Pyrimidine reductive dimerization

Whatever the reason may be behind the strict necessity to deprotonate the flavin donor, the reduced and deprotonated flavin was established in these model studies to be an efficient electron donor, able to reduce nucle-obases and oxetanes. In the model compounds 1 and 2 the pyrimidine dimer translates the electron transfer step into a rapidly detectable chemical cycloreversion reaction [47, 48], Incorporation of a flavin and of a cyclobutane pyrimidine dimer into DNA double strands was consequently performed in order to analyse the reductive electron transfer properties of DNA. 

Flavin-cyclobutane pyrimidine dimer and flavin-oxetane model compounds like 1-3 showed for the first time that a reduced and deprotonated flavin is a strong photo-reductant even outside a protein environment, able to transfer an extra electron to cyclobutane pyrimidine dimers and oxetanes. There then spontaneously perform either a [2n+2n cycloreversion or a retro-Paternd-Buchi reaction. In this sense, the model compounds mimic the electron transfer driven DNA repair process of CPD- and (6-4)-photolyases. 

There is always interest in the photochemistry of the pyrimidine nucleic acid bases and related simple pyrimidinones, due to its importance in genetic mutation. In addition to damaging DNA, photo-induced reactions may also repair the damage, as in the reduction, by FADH, of the thymine glycol 64 back to thymine <06JACS10934>. Another report related to repair of DNA involved a model study, by means of the linked dimer 65, of the involvement of tryptophan in the electron-transfer leading to reversion of thymine oxetane adducts <06OBC291>. 

Another type of ring contractions occurs when 2-phenylpyrimidines (117) are reduced in an aqueous alcoholic-acetate buffer. Whereas pyrimidines generally are reduced in a one-electron reduction to a dimer,1 2-phenylpyrimidines are reduced to 2-phenylpyrroles (118). The following scheme has been suggested173 [Eq. (86)]. 

The polarographic reduction of pyrimidine was discussed in Part I,1 and further details have been clarified.301,302 The first reduction is to a radical that can dimerize and accept another electron in a second step to a dihydro derivative this may at a more negative potential be reduced to a tetra-hydropyrimidine. 

An ab initio study on the structure and splitting of the uracil dimer anion radical (see Scheme 3-66 and keep R = H) gives preference to the one-step mechanism (Voityuk Roesch 1997). Anion radical anions of the pyrimidine dimers cleave with rate constants in excess of 106 sec 1 (Yeh Falvey 1991). However, the cyclobutyl dimer of a quinone, dithymoquinone, also cleaves upon single-electron reduction but much more slowly than the pyrimidine dimers (Robbins Falvey 1993). It is truly an unresolved issue as to why the anion radical cleavage depicted in Scheme 3-66 is so facile. Water participation can probably decrease the barrier of the cycloreversion on physiological conditions (Saettel Wiest 2001). 

We present here a brief account of the specific dimerization, and other related, reactions undergone by a variety of purine and pyrimidine derivatives, and a number of related compound s, during the course of their electrochemical reduction at the surface of a mercury electrode. A characteristic feature of these reactions is the transfer of an electron to the compound, accompanied, or preceded, by its protonation. The resultant free radicals, generated by a one-electron reduction process, rapidly dimerize to products in which each of the monomeric components possesses an additional electron and an additional proton, relative to the parent monomer1 7). (See Scheme 1)... 

Both the foregoing photoadducts exhibit three diffusion-controlled polarographic waves in aqueous medium in the pH range 1-12 92). The first two waves are due to successive one-electron additions, followed by the third, a two-electron reduction step. The pH-dependence of the initial one-electron reduction wave was found to be similar to that for pyrimidone-2. Following electrolysis, at the crest of wave I, of either of the photoadducts, the two one-electron waves disappeared and the reduction products exhibited UV absorption spectra with a band at 375 nm. A similar absorption band is exhibited following sodium borohydride reduction of Cyt(5-4)Pyo, leading to a product identified as 5-(4-pyrimidin-2-one)-3,6-dihydrocytosine. Mass spectroscopy revealed that the products of reduction on wave I were dimers consisting of two molecules of reduced photoadducts 92). 

In aprotic medium, on the other hand, pyrimidine gives a reversible diffusion-controlled le wave at a very negative potential, with formation of a radical anion which is deactivated via two pathways rapid formation of the anionic, probably 4,4 -, dimer, with a rate constant of 8 x 105 L mol-1 sec-1, and proton abstraction from residual water in the medium at a much lower rate constant, 7 L mol-1 sec-1 98). This is rapidly followed by a further le reduction to produce, ultimately, 3,4-dihydropyrimidine 98). In the presence of acid there is also a le reduction wave corresponding to formation of a free radical which, as in aqueous medium, dimerizes, most likely to 4,4 -Z w-(3,4-dihydropyrimidine). Examination of the mechanism of reduction in acetonitrile in the presence of acids supported the conclusion that reduction of pyrimidine in aqueous medium is preceded by its protonation98). 

It must, nonetheless, be emphasized that the products of reduction of pyrimidine have not been unequivocally identified, largely due to their instability in the presence of air (oxygen). Furthermore, the UV absorption spectra of the reduction products of waves I and II (kmax284 nm, smax 1.5 x 103) are suggestive of rapid conversion (proton-catalyzed hydration ) of the products, since both the dimer and the dihydro derivative possess a reduced system of aromatic bonds relative to the parent pyrimidine, as a result of which the UV absorption maximum should be shifted to the violet, whereas it is, in fact, shifted 44 nm to the red (from 240 nm to 284 nm) for both products. Of possible relevance to this is the fact that the reduced rings of 4-aminopyrimidine 102) and nicotinamide 103) undergo acidic hydration to form products absorbing at 280 to 290 nm. 

Following initial studies by Cavalieri Lowy 96), it was shown by Smith Elving74) that the polarographic behaviour of 2-aminopyrimidine in acid medium is similar to that for the parent pyrimidine, which exhibits three waves at the dropping mercury electrode 74). The initial le step involves formation of a free radical which dimerizes and, at the potential of the second le reduction step, 2-amino-3,4-dihydropyrimidine is formed. But, unlike pyrimidine, 2-aminopyrimidines do not undergo a second 2e reduction to tetrahydro derivatives. Wave III (pH 7-9) involves two electrons and two protons, and is due to the combined processes responsible for waves I and II at lower pH. Both Smith Elving 74), and Sugino 104>, found that reduction of 2-aminopyrimi-dine on a mercury electrode 74) and lead cathode 104) resulted in the formation of unstable products. 

Since 2-iminopyrimidine is formally similar, as regards electronic structure, to 2-oxo- and 2-thio- pyrimidine, it might be expected to undergo le reduction with the formation of a photodissociable dimer. It was, in fact, found that the fixed imino form,... 

The experimental observations on the mechanism(s) of electroreduction of 2-thio-pyrimidines have been interpreted on the basis of their electronic structures as calculated with the aid of the CNDO/2 and Huckel procedures l42). The energies of LUMO (lowest unoccupied molecular orbitals), calculated for pyrimidine and its 2-oxo-and 2-thioderivatives, were compared with the reduction half-wave potentials (Table V). These show that the presence of a carbonyl or thione substituent at C2 enhances the electron acceptor properties of the molecule, which are correlated with formation of a dimer susceptible to photooxidation. 

The photochemical conversion of the thiopyrimidine dimers to the parent thio-pyrimidines is most readily followed by the regeneration of the spectra of the latter (Fig. 6), as for the ketopyrimidine dihydrodimers (Fig. 2). The reaction may also be followed electrochemically by measurements of the reduction wave of the regenerated parent pyrimidine. The behaviour of the thiopyrimidine dihydrodimers differs essentially from that of the ketopyrimidine dihydrodimers 140) in that, under aerobic conditions, the rates of conversion of the former are considerably more rapid and exhibit an oxygen effect (Fig. 7a, b). The corresponding quantum yields under aerobic conditions are given in Table VI. Under anaerobic conditions the rates of photooxidation of Dt 3 decrease considerably (Fig. 7a), whereas that of D4 is barely affected and is somewhat more complex (Fig. 7b). 

The foregoing suggests that the structure of the reduction product is a dimer, as shown in Scheme 22, and supported by comparison with the dimer reduction product of pyrimidone-2 (Scheme 2), since the reduced pyrimidine rings in both dimers should be similar. The foregoing interpretation is confirmed by the results of earlier studies on 1 1 photoadducts of alcohols to purine 156157> the products were identified as substituted 1,6-dihydropurines, and the site of addition of the alcohols was readily established as C(6) on the basis of the 1H NMR spectra prior to, and following, selective deuteration at C(6) or C(8) 156,157). 

In neutral aqueous medium the dimer reduction product, but not the product of reduction on wave II, underwent photodissociation to the parent 2-oxopurine, with a quantum yield at 254 nm of 0.03, as compared to 0.1 for photodissociation of pyrimidine-2. 

In bacteria, flavin adenine dinucleotide (FAD) is the prosthetic group of the photolyases that catalyze reductive repair of light-induced pyrimidine dimers in DNA. Riboflavin is the light-emitting molecule in some bioluminescent fungi and bacteria, and is the precursor for synthesis of the dimethylbenzimidazole ring of vitamin B12 (Section 10.7.3). 

Cryptochromes in the human eye have a considerable sequence and structure homology with the photolyases, binding both methylene tetrahydrofolate and FAD. They have the same DNA binding pocket as photolyase, although they do not catalyze the reduction of DNA pyrimidine dimers. They are found in the nucleus of cells of the inner layer of the retina, behind the rods and cones involved in vision (Section 2.3.1), and absorb blue light, with maximum absorbance at 420 nm. 

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