RESUMO
Nucleoside 5'-triphosphate (dNTP) analogues in which the ß,γ-oxygen is mimicked by a CXY group (ß,γ-CXY-dNTPs) have provided information about DNA polymerase catalysis and fidelity. Definition of CXY stereochemistry is important to elucidate precise binding modes. We previously reported the (R)- and (S)-ß,γ-CHX-dGTP diastereomers (X = F, Cl), prepared via P,C-dimorpholinamide CHCl (6a, 6b) and CHF (7a, 7b) bisphosphonates (BPs) equipped with an (R)-mandelic acid as a chiral auxiliary, with final deprotection using H2/Pd. This method also affords the ß,γ-CHCl-dTTP (11a, 11b), ß,γ-CHF (12a, 12b), and ß,γ-CHCl (13a, 13b) dATP diastereomers as documented here, but the reductive deprotection step is not compatible with dCTP or the bromo substituent in ß,γ-CHBr-dNTP analogues. To complete assembly of the toolkit, we describe an alternative synthetic strategy featuring ethylbenzylamine or phenylglycine-derived chiral BP synthons incorporating a photolabile protecting group. After acid-catalyzed removal of the (R)-(+)-α-ethylbenzylamine auxiliary, coupling with activated dCMP and photochemical deprotection, the individual diastereomers of ß,γ-CHBr- (33a, 33b), ß,γ-CHCl- (34a, 34b), ß,γ-CHF-dCTP (35a, 35b) were obtained. The ß,γ-CH(CH3)-dATPs (44a, 44b) were obtained using a methyl (R)-(-)-phenylglycinate auxiliary. 31P and 19F NMR Δδ values are correlated with CXY stereochemistry and pKa2-4 values for 13 CXY-bisphosphonic acids and imidodiphosphonic acid are tabulated.
Assuntos
DNA Polimerase Dirigida por DNA , Nucleotídeos de Desoxicitosina , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância MagnéticaRESUMO
APOBEC3G (Apo3G) is a single-stranded (ss)DNA cytosine deaminase that eliminates HIV-1 infectivity by converting C â U in numerous small target motifs on the minus viral cDNA. Apo3G deaminates linear ssDNA in vitro with pronounced spatial asymmetry favoring the 3' â 5' direction. A similar polarity observed in vivo is believed responsible for initiating localized C â T mutational gradients that inactivate the virus. When compared with double-stranded (ds)DNA scanning enzymes, e.g. DNA glycosylases that excise rare aberrant bases, there is a paucity of mechanistic studies on ssDNA scanning enzymes. Here, we investigate ssDNA scanning and motif-targeting mechanisms for Apo3G using single molecule Förster resonance energy transfer. We address the specific issue of deamination asymmetry within the general context of ssDNA scanning mechanisms and show that Apo3G scanning trajectories, ssDNA contraction, and deamination efficiencies depend on motif sequence, location, and ionic strength. Notably, we observe the presence of bidirectional quasi-localized scanning of Apo3G occurring proximal to a 5' hot motif, a motif-dependent DNA contraction greatest for 5' hot > 3' hot > 5' cold motifs, and diminished mobility at low salt. We discuss the single molecule Förster resonance energy transfer data in terms of a model in which deamination polarity occurs as a consequence of Apo3G binding to ssDNA in two orientations, one that is catalytically favorable, with the other disfavorable.
Assuntos
Citidina Desaminase/metabolismo , DNA de Cadeia Simples/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Desaminase APOBEC-3G , Bacteriófago M13/genética , Sequência de Bases , Sítios de Ligação/genética , Biocatálise , Citidina Desaminase/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA Viral/química , DNA Viral/genética , DNA Viral/metabolismo , Desaminação , Corantes Fluorescentes/química , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
It is difficult to overestimate the importance of nucleoside triphosphates in cellular chemistry: They are the building blocks for DNA and RNA and important sources of energy. Modifications of biologically important organic molecules with fluorine are of great interest to chemists and biologists because the size and electronegativity of the fluorine atom can be used to make defined structural alterations to biologically important molecules. Although the concept of nonhydrolyzable nucleotides has been around for some time, the progress in the area of modified triphosphates was limited by the lack of synthetic methods allowing to access bisCF(2)-substituted nucleotide analogs-one of the most interesting classes of nonhydrolyzable nucleotides. These compounds have "correct" polarity and the smallest possible steric perturbation compared to natural nucleotides. No other known nucleotides have these advantages, making bisCF(2)-substituted analogs unique. Herein, we report a concise route for the preparation of hitherto unknown highly acidic and polybasic bis(difluoromethylene)triphosphoric acid 1 using a phosphorous(III)/phosphorous(V) interconversion approach. The analog 1 compared to triphosphoric acid is enzymatically nonhydrolyzable due to substitution of two bridging oxygen atoms with CF(2) groups, maintaining minimal perturbations in steric bulkiness and overall polarity of the triphosphate polyanion. The fluorinated triphosphoric acid 1 was used for the preparation of the corresponding fluorinated deoxynucleotides (dNTPs). One of these dNTP analogs (dT) was demonstrated to fit into DNA polymerase beta (DNA pol beta) binding pocket by obtaining a 2.5 A resolution crystal structure of a ternary complex with the enzyme. Unexpected dominating effect of triphosphate/Mg(2+) interaction over Watson-Crick hydrogen bonding was found and discussed.
Assuntos
DNA/química , Flúor/química , Organofosfonatos/química , RNA/química , Cristalografia por Raios X , DNA/síntese química , DNA/farmacologia , Ligação de Hidrogênio , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , RNA/síntese química , RNA/farmacologiaRESUMO
The first examples of α-azido bisphosphonates [(RO)(2)P(O)](2)CXN(3) (1, R = i-Pr, X = Me; 2, R = i-Pr, X = H; 3, R = H, X = Me; 4, R = H, X = H) and corresponding ß,γ-CXN(3) dGTP (5-6) and α,ß-CXN(3) dATP (7-8) analogues are described. The individual diastereomers of 7 (7a/b) were obtained by HPLC separation of the dADP synthetic precursor (14a/b).
Assuntos
Azidas/química , Difosfonatos/síntese química , Nucleotídeos/química , Estrutura Molecular , EstereoisomerismoRESUMO
Beta,gamma-fluoromethylene analogues of nucleotides are considered to be useful mimics of the natural substrates, but direct structural evidence defining their active site interactions has not been available, including the influence of the new chiral center introduced at the CHF carbon, as in beta,gamma-fluoromethylene-dGTP, which forms an active site complex with DNA polymerase beta, a repair enzyme that plays an important role in base excision repair (BER) and oncogenesis. We report X-ray crystallographic results for a series of beta,gamma-CXY dGTP analogues, where X,Y = H, F, Cl, Br, and/or CH(3). For all three R/S monofluorinated analogues examined (CHF, 3/4; CCH(3)F, 13/14; CClF 15/16), a single CXF-diastereomer (3, 13, 16) is observed in the active site complex, with the CXF fluorine atom at a approximately 3 A (bonding) distance to a guanidinium N of Arg183. In contrast, for the CHCl, CHBr, and CHCH(3) analogues, both diasteromers (6/7, 8/9, 10/11) populate the dGTP site in the enzyme complex about equally. The structures of the bound dichloro (5) and dimethyl (12) analogue complexes indicate little to no steric effect on the placement of the bound nucleotide backbone. The results suggest that introduction of a single fluorine atom at the beta,gamma-bridging carbon atom of these dNTP analogues enables a new, stereospecific interaction within the preorganized active site complex that is unique to fluorine. The results also provide the first diverse structural data set permitting an assessment of how closely this class of dNTP analogues mimics the conformation of the parent nucleotide within the active site complex.
Assuntos
DNA Polimerase beta/química , DNA Polimerase beta/metabolismo , Guanosina Trifosfato/química , Domínio Catalítico , Cristalografia por Raios X , DNA/química , Guanosina Trifosfato/metabolismo , Halogenação , Humanos , Modelos Moleculares , Ligação Proteica , Especificidade por SubstratoRESUMO
A deuterated cavitand host was examined for its affinity to a series of guests; for halogenated, preorganized guests binding was significantly stronger than the corresponding protium host.
RESUMO
Alpha,beta-difluoromethylene deoxynucleoside 5'-triphosphates (dNTPs, N = A or C) are advantageously obtained via phosphorylation of corresponding dNDP analogues using catalytic ATP, PEP, nucleoside diphosphate kinase, and pyruvate kinase. DNA pol beta K(d) values for the alpha,beta-CF(2) and unmodified dNTPs, alpha,beta-NH dUTP, and the alpha,beta-CH(2) analogues of dATP and dGTP are discussed in relation to the conformations of alpha,beta-CF(2) dTTP versus alpha,beta-NH dUTP bound into the enzyme active site.
Assuntos
Trifosfato de Adenosina/metabolismo , DNA Polimerase beta/metabolismo , Nucleotídeos de Desoxiadenina/síntese química , Nucleotídeos de Desoxicitosina/síntese química , Nucleotídeos de Desoxiguanina/síntese química , Núcleosídeo-Difosfato Quinase/metabolismo , Piruvato Quinase/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Nucleotídeos de Desoxiadenina/química , Nucleotídeos de Desoxicitosina/química , Nucleotídeos de Desoxiguanina/química , Sondas Moleculares , Estrutura Molecular , EstereoisomerismoRESUMO
The mechanism of DNA polymerase beta-catalyzed nucleotidyl transfer consists of chemical steps involving primer 3' OH deprotonation, nucleophilic attack, and pyrophosphate leaving-group elimination, preceded by dNTP binding which induces a large-amplitude conformational change for Watson-Crick nascent base pairs. Ambiguity in the nature of the rate-limiting step and active-site structural differences between correct and incorrect base-paired transition states remain obstacles to understanding DNA replication fidelity. Analogues of dGTP where the beta-gamma bridging oxygen is replaced with fluorine-substituted methylene groups have been shown to probe the contribution of leaving-group elimination to the overall catalytic rate (Biochemistry 46, 461-471). Here, the analysis is expanded substantially to include a broad range of halogen substituents with disparate steric and electronic properties. Evaluation of linear free energy relationships for incorporation of dGTP analogues opposite either template base C or T reveals a strong correlation of log(kpol) to leaving group pKa. Significantly different kpol behavior is observed with a subset of the analogues, with magnitude dependent on the identity of the nascent base pair. This observation, and the absence of an analogous effect on ground state analogue binding (Kd values), points to active-site structural differences at the chemical transition state. Reduced catalysis with bulky halo-containing substrates is manifested in the fidelity of T-G incorporation, where the CCl2-bridging analogue shows a 27-fold increase in fidelity over the natural dGTP. Solvent pH and deuterium isotope-effect data are also used to evaluate mechanistic differences between correct and mispaired incorporation.
Assuntos
Pareamento Incorreto de Bases , DNA Polimerase beta/química , Catálise , Domínio Catalítico , DNA/química , DNA Polimerase beta/genética , DNA Polimerase beta/metabolismo , Nucleotídeos de Desoxiguanina/química , Óxido de Deutério/química , Difosfonatos/química , Guanosina Trifosfato/análogos & derivados , Halogênios/química , Humanos , Concentração de Íons de Hidrogênio , Cinética , Modelos Químicos , Modelos Moleculares , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , TermodinâmicaRESUMO
DNA polymerase catalysis and fidelity studies typically compare incorporation of "right" versus "wrong" nucleotide bases where the leaving group is pyrophosphate. Here we use dGTP analogues replacing the beta,gamma-bridging O with CH2, CHF, CF2, or CCl2 to explore leaving-group effects on the nucleotidyl transfer mechanism and fidelity of DNA polymerase (pol) beta. T.G mismatches occur with fidelities similar to dGTP with the exception of the CH2 analogue, which is incorporated with 5-fold higher fidelity. All analogues are observed to bind opposite template C with Kds between 1 and 4 microM, and structural evidence suggests that the analogues bind in essentially the native conformation, making them suitable substrates for probing linear free energy relationships (LFERs) in transient-kinetics experiments. Importantly, Brnsted correlations of log(kpol) versus leaving-group pKa for both right and wrong base incorporation reveal similar sensitivities (betalg approximately -0.8) followed by departures from linearity, suggesting that a chemical step rather than enzyme conformational change is rate-limiting for either process. The location of the breaks relative to pKas of CF2, O, and the sterically bulky CCl2-bridging compounds suggests a modification-induced change in the mechanism by stabilization of leaving-group elimination. The results are addressed theoretically in terms of the energetics of successive primer 3'-O addition (bond forming) and pyrophosphate analogue elimination (bond breaking) reaction energy barriers.
Assuntos
DNA Polimerase beta/metabolismo , Sequência de Bases , Domínio Catalítico , Cristalografia por Raios X , DNA/biossíntese , DNA/química , DNA/genética , DNA Polimerase beta/química , DNA Polimerase beta/genética , Nucleotídeos de Desoxiguanina/química , Nucleotídeos de Desoxiguanina/metabolismo , Humanos , Técnicas In Vitro , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleotídeos/química , Nucleotídeos/metabolismo , Oxigênio/metabolismo , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , TermodinâmicaRESUMO
The concept that resorcinarenes can be used as templates for the synthesis of large macrocycles is introduced. By way of example, previously inaccessible, aromatic crown ethers compounds are synthesized.