Reaction Of Cytidine Research Articles

Engineering analysis of multienzyme cascade reactions for 3'-sialyllactose synthesis.

Sialo‐oligosaccharides are important products of emerging biotechnology for complex carbohydrates as nutritional ingredients. Cascade bio‐catalysis is central to the development of sialo‐oligosaccharide production systems, based on isolated enzymes or whole cells. Multienzyme transformations have been established for sialo‐oligosaccharide synthesis from expedient substrates, but systematic engineering analysis for the optimization of such transformations is lacking. Here, we show a mathematical modeling‐guided approach to 3ʹ‐sialyllactose (3SL) synthesis from N‐acetyl‐ d‐neuraminic acid (Neu5Ac) and lactose in the presence of cytidine 5ʹ‐triphosphate, via the reactions of cytidine 5ʹ‐monophosphate‐Neu5Ac synthetase and α2,3‐sialyltransferase. The Neu5Ac was synthesized in situ from N‐acetyl‐ d‐mannosamine using the reversible reaction with pyruvate by Neu5Ac lyase or the effectively irreversible reaction with phosphoenolpyruvate by Neu5Ac synthase. We show through comprehensive time‐course study by experiment and modeling that, due to kinetic rather than thermodynamic advantages of the synthase reaction, the 3SL yield was increased (up to 75%; 10.4 g/L) and the initial productivity doubled (15 g/L/h), compared with synthesis based on the lyase reaction. We further show model‐based optimization to minimize the total loading of protein (saving: up to 43%) while maintaining a suitable ratio of the individual enzyme activities to achieve 3SL target yield (61%–75%; 7–10 g/L) and overall productivity (3–5 g/L/h). Collectively, our results reveal the principal factors of enzyme cascade efficiency for 3SL synthesis and highlight the important role of engineering analysis to make multienzyme‐catalyzed transformations fit for oligosaccharide production.

Reaction of Cytidine with Cyanopropargylic Alcohols: Synthesis of Functionalized Cytidine Cyclic Ketals

Cytidine reacts with one or two molecules of cyanopropargylic alcohols under mild conditions (K2CO3, DMF, 20–25 °C, 10–48 h) by the hydroxy groups of the ribose moiety to afford representatives of the two novel families of cytidine cyclic ketals. The first ones (53–76% yields) are formed with participation of the two vicinal hydroxyl groups (at 2′,3′-cis-positions) only. The second ones (7–21% yields) represent the adducts of cytidine with two molecules of cyanopropargylic alcohols involving all the three hydroxyl groups.

New N4-Hydroxycytidine Derivatives: Synthesis and Antiviral Activity

Two series of N4-hydroxycytidine derivatives were synthesized and evaluated as potential antipox virus agents. Acylation of N4-hydroxycytidine (1) with an excess of acyl chloride or imidazolide yielded the corresponding N4-(acyloxy) derivatives. 5'-Phosphonates of 1 were prepared by the reaction of cytidine 5'-phosphonates with aqueous hydroxylamine hydrochloride (pH 6.0). Nucleoside 1 and its N4-(acyloxy) derivatives inhibited replication of pox viruses in cell cultures, N4-(pivaloyloxy)- and N4-(benzoyloxy)cytidines being the most potent. The synthesized 5'-phosphonates were more cytotoxic than the parent nucleoside.

The reactions of cytidine and 2'-deoxycytidine with SO4.- revisited. Pulse radiolysis and product studies.

The reactions of SO4.- with 2'-deoxycytidine 1a and cytidine 1b lead to very different intermediates (base radicals with 1a, sugar radicals with 1b). The present study provides spectral and kinetic data for the various intermediates by pulse radiolysis as well as information on final product yields (free cytosine). Taking these and literature data into account allows us to substantiate but also modify in essential aspects the current mechanistic concept (H. Catterall, M. J. Davies and B. C. Gilbert, J. Chem. Soc., Perkin Trans. 2, 1992, 1379). SO4.- radicals have been generated radiolytically in the reaction of peroxodisulfate with the hydrated electron (and the H. atom). In the reaction of SO4.- with 1a (k = 1.6 x 10(9) dm3 mol-1 s-1), a transient (lambda max = 400 nm, shifted to 450 nm at pH 3) is observed. This absorption is due to two intermediates. The major component (lambda max approximately 385 nm) does not react with O2 and has been attributed to an N-centered radical 4a formed upon sulfate release and deprotonation at nitrogen. The minor component, rapidly wiped out by O2, must be due to C-centered OH-adduct radical(s) 6a and/or 7a suggested to be formed by a water-induced nucleophilic replacement. These radicals decay by second-order kinetics. Free cytosine is only formed in low yields (G = 0.14 x 10(-7) mol J-1 upon electron-beam irradiation). In contrast, 1b gives rise to an intermediate absorbing at lambda max = 530 nm (shifted to 600 nm in acid solution) which rapidly decays (k = 6 x 10(4) s-1). In the presence of O2, the decay is much faster (k approximately 1.3 x 10(9) dm3 mol-1 s-1) indicating that this species must be a C-centered radical. This has been attributed to the C(5)-yl radical 8 formed upon the reaction of the C(2')-OH group with the cytidine SO4(.-)-adduct radical 2b. This reaction competes very effectively with the corresponding reaction of water and the release of sulfate and a proton generating the N-centered radical. Upon the decay of 8, sugar radical 11 is formed with the release of cytosine. The latter is formed with a G value of 2.8 x 10(-7) mol J-1 (85% of primary SO4.-) at high dose rates (electron beam irradiation). At low dose rates (gamma-radiolysis) its yield is increased to 7 x 10(-7) mol J-1 due to a chain reaction involving peroxodisulfate and reducing free radicals. Phosphate buffer prevents the formation of the sugar radical at the SO4(.-)-adduct stage by enhancing the rate of sulfate release by deprotonation of 2b and also by speeding up the decay of the C(5)-yl radical into another (base) radical. Cytosine release in cytidine is mechanistically related to strand breakage in poly(C). Literature data on the effect of dioxygen on strand breakage yields in poly(C) induced by SO4.- (suppressed) and upon photoionisation (unaltered) lead us to conclude that in poly(C) and also in the present system free radical cations are not involved to a major extent. This conclusion modifies an essential aspect of the current mechanistic concept.

Formation of 3,N4-ethenocytidine, 1,N6-ethenoadenosine, and 1,N2-ethenoguanosine in reactions of mucochloric acid with nucleosides.

Mucochloric acid, a genotoxic hydroxyfuranone found in chlorinated drinking water, was reacted with cytidine, adenosine, guanosine, and uridine. HPLC analyses with UV detection at 290 nm showed that one major product peak was formed in the reactions of cytidine, adenosine, and guanosine. The products formed in reactions carried out at 90 degrees C for 24 or 45 h were isolated by HPLC and characterized by 1H and 13C NMR spectroscopy, direct inlet chemical ionization mass spectrometry, and UV absorbance spectroscopy. The structures of the products were assigned to 3,N4-ethenocytidine, 1,N6-ethenoadenosine, and 1,N2-ethenoguanosine. In reactions performed at 37 degrees C and pH 7.0 the same compounds were detected after a reaction time of 7 days. No observable reaction took place between mucochloric acid and uridine.

HPLC as a Tool to Study the Kinetics of Parallel and Consecutive Reactions: Competitive Interconversion, Dephosphorylation, Deamination and Depyrimidination of Cytidine 2′- and 3′-Monophosphates in Aqueous Acid

Abstract The applicability of HPLC as a method to study the kinetics of complicated reaction systems of nucleosides and nucleotides has been demonstrated by using the hydrolytic reactions of cytidine 2′- and 3′-monophosphates as an example.

Synthesis of Novel Polycyclic Nucleoside Analogs

Abstract We have synthesized polycyclic nucleoside derivatives by a novel, one pot procedure by reacting 4-0-TPS-pyrimidine nucleosides with aromatic diamines. The reaction is limited in scope but provides easy access to certain previously unknown heterocyclic ring systems.2 4-0-Triisopro- pylphenylsulfonyl-pyrimidine nucleosides were reacted with aromatic diamines leading to fused, polycyclic ring systems: o-phenylenediamine yielded the pyrimido[1,6-a]benzimidazole, 2.3- diaminonaphthalene gave the naphth[2′,3′:4,5]imidam [1.2-flpyrimidine and 1.8-diaminonaph- thalene led to the pyrimido[l,6-a]perimidine ring system. The reaction is unique because two connected nucleophilic centers react with the pyrimidine nucleoside to form an extended ring system. However, reactions of pyrimidine nucleosides with electrophiles are well known. E.g., reaction of cytidine and adenosine with bromoacetaldehyde yields ethenocytidine and ethenoadenosine) and on reaction of cytidine with 1′-methylthiaminium salts dipyrimido[1...

ChemInform Abstract: An Improved Synthesis of N4‐Aminocytidine.

AbstractThe title compound (III), a mutagenic nucleoside, is prepared by reaction of cytidine (I) with hydrazine (II) under the conditions outlined in the reaction scheme.

An improved synthesis of N4-aminocytidine.

N4-Aminocytidine, a mutagenic nucleoside, was obtained as crystals on reaction of cytidine with a reagent mixture consisting of hydrazine, bisulfite and phosphate buffer at pH 7. The product can be precipitated directly from the reaction mixture, and therefore the procedure is suitable for preparing N4-aminocytidine in large quantities.

N4-aminocytidine, a nucleoside analog that has an exceptionally high mutagenic activity.

The reaction of cytidine with hydrazine to give N4-aminocytidine was greatly promoted by addition of a less-than-stoichiometric amount of bisulfite, and the product was isolated in a good yield. N4-Aminocytidine was strongly mutagenic to bacteria (Salmonella typhimurium TA100 and TA1535, and E. coli WP2 uvrA) and to phage (phi X174 am3). The activity did not require the presence of mammalian microsomal fraction in the system. The mutagenic potency of N4-aminocytidine in these systems was two orders of magnitude greater than that of N4-amino-2'-deoxycytidine, and more than two orders of magnitude greater than that of N4-hydroxycytidine. The greater activity of the riboside than the deoxyriboside was ascribed to the lack of deoxycytidine kinase in these cells. This compound may be useful as a powerful mutagen to induce a transition mutation in microorganisms.

Reaction of cytidine with semicarbazide in the presence of bisulfite. A rapid modification specific for single-stranded polynucleotide.

Semicarbazide reacted rapidly with 5,6-dihydrocytidine-6-sulfonate, which was formed from cytidine by addition of bisulfite across the 5,6-double bond. The transaminated product, 5,6-dihydro-4-semicarbazido-2-ketotopyrimidine-6-sulfonate ribofuranoside, was identified by comparison with that formed by treatment of 4-semicarbazido-2-ketopyrimidine ribofuranoside with bisulfite. The progress of the transamination was monitored spectrophotometrically by use of a strong absorbance of the product in alkali. The reaction between cytidine and the semicarbazide-bisulfite mixture was optimal at pH 4.5. Complete transformation of cytidine into the product required only 5 min with the use of 3M semicarbazide-1M sodium bisulfite, pH 5.0, at the reaction temperature 37 degrees C. The product was stable in unbuffered solution but in phosphate buffers it underwent elimination of bisulfite to give 4-semicarbazido-2-ketopyrimidine ribofuranoside. The rate of the elimination at pH 7.0 and 37 degrees C increased proportionally with the increase of the phosphate concentration. Complete elimination was obtained by treatment with 1 M sodium phosphate for 2 h. When heat-denatured calf-thymus DNA was treated with 3 M semicarbazide-1 M bisulfite at 37 degrees C and pH 5.0 the transamination of reactive cytosine residues was completed by 10 min of incubation. At 20 degrees C, it required 85 min of incubation. Cytosine residues in native DNA did not react at all even by prolonged incubations. The modified DNA samples were further treated with a phosphate buffer at pH 7, producing 4-semicarbazido-2-ketopyrimidine residues in the DNA. Analysis of the base compositions of these samples by perchloric acid hydrolysis showed that the modification was selective to cytosine, which had been expected from studies with monomers. It also showed that the reactive cytosine residues in the denatured DNA, constitute about 80% of the total cytosine, which was consistent with the view that heat-denatured DNA still contains a considerable amount of secondary structure. The semicarbazide-bisulfite modification is expected to be a sensitive method to locate cytosine residues in single-stranded regions of polynucleotides.

Reactions of 2-acyloxyisobutyryl halides with nucleosides. 6. Synthesis and biological evaluation of some 3'-acyl derivatives of 2,2'-anhydro-1-(.beta.-D-arabinofuranosyl)cytosine hydrochloride

The reactions of cytidine with 22 different 2-O-acyloxyisobutyril chlorides lead to the isolation of the corresponding 2,2'-anhydro-1-(3'-O-acyl-beta-d-arabinofuranosyl)cytosine hydrochlorides 9. These compounds, which all show cytotoxicity against HeLa cells in tissue culture, have been examined for antiviral and antileukemic activity. Activity against DNA viruses (vaccinia and Herpes) in tissue culture is maximal in compounds containing acyl groups with 8--12 carbon atoms. Activity against L1210 leukemia in mice varies markedly according to the length of the acyl groups, and high activities were observed in the case of long-chain (C16--C22) esters. The reaction between cytidine and O-acetylsalicyloyl chloride provides an alternate route for the synthesis of 3'-O-Ac cycloC hydrochloride.

Reactions of cytidine with 7-bromomethylbenz(a)anthracene, benzyl bromide, and p-methoxybenzyl bromide. Ratio of amino to 3 substitution.

Reactions of cytidine with 7-bromomethylbenz(a)anthracene, benzyl bromide, and p-methoxybenzyl bromide. Ratio of amino to 3 substitution.

ChemInform Abstract: A NOVEL METHOD FOR THE SYNTHESIS OF CYTIDINE DIPHOSPHATE‐CHOLINE

Cytidine diphosphate choline was synthesized by the reaction of cytidine 5′-phosphate with 2-bromoethyl 2′-(chloromethyl)-4′-nitrophenyl phosphate in the presence of trimethylamine.

A Novel Method for the Synthesis of Cytidine Diphosphate-Choline

Abstract Cytidine diphosphate choline was synthesized by the reaction of cytidine 5′-phosphate with 2-bromoethyl 2′-(chloromethyl)-4′-nitrophenyl phosphate in the presence of trimethylamine.

Reaction of cytidine with ethylating agents.

Reaction of cytidine with ethylating agents.

The reaction of hydroxylamine with 4-thiouridine

Hydroxylamine reacts with 4-thiouridine very easily in neutral aqueous solutions to give N 4- hydroxycytidine . A quantitative conversion of 4-thiouridine into the product was obtained by 21-h incubation in 0.025 M hydroxylamine at pH 7.0 and 37 °C. The reaction was not affected by addition of EDTA or hydroquinone. The optimum pH of the reaction was about 7.2, indicating the mechanism to be a nucleophilic attack of hydroxylamine free base towards C-4 of the undissociated 4-thiouridine. The apparent pseudo-first order rate constant was proportional to the concentration of hydroxylamine, the apparent rate constant in 1 M hydroxylamine at pH 7.0 and 37 °C being 0.292 min −1. The alteration of 4-thiouridine was much faster than the known reactions of cytidine and uridine with hydroxylamine both at pH 7.0 and at pH 5.5. Bis-(4-thiouridine) disulfide also reacts with hydroxylamine to give N 4- hydroxycytidine as a final and sole product. The conversion was complete within 15 min at pH 7.0 and 37 °C, using 1 M hydroxylamine solution. Methoxyamine also reacts with 4-thiouridine in a similar manner, the apparent rate constant at pH 7.0 and 37 °C in 1 M methoxyamine being 0.009 min −1.

A new chemical probe for single-stranded RNA

The reaction of cytidine with sodium bisulfite and methylamine gives transamination to N 4-methylcytidine and deamination to uridine. At pH 7.4, however, transamination takes place exclusively. This reaction was applied to yeast RNA (7 days, 37°) and afforded the specific conversion of 54% of the cytidine residues to N 4-methylcytidine residues. The same procedure converted up to 95% of the cytidines of polycytidylic acid to N 4-methylcytidines. A complex of polycytidylic acid with polyinosinic acid was, however, totally resistant to the reaction. Because of its cytosine specificity, single-strand specificity, possibility of use of radioactive methylamine, mild conditions, and ease of analysis, this reaction seems ideally suited as a chemical probe for single-stranded regions in RNA.

The chemical basis for the mutagenicity of hydroxylamine and methoxyamine

1. The reaction of cytidine with hydroxylamine in poly(C) and TMV RNA causes the formation of predominantly N-4-hydroxycytidine at early stages of the reaction. At pH 4 the reaction proceeds much more slowly than at pH 6 and with greater preference for the primary substitution at N-4 over the C-5,6 addition reaction. 2. The reaction of cytidine in monomeric and polymeric form with methoxyamine shows a similar preference for the N-4 substitution reaction, but it proceeds more rapidly at pH 4 than at pH 5.5. 3. The number of cytosines transformed to the N-4 hydroxylated or methoxylated state in TMV RNA at early stages of the reaction is in approximate agreement with the number of inactivating events observable by bioassay. 4. The predominance of the N-4 substitution reaction, coupled with the recognition that this reaction tends to transform the binding properties of C to those of U, renders it very probable that the mutagenicity of these reagents is also to be attributed to this reaction.

The modification of nucleoties and nucleosides: XI. A modification of cytosine derivatives with succinic anhydride

Acylation of cytosine derivatives with succinic anhydride in an 85% aqueous dimethyl sulfoxide solution was investigated. 1.1. When I-methylcytosine was reacted with succinic anhydride, two products were obtained as crystals. The major product was identified as N4-(β-carboxypropionyl)-1-methylcytosine and the minor one as N4,N4′-succinyl-bis-(1-methylcytosine).2.2. The reaction of cytidine with succinic anhydride gave two main products. The major product was isolated as crystals and identified as N4-(β-carboxypropionyl)cytidine. On incubation at 38° for 3h, the N-acylated nucleoside was practically stable at pH 6 to 8, while 50% of the nucleoside was deacylated at pH 2.5 and 10.5.3.3. N4-(β-Carboxypropionyl)cytidine 2′,3′-cylic phosphate was prepared by acylation of cytidine 2′,3′-cyclic phosphate, resulting in the introduction of an anionic group into the base moiety. It is noted that this compound was much more rapidly hydrolyzed by pancreatic ribonuclease A than N4-acetylcytidine 2′,3′-cyclic phosphate, the rate of hydrolysis being almost identical with that of cytidine 2′,3′-cyclic phosphate.4.4. The possibility of an application of this reaction to the selective modification of particular nucleoside residues in nucleic acids is discussed.