Protonated cytidine

Thymidine phosphate and cytidine phosphate do not phosphoresce in a rigid ethylene glycol-water glass at 77°K109 when directly excited, but thymidine which has lost its proton at Nx does have a triplet which phosphoresces with a decay time of 0.50 sec at high pH uridine also fluoresces with a similar decay time. The quenching of purine fluorescence and appearance of T" fluorescence in UV-irradiated DNA and poly dAT (as well as U" fluorescence in poly rAU) was attributed by Rahn et al.109 to proton transfer from thymine to adenine. This quenches adenine fluorescence and enhances thymine fluorescence. 

X-ray crystal results on cytosine itself10 -13 (anhydrous and monohydrate), its complexes with different partners,15-20 cytidine,21 and cytidine 2, 3 -cyclic phosphate22 all indicate its existence in the lactam-amine form (2). In a number of crystals the cytosine ring is protonated, invariably at N-3.23-32 In cytosine 5-acetic acid33 half of the molecules... 

Differences in the IR spectra of cytosine, cytidine, and cytidylic acid were considered by Angell.41 This author concluded that the cytidylic acids exist in the solid state in a zwitterion form with one of the hydrogen atoms from the phosphate group at N-3 of the cytosine ring (cf.refs.53,54). Previously Miles 44 postulated that the form 7a rather than the form 8 was the structure of cytidine in acid solution. This conclusion is essentially the same as that of Tsuboi et al.i7 who proposed form 7b for the protonated form of cytidine, as 7a and 7b are two canonical forms of the same tautomer (7). 

In general, the problem of tautomerism in nucleic acid bases has been approached by comparing the IR spectra of several isoelectronic model compounds. The model corresponding to the cytosine tautomers 4 or 5 have not yet been investigated. The IR spectroscopy studies cannot therefore definitely rule out these tautomers. It seems, however, that they do rule out form 6 for cytosine and cytidine and indicate that the dominant tautomer of the compounds in aqueous solution is the lactam-amino form 2, and that the protonated cations have the structure 7. 

Pyrimidines and purines, which contain the -NH2 group, are weakly basic. The cationic protonated conjugate acid forms of cytidine, adenosine, and guanosine have pKa values of 4.2,3.5, and 2.7, respectively. Similar values are observed for the 5 -nucleotides. In these compounds it is not the -NH2 group that binds the proton but an adjacent nitrogen atom in the ring (Eq. 5-3). 

Comparison of the spectrum of uridine with data of 4-thiouridine, 2,4-dithiouridine, thymidine, 4-thiothymidine, cytidine and 2-thiocytidine allows the assignment of all carbon resonances of these pyrimidine moieties. Further confirmation of these assignments is obtained from the proton off-resonance decoupled 13C-spectra of these nucleosides, as has been demonstrated for thymidine The pyrimidine CH3 and CH resonances of the proton broadband-decoupled spectrum are split into a quartet and a doublet, respectively, in the proton off-resonance spectrum, and can thus be easily assigned. 

With cytidine cyclic phosphate as a substrate the step 2 process alone is measured. The procedure of Crook et al. (395) relies on the increase in absorbance at 286 nm which occurs on hydrolysis of the cyclic phosphate ring. Cleavage of the ring also results in proton release which is the basis of the titrimetric procedure described by Stark and Stein (132). The assays employing C>p are usually carried out in 0.35 M NaCl. The ionic strength for maximum activity is much higher for this substrate than for RNA. A brief summary of the above procedures is given by Klee (396). 

Sugiyama H, Fujimoto K, Saito I (1995) Stereospecific 1,2-hydride shift in ribonolactone formation in the photoreaction of 2 -iododeooxyuridine. J Am Chem Soc 117 2945-2946 Sugiyama H, Fujimoto K, Saito I (1997) Preferential Cl hydrogen abstraction by a uracilyl radical in a DNA-RNA hybrid. Tetrahedron Lett 38 8057-8060 Svoboda P, Harms-Ringdahl M (1999) Protection or sensitation by thiols or ascorbate in irradiated solutions of DNA or deoxyguanosine. Radiat Res 151 605-616 Symons MCR(1990) ESR spectra for protonated thymine and cytidine radical anions their relevance to irradiated DNA. Int J Radiat Biol 58 93-96... 

Fig. 3. Stereoviews of the central trinucleotide d(CA G ) d(CTG) of the A G -platinated duplex d(CTCA G CCTC)d(GAGGCTGAG) (2). Top energy-minimized model in which the thymine conserves its Watson-Crick imino hydrogen bond with A, whereas the amino hydrogen bond is disrupted. Bottom energy-minimized model in which the thymine retains its Watson-Crick amino hydrogen bond with A, whereas the imino hydrogen bond is disrupted. The H(2 ) proton of the cytidine of the platinated strand lies in the shielding cone of the five-membered ring of A in both models, which accounts for the strong upfield shift observed for its NMR signal (reproduced from [70] with permission).

In uridine and cytidine (Scheme 10.4), deuterium exchange occurs at C-5 after initial attack at C-6 by a nucleophile (D20 or OD ). When that nucleophile is able to abstract the C-6 proton to form a delocalized anion,... 

High-frequency, p.m.r. spectroscopy has proved to be an extremely important tool in studies of the structures, conformations, and inter-and intra-molecular base-stacking interactions of nucleosides and nucleotides. The temperature dependence of the chemical shift of the base proton at position 6 (H-6) has been studied at 220 MHz for uridine (36), cytidine (38), and a number of their mono- (37, 39) and... 

So far, two different mechanisms of single strand break formation based on adiabatically stable anions have been proposed. The first mechanism, suggested by the Leszczynski group, assumes the formation of stable anions of 3 - and 5 -phosphates of thymidine and cytidine in which the cleavage of the C-O bond take place via the SN2-type process. The second reaction sequence, proposed by us, starts from the electron induced BFPT process followed by the second electron attachment to the pyrimidine nucleobase radical, intramolecular proton transfer, and the C-O bond dissociation. In both mechanisms the bottleneck step is associated with very low kinetic barrier which enables the SSB formation to be completed in a time period much shorter than that required for the assay of damage. 

---