Dinucleoside monophosphate

A method for the direct spectrophotometric determination of dinucleoside monophosphates has been developed which relies on changes in u.v. absorbance after enzymic hydrolysis. - Hydrolytic fission of the dinucleoside monophosphate with a phosphodiesterase causes a change in the u.v. absorbance of the solution allowing the 5 -nucleoside to be estimated. Addition of a phosphomonoesterase to the hydrolysate causes a further change in u.v. absorbance, allowing the 3-nucleoside to be estimated. 

In ref. [123], 1-triisopropylbenzenesulfonyl- or mesitylenesulfonyl-5-(pyridine-2-yl)-tetrazole (VIII) was successfully used as a coupling agent for the synthesis of protected di- and trinucleotides. In comparison with the sulfonic acid tetrazolides these are able to achieve a stereoselective synthesis of dinucleoside monophosphate aryl esters. 311 1241... 

Interestingly, it was recently shown that the one-electron oxidation reaction promoted by photoexcited menadione (MQ) gives rise to A -formylade-nine (52) and A -acetyladenine (53) residues (Scheme 5) in several dinucleoside monophosphates including d(ApA), d(CpA) and d(ApC) [92]. 

The HPLC-MS/MS assay was also successfully applied to the measurement of UV-induced dimeric pyrimidine photoproducts [123, 124]. The latter lesions were released from DNA as modified dinucleoside monophosphates due to resistance of the intra-dimer phosphodiester group to the exonuclease activity during the hydrolysis step [125, 126]. The hydrolyzed photoproducts exhibit mass spectrometry and chromatographic features that allow simultaneous quantification of the three main classes of photolesions, namely cyclobutane dimers, (6-4) photoproducts, and Dewar valence isomers, for each of the four possible bipyrimidine sequences. It may be added that these analyses are coupled to UV detection of normal nucleosides in order to correct for the amount of DNA in the sample and obtain a precise ratio of oxidized bases or dimeric photoproducts to normal nucleosides. 

In this regard, recent partial MM relaxation calculation on DNA and RNA intercalation models merit special attention. Kollman (309) performed preliminary studies on the interaction of cationic intercalators like ethidium bromide (118) with two base-paired dinucleoside monophosphates, CpG (119), and GpC. Their MM scheme minimizes 14 torsional (only for 119) and 12 intermolecular... 

Receptor models 71a and 71b for dinucleotides (dinucleoside monophosphates) have been developed out of 69 by replacing the naphthoyl residue, which is not involved in any host-guest interactions in the complexes, by a second carbazolyl cleft. The adenine selectivity of the two pincers was reflected in single extraction experiments dissolved in CH2CI2,71a removes one full equivalent of both adenylyl(3 -+ 5 )adenosine (ApA) and 2 -deoxyadenylyl(3 ->5 )-2 -deoxyadenosine [d(ApA)] 72 out of aqueous solutions of guest (8-fold excess) but only half the amount of d(ApG) [103]. 

Most of the properties of RNase Ti are summarized in Tables II and IV. It is a very acidic protein, active between pH 4 and 8.5 it is most active at pH 7.5 for RNA digestion (12) and at pH 7.2 for the hydrolysis of guanosine 2, 3 -cyclic phosphate (18). The purified enzyme possesses a specific activity of about 1.6 X 10 units/mg of protein. The molecular activity (standard units/jumole enzyme) has not been determined for the cleavage of a definite dinucleoside monophosphate such as GpC or for the hydrolysis of guanosine 2, 3 -cyclic phosphate. 

The base specificity has been extensively investigated by the 3 -terminal analysis of digestion products of RNA and polyribonucleotides and by the studies on the susceptibility of dinucleoside monophosphates or nucleoside 2, 3 -cyclic phosphates to the enzyme. The results are summarized in Table V (24-87). From the base specificity study, the es-... 

Imazawa et al. (97) also measured the Km and Fma values at pH 6.0 with various dinucleoside monophosphates, XpY (Table X) Km values increased in the order of A C < G < U for both X and Y, while the Fmax values decreased in the order of U > G > C > A for X and of C > G > U > A for Y. When either X or Y in XpY was adenylyl residue, their Km values were smaller than those of others. This finding also supports the consideration mentioned above. 

Both 1-methylApU and glyoxalGpU are more resistant to RNase U2 than ApU and GpU (SO). Furthermore, it was found that RNase U2 does not attack the dinucleoside monophosphates in which N-l of gua-... 

The observation that the final digestion products of RNA by the enzyme are four nucleoside 2, 3 -cyclic phosphates led Ukita and coworkers (121) to utilize the enzyme for the synthesis of various dinucleoside monophosphates. Indeed they have successfully synthesized UpU, CpU, ApU, GpU, UpC, CpC, ApC, and GpC in good yields (from 20 to 75% based on initial nucleoside 2, 3 -cyclic phosphates) by the reaction... 

Wechter (34) and Richards et al. (35) showed that venom exonuclease is capable of hydrolyzing dinucleoside monophosphates w ith one or both nucleosides containing arabinose. Even though only four different sugars have been tested, it would appear that venom exonuclease is totally blind to sugar, at least in the qualitative sense. Other enzymes capable of hydrolyzing DNA and RNA are incapable of hydrolyzing derivatives of arabinose, e.g., micrococcal nuclease (SO). 

Patterson LK, Bansal KM, Bogan G, Infante GA, Fendler EJ, Fendler JH (1972) Micellar effects on CI2 reactivity. Reactions with surfactants and pyrimidines. J Am Chem Soc 94 9028-9032 Paul CR, Belfi CA, Arakali AV, Box HC (1987a) Radiation damage to dinucleoside monophosphates mediated versus direct damage. Int J Radiat Biol 51 103-114 Paul CR, Arakali AV, Wallace JC, McReynolds J, Box HC (1987b) Radiation chemistry of 2 deoxycytidy-lyl-(3 5 ) -2 deoxyguanosine and its sequence isomer in N20- and 02-saturated solutions. Radiat Res 112 464-477... 

The double helices thus constructed are tested for geometric acceptability using semiempirical potential energy functions that reflect the extent of intrastrand base stacking and interstrand base pairing in a miniature dinucleoside monophosphate duplex of the same conformation. As described elsewhere (22), the base stacking potential Vs is chosen to display a minimum at a close to ideal base separation distance of 3.3 /( Energies within... 

Sequence Specificity Optical studies on the binding of intercalative drugs to dinucleoside monophosphates demonstrated that actinomycin exhibits a purine(3 -5 )pyrimidine specificity (57-60) while ethidium exhibits a pyrimidine(3 -5 )purine (61-63)... 

Whereas each adenine residue in a dinucleoside monophosphate undergoes reduction, the level and ease of reduction are apparently radically reduced with an increase in length of the chain. It is claimed that, for longer oligonucleotides, the maximal number of adenine residues reduced is three. In an analogous study on the mechanism... 

Many extensive studies110-126 at 60, 100, or 220 MHz of dinucleotides and dinucleoside monophosphates have confirmed quite conclusively that the magnetic anisotropy of one base moiety influences the chemical shifts of the protons of the other base and, therefore, that these compounds exist in one or more folded conformations, in which the rings of the bases are stacked in parallel planes. Intra- and inter-molecular base-stacking both appear to be extremely common,103,122-126 and the chemical shifts experienced by H-l, H-2, and H-8 of adenylyl-(3 - 5 )-adenosine (44) on inter-... 

The pyrimidine nucleobases have the highest quantum yields for photoreactivity, with thymine uracil > cytosine. The purine nucleobases have much lower quantum yields for photochemistry, but can be quite reactive in the presence of oxygen. As can be seen from Figure 9-3, thymine forms primarily cyclobutyl photodimers (ToT) via a [2ir + 2tt cycloaddition, with the cis-syn photodimer most prevalent in DNA. This is the lesion which is found most often in DNA and has been directly-linked to the suntan response in humans [65]. A [2Tr + 2Tr] cycloaddition reaction between the double bond in thymine and the carbonyl or the imino of an adjacent pyrimidine nucleobase can eventually yield the pyrimidine pyrimidinone [6 1]-photoproduct via spontaneous rearrangement of the initially formed oxetane or azetidine. This photoproduct has a much lower quantum yield than the photodimer in both dinucleoside monophosphates and in DNA. Finally, thymine can also form the photohydrate via photocatalytic addition of water across the C5 = C6 bond. 

The fluorescence decay of dinucleoside monophosphates containing the modified base ethanoadenine (EAD) (see Table 4) and the natural bases guanine and uracil have been investigated by Kubota et al. [31c]. The fluorescence quantum yields are lower for the conjugates than for the nucleotide of EAD (<0.1 versus 0.52). The observation of multiple exponential fluorescence decay was attributed to multiple conformations. A small component with a decay time similar to that of unmodified EAD was attributed to an extended conformation and two shorter components to loosely stacked folded conformations. While the mechanism of fluorescence quenching was not addressed, an electron transfer mechanism in which singlet EAD can serve as an electron acceptor (with guanine) or electron donor (with uracil) is... 

The separation of a, / , y and 5-tocopherols on LH-20 in chloroform was reported [205], separation being related to structure of the to-copherols and independent of molecular size. The correlations between the differences in conformation and elution behaviour of 2, 5 - and 3, 5 -dinucleoside monophosphates on LH-20 has been studied [206]. Separation of the dinucleoside monophosphates was apparently related to conformation which affected their adsorption to the LH-20. The different degrees of interaction of biogenic amines, aromatic amino acids, and phenylphrine with Bio-Gel P-2 were used to separate these species [207]. 

Papers which report the synthesis of dinucleoside monophosphates or their analogues as model studies for oligonucleotide synthesis are covered in section 4. 

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