Cytosine formation

An enzyme nucleophilic group, most likely SH in the context of the Pro-Cys doublet conserved in all known m C-MTases, is believed to assist the chemical catalysis by attacking the C-6 of the substrate cytosine. Formation of a covalent intermediate apparently renders the otherwise inert C-5 electron rich and facilitates the electrophilic transfer of a methyl group from AdoMet. In the absence of AdoMet, M Hhal catalyzes the exchange of the 5-H of the substrate cytosine for protons of water at a rate about sevenfold higher than the rate of methylation. 

The direct formation of dipyrimidin-5-yl sulfides occurs on treatment of appropriate 5-unsubstituted pyrimidine substrates with sulfur mono- or di-chloride. Thus, reaction of uracil (83 R = H) with sulfur monochloride in boiling formic acid gives diuracil-5-yl sulfide in good yield sulfur dichloride gives a poor yield. Simple derivatives of uracil and barbituric acid undergo similar reactions but not cytosine, isocytosine, 2,4-bismethylthiopyrimidine or pyrimidine-4,6-dione (59). The mechanism is unknown (72AJC2275). 

The reaction mechanism of the DNA (cytosine-5)-methyltransferase-catalyzed cytosine methylation was investigated at the MP2 and DFT levels [98JA12895]. This system has been modeled by 1-methylcytosine 117, methylthiolate, and trimethylsulfonium. The cytosine methylation is initiated by an attack of the anionic methylthiolate at Cg of the cytosine ring (Scheme 77). The formation of the methylthiolate adduct 118 of the neutral 117 was found to be endothermic in the gas phase and in solution. However, the MP2 and DFT results differ... 

The molyhdopterin cofactor, as found in different enzymes, may be present either as the nucleoside monophosphate or in the dinucleotide form. In some cases the molybdenum atom binds one single cofactor molecule, while in others, two pterin cofactors coordinate the metal. Molyhdopterin cytosine dinucleotide (MCD) is found in AORs from sulfate reducers, and molyhdopterin adenine dinucleotide and molyb-dopterin hypoxanthine dinucleotide were reported for other enzymes (205). The first structural evidence for binding of the dithiolene group of the pterin tricyclic system to molybdenum was shown for the AOR from Pyrococcus furiosus and D. gigas (199). In the latter, one molyb-dopterin cytosine dinucleotide (MCD) is used for molybdenum ligation. Two molecules of MGD are present in the formate dehydrogenase and nitrate reductase. 

Schimanski, A., Ereisinger, E., Erxleben, A. and Lippert, B. (1998) Interactions between [AuX4] (X = Cl, CN) and cytosine and guanine model nucleobases salt formation with (hemi-) protonated bases, coordination, and oxidative degradation of guanine. Inorganica Chimica Acta, 283, 223. 

P212121 Z — 8 Dx= 1.57 R = 0.085 for 1,743 intensities. The two independent molecules have similar conformations. The glycosyl dispositions are anti (90.1°, 91.2°), and the D-ribosyl groups are 3T4 (24.0°, 34.1° 15.6°, 35.5°). The exocyclic, C-4 -C-5 bond orientations are gauche+ (63.1°, 53.8°). The orientation of the methyl groups in both molecules is such that it is directed away from the imidazole moiety of the base, that is, the 0-6-C-7 bond is trans to the C-5-C-6 bond this arrangement constitutes an obstacle to formation of Watson-Crick hydrogen-bonds to the complementary base cytosine. In molecule A, 0-6 and C-7 are displaced from the purine plane by 79 and 87 pm, and, in molecule B, by 49 and 16 pm. The bases are stacked. 

Other degradation products of the cytosine moiety were isolated and characterized. These include 5-hydroxy-2 -deoxycytidine (5-OHdCyd) (22) and 5-hydroxy-2 -deoxyuridine (5-OHdUrd) (23) that are produced from dehydration reactions of 5,6-dihydroxy-5,6-dihydro-2 -deoxycytidine (20) and 5,6-dihydroxy-5,6-dihydro-2 -deoxyuridine (21), respectively. MQ-photosen-sitized oxidation of dCyd also results in the formation of six minor nucleoside photoproducts, which include the two trans diastereomers of AT-(2-de-oxy-/j-D-eryf/iro-pentofuranosyl)-l-carbamoyl-4 5-dihydroxy-imidazolidin-2-one, h/1-(2-deoxy-J8-D-crythro-pentofuranosyl)-N4-ureidocarboxylic acid and the a and [5 anomers of N-(2-deoxy-D-eryfhro-pentosyl)-biuret [32, 53]. In contrast, formation of the latter compounds predominates in OH radical-mediated oxidation of the pyrimidine ring of dCyd, which involves preferential addition of OH radicals at C-5 followed by intramolecular cyclization of 6-hydroperoxy-5-hydroxy-5,6-dihydro-2 -deoxycytidine and subsequent generation of the 4,6-endoperoxides [53]. 

Fig. 8 Long range charge transport between dppz complexes of Ru(III) and an artificial base, methyl indole, in DNA. The methyl indole is paired opposite cytosine and separated from the intercalating oxidant by distances up to 37 A. In all assemblies, the rate constant for methyl indole formation was found to be coincident with the diffusion-controlled generation of Ru(III) (> 107 s )> indicating that charge transport is not rate limiting over this distance regime...

Many years later, Schwartz (Schwartz and Goverde, 1982 Voet and Schwartz, 1983) discovered that the synthesis of adenine via polymerisation of HCN can be accelerated by adding formaldehyde and other aldehydes. Reactions in the gas phase (nitrogen/methane atmosphere) promoted by electrical discharges led to the formation of cyanoacetylene in relatively good yields the latter reacts with urea to give various products, including cytosine (Sanchez et al., 1968). 

The same problem, the stability of the nucleobases, was taken up by Levi and Miller (1998). They wanted to show that a synthesis of these compounds at high temperatures is unrealistic, and thus they took a critical look at the high temperature biogenesis theories, such as the formation of biomolecules at hydrothermal vents (see Sect. 7.2). The half-life of adenine and guanine at 373 K is about a year, that of uracil about 12 years and of the labile cytosine only 19 days. Such temperatures could have easily been reached when planetoids impacted the primeval ocean. 

Reports of the synthesis of cytosine from cyanoacetylene (or its hydrolysis product cyanoacetaldehyde) with cyanate, cyanogens or urea show that these substances react faster with nucleophilic compounds to give side products than to give the required main product. In addition, the formation of cytosine requires concentrations which are unrealistic in prebiotic environments. 

The interstrand cross-link also induces DNA bending.72 X-ray and NMR studies on this adduct show that platinum is located in the minor groove and the cytosines of the d(GC) base pair involved in interstrand cross-link formation are flipped out of the helix stack and a localized Z-form DNA is observed.83-85 This is a highly unusual structure and very distorting—implications for differential repair of the two adducts have been addressed. Alternatively, the interstrand cross-link of the antitumor inactive trans-DDP is formed between a guanine (G) and its complementary cytosine (C) on the same base p a i r.86,87/ nms- D D P is sterically incapable of producing 1,2-intrastrand adducts and this feature has been cited as a dominant structural reason for its lack of antitumor efficacy. It is clear that the structural distortions induced on the DNA are very different and likely to induce distinctly different biological consequences. 

Two types of addition to pyrimidine bases appear to exist. The first, the formation of pyrimidine photohydrates, has been the subject of a detailed review.251 Results suggest that two reactive species may be involved in the photohydration of 1,3-dimethyluracil.252 A recent example of this type of addition is to be found in 6-azacytosine (308) which forms a photohydration product (309) analogous to that found in cytosine.253 The second type of addition proceeds via radical intermediates and is illustrated by the addition of propan-2-ol to the trimethylcytosine 310 to give the alcohol 311 and the dihydro derivative 312.254 The same adduct is formed by a di-tert-butyl peroxide-initiated free radical reaction. Numerous other photoreactions involving the formation by hydrogen abstraction of hydroxyalkyl radicals and their subsequent addition to heterocycles have been reported. Systems studied include 3-aminopyrido[4,3-c]us-triazine,255 02,2 -anhydrouri-dine,256 and sym-triazolo[4,3-fe]pyridazine.257 The photoaddition of alcohols to purines is also a well-documented transformation. The stereospecific addition of methanol to the purine 313, for example, is an important step in the synthesis of coformycin.258 These reactions are frequently more... 

Figure 1.45 Reaction of bisulfite with cytosine bases is an important route of derivatization. It can lead to uracil formation or, in the presence of an amine (or hydrazide) containing compound, transamination can occur, resulting in covalent modification.

Figure 27.2 Treatment of cytosine bases with bisulfite results in a multi-step deamination reaction, ultimately leading to uracil formation.

Since the site of modification on cytosine bases is at a hydrogen bonding position in double helix formation, the degree of bisulfite derivatization should be carefully controlled. Reaction conditions such as pH, diamine concentration, and incubation time and temperature affect the yield and type of products formed during the transamination process. At low concentrations of diamine, deamination and uracil formation dramatically exceed transamination. At high concentrations of diamine (3M), transamination can approach 100 percent yield (Draper and Gold, 1980). Ideally, only about 30-40 bases should be modified per 1,000 bases to assure hybridization ability after derivatization. 

Biotin-dUTP derivatives are formed by modification of the C-5 position of uridine. This location is not involved in hydrogen bonding activity with complementary DNA strands, thus hybridization efficiency is not immediately compromised. By contrast, biotin-dCTP or biotin-dATP derivatives involve modification of the bases at the N-4 position of cytosine and the N-6 position of adenine, locations directly involved in hydrogen bond formation with complementary bases. Thus, DNA biotinylation through the use of modified deoxynucleoside triphosphates to be incorporated into existing DNA strands may result in better activity of the probe if dUTP is used over dATP or dCTP. 

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