Cytidine 5 - , alkaline

Facile cleavage under alkaline conditions has also been observed for ribitol192 and glycerol200 esters of cytidine 5 -pyrophosphate. Cytidine 5 -(3,6-dideoxy-a-D-arahtno-hexopyranosyl pyrophosphate)... 

The foregoing proposed mechanism of reduction of cytosine, cytidine and their derivatives in acid and moderately alkaline media are further supported by the results of a recent study, by means of a.c. polarography, of the orientation of cytosine and cytidine at the mercury electrode. It was shown87) that, whereas at acid pH (about 5) both of these are adsorbed via the positive charge on the ring N3, so that the N3 = C4 bond is adjacent to the electrode surface, the orientation in alkaline medium (pH 9) is modified so that the C5 = C6 bond is located at the surface. This is consistent with the proposed mechanisms of reduction. 

As far as the reactivity of the above-described Mannich bases is concerned, studies of prcxlucts 469 indicate a much better resistance to alkaline than to acid hydrolysis the nature of the nucleoside also plays a role, as the cytidine derivatives arc much more stable than the purine derivatives. Similar investigations have been madc into the deaminomethylation of Mannich bases of type 466. 

Lippke JA, Gordon LK, Brash DE, Haseltine WA. Distribution of UV light-induced damage in a defined sequence of human DNA detection of alkaline-sensitive lesions at pyrimidine nucleoside-cytidine sequences. Proc. Natl. Acad. Sci. U.S.A. 1981 78 3388-3392. 

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... 

The possibility that, during the alkaline hydrolysis of ribonucleic acid, tri-esters (18) - could be intermediates was ruled out on mechanistic groxmds."f > Furthermore, it was shown that the tri-ester, uridine 2 3 -(methyl phosphate) (18, R = CH3), synthesized by methylation of uridine 2 3 -cychc phosphate with diazomethane, is resistant to ribo-nuclease. f However, it has been observed that, when the benzyl ester of cytidylic acid 6 is partially hydrolyzed in acid, a significant proportion of the o isomer of this phosphoric diester is formed." ) In addition, the dinucleoside phosphates, such as adenylyl-(3 —>5 )-cytidine (14a), are also isomerized to the corresponding (2 - 5 )-dinucleoside phosphates by treatment with acid. " These studies suggest that cyclic triesters (or derivatives thereof) may be intermediates in isomerization of these phospho-diesters in acidic mediiun. 

It remains to be explained why treatment of the protected nucleotide (153) with ammonium hydroxide produces cytidine 2 -phosphate only, whereas similar treatment of (156) gives a mixture of the 2 - and 3 -isomers. A possible explanation may reside in differences in the lability of the benzoyl group on 0-2 or 0-3 of ribonucleosides, the 2 -benzoate being the more labile to ammonium hydroxide. Such a hypothesis is in accordance with the observation that partial debenzoylation of iV -benzoyl-tri-0-benzoylcytidine yields a mixture containing (152), but not (155). It had been noted that, in ribonucleosides, the 2 -hydroxyl group is more acidic than the 3 - and 5 -hydroxyl groups. It would be expected that 2 -0-acyl derivatives of ribonucleosides should be relatively more susceptible to alkaline hydrolysis than the 3 - or 5 -0-acyl derivatives. 

Under basic conditions (0.2 M barium hydroxide, 100°, 30 minutes), the nucleoside 3 5 -cycIic phosphates are converted into the 3 - and 5 -phosphates in the ratio of about 5 1. For cytidine 3 5 -cyclic phosphate, some concomitant deamination to the uridine nucleotide analogs is also observed (as noted with cytidine 3 -phosphate), so that the nucleotides obtained are similar to those found in the alkaline hydrolyzate of uridine 3 5 -cyclic phosphate. In addition, one of the nucleotides obtained in these hydrolyzates differs from the 3 - or 5 -phosphates in its ion-exchange chromatographic behavior. The presence of an unidentified nucleoside monophosphate in small proportion was also observed when 2 -deoxy-cytidine 3 5 -cyclic phosphate was hydrolyzed with barium hydroxide under similar conditions. 

Molar extinction coefficients for all pH values were calculated from h3Tpochromicities at 260 mpt upon alkaline hydrolysis using the following values of E25Q x 10 (pH 7) 15.4, 7.3, 11.6, and 9.9, for 2 (3 )-monophosphates of adenosine, cytidine, guanosine, and uridine, respectively. All absorbance measurements were made at 20°C. The values in the Note column, when given, are ain absorbance ratios relative to... 

In the one-pot system, reactions were supplemented with pyrophosphatase to improve conversion rates of the CSS-catalyzed step. Also, addition of alkaline phosphatase at the final stage of the conversions considerably improved the efficiency of sialoconjugate formation, probably by preventing SiaT inhibition by released CMP (Kj<0.3mM) [65]. Thus, it proved more favorable to uncouple the two forward steps by first setting up for the CSS-catalyzed activation, and then supplementing for the sialyltransfer [33, 34]. An alternative solution to the inhibition problem would be an in situ regeneration of CMP to cytidine diphosphate (CDP)/CTP which, however, requires two extra enzymatic steps [66]. 

Incubate a solution of polymer 25 (22 mg, approx 20 pmol of substrate), cytidine-5 -monophospho-N-acetyl neuraminic acid (CMP-NeuAc, Sigma C 8271) (15 mg, 24.4 xmol), a-(2—>3)-sialyltransferase (from rat liver, Sigma S 2769) (0.3 unit), bovine serum albumin (BSA, 4 mg), and calf intestinal alkaline phosphatase (CIAP, Sigma P 7923) (20 unit) in 50 mM sodium cacodylate buffer (pH 7.4, 2.0 mL) containing manganese(II) chloride (0.62 mg) and Triton CF-54 (10 xL) at 37°C for 72 h. 

It had previously been shown by a number of workers, and in a number of systems (138, 809-818) that the AMP moiety of labeled ATP was incorporated into soluble RNA, and that this incorporation occurred predominantly in a terminal position (as shown by recovery of the label, after alkaline hydrolysis of the RNA, as adenosine rather than as 2 - and 3 -AMP). Hecht and co-workers (193,813,814) showed that the incorporation of ATP was very small when preincubated RNA was used, but could be restored completely by either prior or simultaneous addition of C3rtidine triphosphate (CTP). This suggested that the terminal AMP had to be attached to a cytidine monophosphate (CMP) end group. This was further corroborated by the fact that in the absence of ATP, but not in its presence, most of the incorporated CTP was, itself, terminal. For every mole of ATP incorporated, there seemed to be two moles of CTP taken up. The terminal sequence of sRNA, therefore, emerged as adenylyl-5 3 -cytidylyl-5 -3 -cytidylyl-5 -3 -RNA (RNApCpCpA) and that the presence of this end group was, indeed, necessary for attachment of amino acids was shown by further detailed experiments of Hecht et al. (188). 

The binding of methyl p-D-ribofuranoside, cytidine and adenosine to a column bearing the substituted phenylboronic acid ligand (7) was examined. The nitro-group enhanced complexation between the boronic acid group and the cts-diol groups in these ribosides so that it occurred at less alkaline pH values and increased the speed of complexation - decomplexation. ... 

When the second substituent in the phosphate is another phosphate instead of an alkyl group, formation of the cyclic compound occurs under mild alkaline treatment. Thus, uridine diphosphate glucose yields uridine monophosphate and glucose cyclic i 2-phosphate and 5-phosphoribosyl P5U ophosphate gives ribose 5-phosphate cyclic i 2-phosphate and inorganic phosphate. Similar changes occur with cytidine diphosphate ribitol and cytidine diphosphate glycerol . 

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