Article Oxidative Repair of Pyrimidine Cyclobutane Dimers • by Nitrate Radicals (NO3 ): A Kinetic and Computational Study Tomas Haddad, Joses G. Nathanael , Jonathan M. White and Uta Wille * School of Chemistry, Bio21 Institute, The University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010, Australia; [email protected] (T.H.); [email protected] (J.G.N.); [email protected] (J.M.W.) * Correspondence: [email protected] Received: 14 April 2020; Accepted: 6 May 2020; Published: 9 May 2020 Abstract: Pyrimidine cyclobutane dimers are hazardous DNA lesions formed upon exposure of DNA to UV light, which can be repaired through oxidative electron transfer (ET). Laser flash photolysis and computational studies were performed to explore the role of configuration and constitution at the cyclobutane ring on the oxidative repair process, using the nitrate radical (NO3•) as oxidant. The rate coefficients of 8–280 107 M 1 s 1 in acetonitrile revealed a very high reactivity of the cyclobutane × − − dimers of N,N’-dimethylated uracil (DMU), thymine (DMT), and 6-methyluracil (DMU6-Me) towards NO3•, which likely proceeds via ET at N(1) as a major pathway. The overall rate of NO3• consumption was determined by (i) the redox potential, which was lower for the syn- than for the anti-configured dimers, and (ii) the accessibility of the reaction site for NO3•. In the trans dimers, both N(1) atoms could be approached from above and below the molecular plane, whereas in the cis dimers, only the convex side was readily accessible for NO3•. The higher reactivity of the DMT dimers compared with isomeric DMU dimers was due to the electron-donating methyl groups on the cyclobutane ring, which increased their susceptibility to oxidation. On the other hand, the approach of NO3• to the dimers of DMU6-Me was hindered by the methyl substituents adjacent to N(1), making these dimers the least reactive in this series. Keywords: pyrimidine cyclobutane dimers; nitrate radicals; kinetic studies; DFT calculations; oxidation 1. Introduction Exposure to UV light from the sun (λ = 180–400 nm) causes hazardous DNA lesions in the human body, which can lead to melanoma, basal cell, and squamous cell skin cancer [1]. The primary photo adducts implicated in skin cancer are pyrimidine cyclobutane dimers, which are formed through [2 + 2] cycloaddition between adjacent thymine nucleobases in the same oligonucleotide strand (Scheme1, path A). A minor pathway (ca. 25%) proceeds through [2 + 2] photocycloaddition between the alkene and carbonyl moiety to give an oxetane intermediate that undergoes ring-opening to the 6–4 adduct (Scheme1, path B) [ 2]. Cytosine forms the same type of dimers as thymine, but deamination occurs within only a few hours to yield the highly mutagenic uracil dimer (not shown) [3]. In general, pyrimidine cyclobutane dimers can exist in four isomeric structures with head-to-head (syn, s), head-to-tail (anti, a), as well as cis (c) and trans (t) configuration at the cyclobutane ring. However, because of geometrical constraints, only the cis,syn dimer is formed in DNA [4]. Such pyrimidine dimers can prevent DNA polymerases from transcribing DNA, thereby promoting the accumulation of errors [5]. Chemistry 2020, 2, 27; doi:10.3390/chemistry2020027 www.mdpi.com/journal/chemistry Chemistry 2020, 2, 27 1 of 16 Chemistry 2020, 2, x 2 O O O O R R R R HN NH [2+2] HN 55’NH 66’ path (A) O N N O O N N O H H Rb Rb Rb Rb cis,syn cyclobutane dimer O O O R R R HN HN OH HN [2+2] O O N O path (B) O N O N O N R R NH NH Rb Rb N Rb R R N O b N O oxetane (6-4) photoproduct R Rb b Scheme 1. Light-inducedLight-induced dimerization of pyrimidines. The The deoxyribose deoxyribose phosphate backbone of DNA is abbreviated as RRb;; thymine thymine (R (R = Me),Me), uracil uracil (R (R == H).H). The endogenousendogenous repair repair mechanism mechanism of damagedof dama DNAged DNA nucleotides nucleotides in placental in placental mammals mammals involves excisioninvolves andexcision replacement and replacement of the affected of the base affected or strand base clusteror strand that cluster is orchestrated that is orchestrated by a large by system a large of reparativesystem of enzymesreparative [6 ,enzymes7]. Other [6,7]. species, Other such specie as plantss, such or fish,as plants employ or the fish, enzyme employ photolyase the enzyme and itsphotolyase reducing and cofactor its reducing FADH (reducedcofactor flavineFADH adenine(reduced dinucleotide) flavine adenine to repair dinucleotide) dimer lesions to repair through dimer a reductivelesions through one-electron a reductive transfer one-electron (ET) process transfer [8,9]. (ET) process [8,9]. In additionaddition toto repairrepair by by excision excision or or enzymatic enzymatic reduction, reduction, pyrimidine pyrimidine cyclobutane cyclobutane dimers dimers can alsocan bealso oxidatively be oxidatively cleaved cleaved into theirinto their monomers, monomers, for example, for example, by oxidizing by oxidizing radicals radicals and radicaland radical ions ions [10]. Heelis[10]. Heelis et al. et showed al. showed that the that reaction the reaction of the of thymine the thymine cyclobutane cyclobutane dimer withdimer sulfate with sulfate radical radical anions (SO ) or hydroxyl radicals ( OH, this radical is commonly formed in vivo and can lead to oxidative anions4•− (SO4•−) or hydroxyl radicals• (•OH, this radical is commonly formed in vivo and can lead to damageoxidative in damage biomolecules in biomolecules [11]) led to [11]) the recoveryled to the of recovery the monomeric of the pyrimidinemonomeric andpyrimidine suggested and a mechanismsuggested a initiatedmechanism by theinitiated abstraction by the of abstraction a hydrogen of atoma hydrogen at C(6) atom (see below) at C(6) [ 12(see]. Inbelow) experiments [12]. In involvingexperiments the cisinvolving,syn-configured the cisN,,Nsyn’-dimethyluracil-configured N cyclobutane,N’-dimethyluracil dimer ( c,scyclobutane-DMU<>DMU), dimer Yan et(c, al.s- showedDMU<>DMU), that oxidative Yan et al. repair showed and formationthat oxidative of N repair,N’-dimethyluracil and formation (DMU) of N,N occurred’-dimethyluracil by reaction (DMU) with •occurredOH (possibly by reaction through with initial •OH hydrogen (possibly abstraction) through initial and hydrogen bromide radical abstraction) anions and (Br 2bromide•−, presumably radical through initial oxidation) but not with the azide radical (N )[13], which could be due to the only anions (Br2•−, presumably through initial oxidation) but not 3with• the azide radical (N3•) [13], which moderately high oxidizing ability of this radical (E0 (N /N ) = 1.35 V vs. NHE [14])). Comparable could be due to the only moderately high oxidizing ability3• 3 −of this radical (E0 (N3•/N3−) = 1.35 V vs. resultsNHE [14])). were Comparable obtained by Itoresults et al., were who obtained found in by the Ito reaction et al., who of the found C(5)–C(5 in the0) linked reaction thymine of the dimerC(5)– with OH, SO and N that repair only occurred in the case of the former two radicals [15]. C(5′) •linked thymine4•− dimer3• with •OH, SO4•− and N3• that repair only occurred in the case of the former two radicalsIn previous [15]. published and unpublished work, we performed product studies of the oxidative repairIn of previous pyrimidine published cyclobutane and dimersunpublished by nitrate work, radicals we performed (NO3•), which prod areuct importantstudies of oxidants the oxidative in the nighttime troposphere. NO was generated through photo-induced• ET from cerium(IV) ammonium repair of pyrimidine cyclobutane3• dimers by nitrate radicals (NO3 ), which are important oxidants in nitrate (CAN) at λ = 350 nm in acetonitrile• in the presence of various stereoisomeric N,N’-dimethylated the nighttime troposphere. NO3 was generated through photo-induced ET from cerium(IV) cyclobutaneammonium nitrate dimers (CAN) of uracil at λ (DMU), = 350 nm thymine in acetonitrile (DMT), in and the the presence unnatural of various pyrimidine stereoisomeric 6-methyl uracilN,N’- 6-Me (DMUdimethylated), according cyclobutane to Reaction dimers of (1) uracil [16,17 (DMU),]: thymine (DMT), and the unnatural pyrimidine 6- methyl uracil (DMU6-Me), according to reaction (1) [16,17]: (NH ) Ce(NO ) + hν (350 nm) NO • + (NH ) Ce(NO ) (1) 4 2 3 6 ! 3 4 2 3 5 (NH4)2Ce(NO3)6 + hν (350 nm) → NO3• + (NH4)2Ce(NO3)5 (1) Table1 shows the major products formed after a reaction time of two hours with a reactant ratio Table 1 shows the major products formed after a reaction time of two hours with a reactant ratio of [dimer]:[CAN] = 5. The data were obtained by gas chromatographic (GC) analysis of the reaction of [dimer]:[CAN] = 5. The data were obtained by gas chromatographic (GC) analysis of the reaction mixture from the relative peak areas [17] and, therefore, provided only a qualitative picture. In the mixture from the relative peak areas [17] and, therefore, provided only a qualitative picture. In the case of c,s-DMU<>DMU, the major products were the monomer DMU and the partially cleaved case of c,s-DMU<>DMU, the major products were the monomer DMU and the partially cleaved dimer dimer 1, whereas only small amounts of the intact dimer remained (entry 1). Cyclobutane cleavage 1, whereas only small amounts of the intact dimer remained (entry 1). Cyclobutane cleavage was was considerably less efficient with t,s-DMU<>DMU, where mainly 1 and smaller amounts of DMU considerably less efficient with t,s-DMU<>DMU, where mainly 1 and smaller amounts of DMU were were obtained (entry 2). Likewise, repair of c,a-DMU<>DMU, t,a-DMU<>DMU, c,s-DMT<>DMT, obtained (entry 2). Likewise, repair of c,a-DMU<>DMU, t,a-DMU<>DMU, c,s-DMT<>DMT, and c,a- and c,a-DMT<>DMT was also incomplete with c,a-DMT<>DMT being the least efficiently cleaved DMT<>DMT was also incomplete with c,a-DMT<>DMT being the least efficiently cleaved dimer under these conditions (entries 3–6).