoromorpholidate electrophile in pyridine solvent. This protocol was attractive from the point of view that it can be executed on completely deprotected precursors. This method suffers, however, from the lengthy reaction times required for reasonable conversion, although a tetrazole modification, introduced by Wong, had been shown to shorten reaction times considerably. The major drawback of this method, for our own purposes, is that it would require construction of either a carbohydrate-derived phosphoromorpholidate or the preparation of a phosphoromorpholidate intermediate deriving from our rather expensive undecaprenyl monophosphate precursor. 

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