Density Functionaltheory Study On The Mechanisms Of DNA Damage Induced By Nitrosoureas

Nitrosoureas are an important family of anticancer agents, which are mainly usedin the clinical treatment of brain tumors, leukemia and Hodgkin lymphoma since theyhave the ability to cross the blood-brain barrier. However, it has been demonstratedthat nitrosoureas could induce the secondary tumor, which greatly hinders theapplication and development of nitrosoureas as anticancer agents. It is generallybelieved that the carcinogenesis of nitrosoureas are related to their abilities to inducethe DNA damage, therefore the researches on the mechanism of DNA damageinduced by nitrosoureas are significant for the design of new anticancer drugs withhigh activity and low toxicity. In this work, the influence of alkyl group ofnitrosoureas on the mechanism of DNA damage was comprehensively investigatedusing Density Functional Theory （DFT） method from three aspects: the mechanism ofDNA alkylation by nitrosoureas; the DNA nucleophilic sites alkylation selectivity ofnitrosoureas and the Single Strand Scission （SSS） reaction induced by DNA alkylationproducts. Some energy parameters for the quantitative calculation of the DNAalkylation damage and the SSS damage were proposed in this work, which provides atheoretical foundation for the further investigation on the quantitative analysis of thecarcinogenic mechanism of nitrosoureas. All calculations in this work were carriedout with the hybrid functional Becke, Three-parameter, Lee-Yang-Parr （B3LYP）. Fullgeometry optimizations were performed for all structures on6-31++G（d,p） basis set.The Conductor-like Polarizable Continuum Model （CPCM） was employed for allcalculations to simulate the solvent effect of water solution in the cellularenvironment.（1） Research on the mechanism of DNA alkylation by nitrosoureasNitrosoureas have been proved to decompose directly into （E）-diazoniumhydroxide and isocyanate without metabolism.（E）-diazonium hydroxide and itsdecomposition products, diazonium cation and carbocation, are considered as DNAalkylating agents. In this work, the N7atom in guanine was selected as the model ofDNA nucleophilic site. Four alkylnitrosoureas, including Methylnitrosourea,Ethylnitrosourea, iso-Propylnitrosourea and Benzylnitrosourea, which havesignificantly different carcinogenicity in animal experiments, were selected as themodel of nitrosoureas.The four reaction steps, such as the O-N bond cleavagereactions of the （E）-diazonium hydroxides, the N_α-C_αbond cleavage reactions of thediazonium cations and alkylation reactions of the （E）-diazonium hydroxides and thediazonium cations, were investigated to analyze the influence of alkyl group structureon the pattern of ultimate alkyalting agent and the alkyaltion reactivity of nitrsoureas.The results are summarized as follows:1. The activation energy of （E）-diazonium hydroxide and diazonium cation in the alkylation reaction decreases with the increaseof steric hinderance of the substituent on C_αatom. This is because that the biggersteric hinderance effect of the substituent has, the greater stabilizing effect on C_αatomprovided by substituent group will be.2. All the activation energies in the alkylationreactions of the four （E）-diazonium hydroxides are higher than those in thecorresponding O-N bond cleavage reactions, which suggests that the diazoniumcation should be the necessary intermediate in the DNA alkylation reactions by thefour nitrosoureas.3. Along with the increase of steric hinderance of alkyl group, theactivation energies in the alkylation reactions of diazonium cations decrease to thosein the N_α-C_αbond cleavage reactions. All the activation energies of methyl-, ethyl-,and iso-propyl diazonium cations in the alkylation reactions are lower than those inthe N_α-C_αbond cleavage reactions, except the benzyl diazonium ion. Therefore, theultimate alkylating agents of methyl-, ethyl-, and iso-propylnitrosourea are diazoniumcations, as well as that of benzylnitrosourea is nitrogen-separated ion pair.（2） Research on the DNA nucleophilic sites alkylation selectivity of nitrosoureasFirstly, n-propyl diazonium cation was selected as the model compound ofnitrosourea for the research on the deoxyguanosine nucleophilic sites alkylationselectivity of nitrosourea. It was found that the calculated relative energy differences（ETS-COMP） between the transition states and the complexes in the alkylationreactions of the nucleophilic sites, can be used for the quantitative prediction ofalkylation selectivity. Subsequently, the alkyaltion selectivities of methyl-, ethyl-andn-propyl diazonium cations were compared. The result shows that the alkylationselectivity decreased with the increase of the steric hindrance of alkyl group, whichindicates that the steric hindrance of alkyl group is a key factor influencing thealkylation selectivity. Finally, n-propyl diazonium cation and GC_αbase pair wereselected as model compounds to explore the effect of DNA duplex structure on thealkylation selectivity. The results exhibit that the complementary cytosine in GCbasepair can enhance the alkylation reactivities of N7, N₃and N₂atoms of deoxyguanosine,but decrease the reactivity of O6atom. The results also show that the alkyaltionreactions of N₃and N₂atoms, which occur in the direction perpendicular to the purinering, can be hindered by the adjacent base in the base stacking direction of DNAduplex structure.（3） Research on the SSS reaction induced by DNA alkylation productsN7-alkyl-3-methyldeoxyguanosine and phosphate triester were selected as themodel compounds of DNA alkylation products on N7atom of guanine and phosphategroup, respectively, to investigate the mechanism of SSS induced by DNA alkylation.The results of the research are concluded as follows:1. In the SSS reaction induced byN7R-3MedG, the calculated activation energy of depurination reaction is markedly lower than that of the reaction on apurinic site leading to the further SSS, which is therate-controlling step. Because the reactivity of the rate-controlling step is not affectedby alkyl substituent, the reaction rate of SSS induced by various N7R-3MedG shouldbe the same.2. There is a significant propotional linear relationship between theelectron-withdrawing ability （*） of alkyl substituent and the reactivity ofdepurination reaction, which illustrates that the stronger electron-withdrawing abilityof alkyl substituent is, the more easily will the apurinic site generate.3. In the SSSreactions induced by phosphate triesters, the mechanisms of the reactions can beaffected by alkyl substituent. For the general phosphate triesters, the SSS reactions areinitiated by the attacking of water molecule on the central phosphorus atom,generating an acyclic Pentacoordinate Phosphorus Intermediate （PPI）, which thenhydrolyze and lead to the SSS subsequently. When the alkyl substituent of phosphatetriester is hydroxylated, the substituted hydroxyl group can attack on the phosphorusatom instead of the water molecule, resulting in the formation of a cyclic PPI.4. Theformation reactivity of PPI is a key affecting factor for the reaction rate of SSS. Theformation reactivity of the cyclic PPI is significantly higher than that of the acyclicone, which indicates that the SSS reaction of the hydroxyl-substituted phosphatetriester should be more rapid than other alkyl phosphate triesters. Moreover, theformation of PPI can be catalyzed by hydroxide anion, which expresses that the SSSreaction rate in alkaline condition should be faster than that in neutral condition.