Quantum Molecular Modeling of the Interaction Between Guanine and Alkylating Agents - 1 - Sulfur Mustard

Abstract

Interaction between Guanine and the episulfonium form of Sulfur mustard (HD) was studied using the ab initio LCAO-MO method at the HF/6–31G level. The alkylation mechanism on guanine-N7 was analyzed by using a supermolecular modeling. Our stereostructural results associated with the molecular electrostatic potentials and HOMO-LUMO properties, show that in vacuum the alkylation of the N7 of guanine by HD in the agressive episulfonium form is a direct process without transition state and of which the pathway is determined.     

Kinetics of DNA alkylation, depurination and hydrolysis of anti diol epoxide of benzo(a)pyrene and the effect of cadmium on DNA alkylation

Anti benzo[a]pyrene diol epoxide (BPDE) alkylates guanines of DNA at N7 in the major groove and at the exocyclic amino group in the minor groove. In this report we investigated the rates of BPDE hydrolysis, DNA alkylation and subsequent depurination of BPDE-adducted pBR322 DNA fragment using polyacrylamide gel electrophoresis. Preincubation studies showed that it hydrolyzed completely in triethanolamine buffer in <2 min. The depurination kinetics showed that a fraction of the N7 alkylated guanine depurinated rapidly; however a significant amount of N7 guanine alkylation remained stable to spontaneous depurination over a 4-h period. Similar results were obtained for the hydrolysis and alkylation rates of syn isomer but it required nearly 500 times more concentration to induce similar levels of N7 guanine alkylation. Cadmium ion strongly inhibited the N7 guanine alkylation of both isomers. But the minor groove alkylation was not affected as demonstrated by postlabeling assay which confirmed the presence of heat-and cadmium-stable minor groove adducts in BPDE-treated calf thymus DNA. Based on these and our earlier findings, we propose a mechanism for the synergistic effect of cadmium in chemically induced carcinogenesis.     

Informational parameters of an exact DNA base sequence

A series of hierarchical chemical reactivity calculations have been performed to elucidate the alkylation properties of a methyldiazonium ion toward DNA base sites. Both MINDO/3 and CNDO/2 approximate methods have been employed. For the isolated bases the O6 of guanine is predicted to be the most reactive site. This prediction may also be relevant to single-stranded DNA chains containing guanine. For base-pairs, the N7 and O6 sites on guanine are about equally favored for alkylation. The previous study of aziridinium ion alkylation gave about the same results with N7 guanine modestly favored as the preferred site of alkylation for base-pairs. In composite we conclude that N7 guanine and/or O6 guanine will be the preferred sites for alkylation by a methyldiazonium ion but cannot distinguish between these two in terms of chemical specificity.     

DNA sequence selectivity of guanine-N7 alkylation by nitrogen mustards.

Nitrogen mustards alkylate DNA primarily at the N7 position of guanine. Using an approach analogous to that of the Maxam-Gilbert procedure for DNA sequence analysis, we have examined the relative frequencies of alkylation for a number of nitrogen mustards at different guanine-N7 sites on a DNA fragment of known sequence. Most nitrogen mustards were found to have similar patterns of alkylation, with the sites of greatest alkylation being runs of contiguous guanines, and relatively weak alkylation at isolated guanines. Uracil mustard and quinacrine mustard, however, were found to have uniquely enhanced reaction with at least some 5'-PyGCC-3' and 5'-GT-3' sequences, respectively. In addition, quinacrine mustard showed a greater reaction at runs of contiguous guanines than did other nitrogen mustards, whereas uracil mustard showed little preference for these sequences. A comparison of the sequence-dependent variations of molecular electrostatic potential at the N7-position of guanine with the sequence dependent variations of alkylation intensity for mechlorethamine and L-phenylalanine mustard showed a good correlation in some regions of the DNA, but not others. It is concluded that electrostatic interactions may contribute strongly to the reaction rates of cationic compounds such as the reactive aziridinium species of nitrogen mustards, but that other sequence selectivities can be introduced in different nitrogen mustard derivatives.     

Deprotecton Studies of Ethanoic Acid 5,6,7,9-Tetrahydro-3-/2-(Acetoxyemxy Iethyl-5-0-Acetyl-9-OXO-3H-IMIDAZO/1,2a/ Purine-6,7-Dioyol Ester (1) and Related Glyoxal-Guaniee Adduts

Abstract

Selective deprotection of fully protected 1 was achieved via sodium methoxide or cyanide catalyzed removal of acetate groups. The glyoxal protecting group diminished nucleophylisity of N7 regardless of the blocking of N1 position in guanine.     

Molecular Dynamics Simulations of Chlorambucil/DNA Adducts. A Structural Basis for the 5′ -GNC Interstrand DNA Crosslink Formed by Nitrogen Mustards

Abstract

The alkylation of DNA by chlorambucil has been studied using a computational approach. Molecular dynamics simulations were performed on the fully solvated non-covalent complex, two monoadducts and a crosslinked diadduct of chlorambucil with the d(CGG₃G₂CGC).- d(GCG₁CCCG) duplex, in which the N7 atoms of G₁, G₂ and G₃ are potential alkylation sites. The results provide a structural basis for the preference of nitrogen mustards to crosslink DNA duplexes at a 5′-GNC site (a 1,3 crosslink, G₁ -G₃) rather than at a 5′-GC sites (a 1,2 crosslink, G₁ -G₂).

In the non-covalent complex simulation the drug reoriented from a non-interstrand crosslinking location to a position favorable for G₁ -G₃ diadduct formation. It proved possible to construct a G₁ -G₃ diadduct from a structure from the non-covalent simulation, and continue the molecular dynamics calculation without further disruption of the DNA structure. A crosslinked diadduct developed with four B_II conformations on the 3′ side of each alkylated guanine and of their respective complementary cytosine. In the first monoadduct simulation the starting point was the same DNA conformation used in the crosslinked diadduct simulation with alkylation at G₁. In this simulation the DNA deformation was reduced, with the helix returning to a more canonical form. A second monoadduct simulation was started from a canonical DNA conformation alkylated at G₃. Here, no significant motion towards a potential crosslinking conformation occurred. Collectively, the results suggest that crosslink formation is dependent upon the drug orientation prior to alkylation and the required deformation of the DNA to permit 1,3 crosslinking can largely be achieved in the non-covalent complex.     

Effect of ionic strength and cationic DNA affinity binders on the DNA sequence selective alkylation of guanine N7-positions by nitrogen mustards

Large variations in alkylation intensities exist among guanines in a DNA sequence following treatment with chemotherapeutic alkylating agents such as nitrogen mustards, and the substituent attached to the reactive group can impose a distinct sequence preference for reaction. In order to understand further the structural and electrostatic factors which determine the sequence selectivity of alkylation reactions, the effect of increased ionic strength, the intercalator ethidium bromide, AT-specific minor groove binders distamycin A and netropsin, and the polyamine spermine on guanine N7-alkylation by L-phenylalanine mustard (L-Pam), uracil mustard (UM), and quinacrine mustard (QM) was investigated with a modification of the guanine-specific chemical cleavage technique for DNA sequencing. For L-Pam and UM, increased ionic strength and the cationic DNA affinity binders dose dependently inhibited the alkylation. QM alkylation was less inhibited by salt (100 mM NaCl), ethidium (10 microM), and spermine (10 microM). Distamycin A and netropsin (100 microM) gave an enhancement of overall QM alkylation. More interestingly, the pattern of guanine N7-alkylation was qualitatively altered by ethidium bromide, distamycin A, and netropsin. The result differed with both the nitrogen mustard (L-Pam less than UM less than QM) and the cationic agent used. The effect, which resulted in both enhancement and suppression of alkylation sites, was most striking in the case of netropsin and distamycin A, which differed from each other. DNA footprinting indicated that selective binding to AT sequences in the minor groove of DNA can have long-range effects on the alkylation pattern of DNA in the major groove.     

Tautomerism and Regioselectivity in Ribosylation of Guanine

Abstract

N²-acetyl- and 9, N²-diacetylguanines were subjected to reaction with tetraacetylribose in the presence of p-toluenesulfonic acid. Unlike the ribosylation of diacetylguanine, which gives 7-riboside as a kinetic product, the reaction of monoacetylguanine produces directly a mixture of 7- and 9-ribosides. This reflects N⁷H ? N⁹H tautomerism of the guanine substrate and supports the hypothesis that only the unsubstituted nitrogens of the imidazolium portion of guanine (either N7 or N9) react directly with a sugar cation.     

Crystal structure of 9-amino-N-[2-(4-morpholinyl)ethyl]-4-acridinecarboxamide bound to d(CGTACG)2: implications for structure–activity relationships of acridinecarboxamide topoisomerase poisons

The structure of the complex formed between d(CGTACG)₂ and 9-amino-N-[2-(4-morpholinyl)ethyl]-4-acridinecarboxamide, an inactive derivative of the antitumour agents N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) and 9-amino-DACA, has been solved to a resolution of 1.8 Å using X-ray crystallography. The complex crystallises in the space group P6₄ and the final structure has an overall R factor of 21.9%. A drug molecule intercalates between each of the CpG dinucleotide steps with its side chain lying in the major groove, and its protonated morpholino nitrogen partially occupying positions close to the N7 and O6 atoms of guanine G2. The morpholino group is disordered, the major conformer adopting a twisted boat conformation that makes van der Waals contact with the O4 oxygen of thymine T3. A water molecule forms bridging hydrogen bonds between the 4-carboxamide NH and the phosphate group of guanine G2. Sugar rings are found in alternating C3′-exo/C2′-endo conformations except for cytosine C1 which is C3′-endo. Intercalation perturbs helix winding throughout the hexanucleotide compared with B-DNA, steps 1 and 2 being unwound by 10 and 8°, respectively, while the central TpA step is overwound by 11°. An additional drug molecule lies at the end of each DNA helix linking it to the next duplex to form a continuously stacked structure. The protonated morpholino nitrogen of this ‘end-stacked’ drug hydrogen bonds to the N7 atom of guanine G6, and its conformationally disordered morpholino ring forms a C–H···O hydrogen bond with the guanine O6 oxygen. In both drug molecules the 4-carboxamide group is internally hydrogen bonded to the protonated N10 atom of the acridine ring. We discuss our findings with respect to the potential role played by the interaction of the drug side chain and the topoisomerase II protein in the poisoning of topoisomerase activity by the acridinecarboxamides.     

High-performance liquid chromatographic determination of N-[2- (hydroxyethyl)-N-(2-(7-guaninyl)ethyl)]methylamine, a reaction product between nitrogen mustard and DNA and its application to biological samples

Nitrogen mustard (HN2) is a bifunctional alkylating agent which is thought to cause cytotoxicity by covalently binding to DNA. Most studies to date have looked at qualitatively determining the presence of DNA–HN2 adducts from reactions with native DNA. The adduct which is predominately formed in these reactions is N-[2-(hydroxyethyl)-N-(2-(7-guaninyl)ethyl]methylamine (N7G). A simple and sensitive reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of N7G from DNA using ultraviolet detection is described. DNA samples having been exposed to HN2 treatment were hydrolyzed and preseparated from high-molecular-mass material by filtration using a molecular mass cut-off of 3000. The mobile phase consisted of methanol–26 mM ammonium formate, pH 6.5 (24:76, v/v). N7G, as well as the internal standard, methoxyphenol, were separated within 30 min. The recovery of N7G after hydrolysis of the DNA reaction product was quantitative and limits of detection and quantification of 10 and 20 ng/ml, respectively, were calculated. The method was validated in DNA–HN2 dose response experiments. The N7G reaction product appears to be the first reaction product formed at lower ratios of HN2/DNA but its production plateaus at higher ratios of HN2/DNA probably due to increased formation of hitherto unknown adducts. The method is simple and sensitive and for this reason, may be suited for the determination of DNA/HN2 reaction products.     

Mapping of Altromycin B-DNA Adduct at Nucleotide Resolution in the Human Genomic DNA by Ligation-mediated PCR

The ligation-mediated PCR was used to map DNA alkylation sites induced by altromycin B at nucleotide resolution in genomic DNA purified from cultured human colon carcinoma. Altromycin B, one of the pluramycin group of antitumor antibiotics, is characterized as intercalator with the added ability to alkylate N7 guanine. DNA adducts formed in genomic DNA were cleaved into DNA strand breaks by hot piperidine treatment, and fragments containing ligatable breaks were then amplified in a single-sided, ligation-mediated PCR. The alkylation sites observed in exon 9 of the p53 gene revealed that the most high reactivity sites for altromycin B were found to be N7 of guanine in a 5-AG* sequence. Determination of the DNA alkylation sites in naked radiolabeled plasmid DNA also showed that altromycin B preferred N7 of guanine in a 5-AG* sequence. Thus, it can be concluded that the sequence selective DNA adduct formation induced by the intercalating alkylator, altromycin B, in genomic DNA is similar to that observed in naked plasmid DNA.     

An Efficient Synthesis Of Acyclic N7- and N9-Adenine Nucleosides Via Alkylation With Secondary Carbon Electrophiles to Introduce Versatile Functional Groups At the C-1 Position of Acyclic Moiety

The introduction of versatile functional groups, allyl and ester, at the C-1 position of the acyclic chain in acyclic adenine nucleosides was achieved for the first time directly by alkylation of adenine and N⁶-protected adenine. Thus, the C-1′-substituted N⁹-adenine acyclic nucleoside, adenine-9-yl-pent-4-enoic acid ethyl ester (11), was prepared by direct alkylation of adenine with 2-bromopent-4-enoic acid ethyl ester (6), while the corresponding N⁷-regioisomer, 2-[6, (dimethylaminomethyleneamino)-purin-7-yl]-pent-4-enoic acid ethyl ester (10), was obtained in one step by the coupling of N,N-dimethyl-N′- (9H-purin-6-yl)-formamidine (9) with 2-bromopent-4-enoic acid ethyl ester (6). The functional groups, ester and allyl, were converted to the desired hydroxymethyl and hydroxyethyl groups, and subsequently to phosphonomethyl derivatives and corresponding pyrophosphorylphosphonates.     

模拟氮沉降对天山云杉细根分解及其养分释放的影响

采用野外模拟试验,设计4种氮处理——对照(不施氮,CK)、低氮(施氮5kg·hm-2·a-1,LN)、中氮(施氮10kg·hm-2·a-1,MN)、高氮(施氮15kg·hm-2·a-1,HN),研究氮沉降对天山云杉细根分解及养分释放的影响。结果表明:(1)不同氮处理分解2年后天山云杉细根残留率依次为74.044%(HN)、71.967%(MN)、68.156%(CK)、61.933%(LN),且差异显著。(2)天山云杉的细根月分解速率在试验前期不同氮处理下规律不明显;而在试验后期呈现为对照中氮低氮高氮。(3)4种氮处理下天山云杉细根分解50%需要的时间依次为3.31年(LN)、3.67年(CK)、4.28年(MN)、4.64年(HN),分解95%需要的时间依次为14.39年(LN)、15.93年(CK)、18.58年(MN)和20.17年(HN)。(4)天山云杉细根C元素迁移模式总体表现为直接释放,N元氮为富集-释放模式,残留率呈现波动式下降趋势。(5)不同氮处理下天山云杉细根分解率与C元素浓度间均呈线性负相关关系;对照和低氮处理下,天山云杉细根分解率与N元素浓度间均为线性负相关关系,中氮和高氮处理下,细根分解率随N元素浓度的增加呈先增加后降低的趋势。     

Synthesis of Peptide Nucleic Acid Monomers

Abstract

The chemical synthesis of peptide nucleic acid (PNA) monomers is described using Fmoc (backbone), anisoyl (cytosine, adenine), 4-tert-butylbenzoyl (cytosine) and isobutyryl/diphenylcarbamoyl (guanine) protecting group combinations. For the guanine monomer the alkylation was realized both in a Mitsunobu [DIAD, triphenylphosphine or (4-dimethylaminophenyl)diphenylphosphine, tert-butyl glycolate] and in a low-temperature, sodium-hydride mediated alkylation (tert-butyl bromoacetate) to give the N⁹ -substituted derivative.     

模拟N沉降下三种林分土壤营养动态分析

通过模拟N沉降实验,设置对照(CK,0 g N m~(-2)a~(-1));低氮(LN,5 g N m~(-2)a~(-1));中氮(MN,10 g N m~(-2)a~(-1));高氮(HN,15 g N m~(-2)a~(-1))4种N处理,以NH_4NO_3为外源N来研究福建省三明格氏栲自然保护区内板栗人工林、观光木人工林及米槠天然林0—10 cm土层养分变化动态。结果表明:N沉降会使板栗人工林土壤显著酸化,P含量降低,在一些时间段内,中高水平的N沉降会显著降低有机C、全N和速效N含量,中或低水平N沉降会显著降低土壤全P和速效P含量,而从第6个月起只有LN处理会显著降低土壤K含量。N沉降总体上会不同程度地提高观光木人工林土壤p H值、有机C、全N和速效N含量,有时影响会达显著或极显著水平;比较而言,LN和HN处理更会造成土壤全P的富集,而MN处理对速效P的影响更显著;LN和HN处理也会显著增加K含量,且以LN处理的效果更稳定。总体上N沉降量越大米槠天然林土壤酸化越显著;N沉降会使其有机C和速效P量显著波动;实验期间,HN处理会显著降低土壤全N和速效N量,而LN与MN处理则会使速效N和K含量增加;在4种处理下全P含量会呈相同趋势波动,差异不显著。     

Aflatoxin B1 and DNA Adducts. Proposed Model for Surface Noncovalent and Covalent Complexes with N(7) of Guanine. II.

Abstract

An alternate model for surface noncovalent and surface covalent binding of aflatoxin B₁ to N(7) of guanine in DNA is proposed. This model considers the out-of-plane motions of C(8) of aflatoxin B₁ in those interactions.

The covalent intercalated fit of aflatoxin B₁ into DNA arises from steric adjustments made by DNA at the covalent intercalation site as well as local strain in the bond angles about N(7) of guanine and C(8) of aflatoxin B₁. The bond angle about N(7) deviates modestly from the sp² value toward the sp³ value.

This study suggests that the surface covalent aflatoxin B₁ -DNA complex serves only a minor role in aflatoxin's precarcinogenic interaction with DNA and is a likely correctable error.     

模拟氮沉降对华西雨屏区天然常绿阔叶林凋落叶分解过程微生物生物量的影响

为进一步深化氮沉降对凋落物分解影响的研究,2016年3月—2017年3月,在华西雨屏区天然常绿阔叶林内,用凋落叶分解袋法研究了模拟氮沉降对凋落叶分解过程中微生物生物量碳(MBC)、微生物生物量氮(MBN)和微生物生物量磷(MBP)的影响。实验设置了对照(0 g N m~(-2)a~(-1))、低氮(5 g N m~(-2)a~(-1))、中氮(15 g Nm~(-2)a~(-1))和高氮沉降(30 g N m~(-2)a~(-1)) 4个处理。结果表明:低氮和中氮处理显著增加了凋落叶分解过程中的MBC和MBN,以低氮处理增加幅度最高;低氮和中氮处理对凋落叶分解过程中的MBP影响不显著;高氮处理显著降低了分解过程中的MBC、MBN和MBP。随模拟氮沉降量的递增,凋落叶分解过程中微生物生物量碳氮比逐渐减少,微生物生物量碳磷比呈现先增加后下降的趋势。研究结果说明,氮沉降影响了华西雨屏区天然常绿阔叶林凋落物分解过程中微生物生物量,进而改变了凋落物的分解过程。     

Stereoselective Approach to the Z-Isomers of Methylenecyclopropane Analogues of Nucleosides: A New Synthesis of Antiviral Synguanol

Stereoselective synthesis of antiviral synguanol (1) is described. Reaction of 6-benzyloxy-2-(dimethylaminomethyleneamino)purine (10) with ethyl (cis,trans)-2-chloro-2-(chloromethyl) cyclopropane-1-carboxylate (2c) under the conditions of alkylation-elimination gave (Z)-6- benzyloxy-2-formylamino-9-[(2-carbethoxycyclopropylidene)methyl]purine (11) but no E,N⁹-isomer. Minor amounts of (Z)-6-benzyloxy-2-formylamino-7-[(2-carbethoxy-cyclopropylidene)methyl]purine (13) were also obtained. Hydrolysis of compounds 11 and 13 in 80% acetic acid afforded (Z)-9-[2-(carbethoxycyclopropylidene)methyl]guanine (14) and (Z)-7-[2-(carbethoxy- cyclopropylidene)methyl]guanine (15). Reduction of 14 furnished synguanol (1). Reaction of N⁴-acetylcytosine (7) with ester 2c led to (Z,E)-1-(2-carbethoxycyclopropropylidenemethyl)cytosine (8, Z/E ratio 6.1:1). Basicity of purine base, lower reactivity of alkylation intermediates as well as interaction of the purine N³ or cytosine O² atoms with the carbonyl group of ester moiety seem to be essential for the observed high stereoselectivity of the alkylation-elimination. The Z-selectivity is interpreted in terms of E1cB mechanism leading to a transitory “cyclic” cyclopropenes which undergo a cyclopropene-methylenecyclopropane rearrangement.     

降水和氮沉降增加对草地土壤酶活性的影响

为探究降水和氮沉降增加对草地生态系统土壤酶活性的影响,于2014年生长季在内蒙古温带典型羊草草原开展了野外原位控制实验。试验共设置降水(对照,W0,自然降水;W15,增加15%的年均降水量)、施氮(对照,CK,0 kg N hm~(-2)a~(-1);低氮,LN,25 kg N hm~(-2)a~(-1);中氮,MN,50 kg N hm~(-2)a~(-1);高氮,HN,100 kg N hm~(-2)a~(-1))及其交互作用等8个不同的处理水平来模拟降水和氮沉降增加的全球变化情景,分别定量探讨了不同水、氮添加条件下草地表层土壤中与氮循环相关的蛋白酶,脲酶,硝酸还原酶,亚硝酸还原酶活性的月动态变化及其与土壤理化性质之间的相关性。研究结果表明:在自然降水条件下,不同施氮水平蛋白酶、脲酶和硝酸还原酶活性无显著差异,亚硝酸还原酶活性相比于对照显著降低;在增加降水条件下,不同施氮水平对蛋白酶和硝酸还原酶活性未产生显著性影响,高氮水平显著降低脲酶和亚硝酸还原酶活性。不同施氮水平是否添加降水对亚硝酸还原酶活性无影响,而增添降水使低氮处理的蛋白酶活性和中、高氮处理水平的硝酸还原酶活性增加、高氮处理的脲酶活性降低。降水在影响蛋白酶和硝酸还原酶活性方面具有主效应,氮沉降在影响亚硝酸还原酶活性方面具有主效应,而降水和施氮处理未表现出明显地交互作用。土壤亚硝酸还原酶活性与土壤碳氮比和NH~+_4-N含量极显著正相关,与NO-3-N含量显著正相关。     

Regioselective N1-Alkylation of Guanosine Derivatives Protected at N 2 by an N,N-Dialkyl Amidine Group

Abstract

Under Mitsunobu reaction conditions or in the presence of eiectrophilic alkylating reagents, alkylation of guanosine derivatives protected by an N,N-dialkyl amidine at the exocyclic amino group occurs selectively on nitrogen N ¹ of the purine base.