四甲基吡啶卟啉与核酸相互作用的稳态和时间分辨光谱

采用稳态吸收和荧光光谱、圆二色谱和皮秒时间分辨荧光光谱手段, 研究了5,10,15,20-四[4-(N-甲基吡啶)]卟啉(TMPyP4)与腺嘌呤(A)、鸟嘌呤(G)、胸腺嘧啶(T)和胞嘧啶(C)等4种碱基, 以及相应的核苷、核苷酸和单链DNA的结合能力和光谱学性质. 研究结果发现, 嘌呤与TMPyP4的结合能力比嘧啶的强. 对于某一碱基系列, 结合能力强弱顺序依次为: 碱基~核苷<核苷酸<单链DNA. 时间分辨荧光谱研究发现, 除鸟嘌呤外, 核酸和TMPyP4复合物的荧光动力学均含有快(1~2 ns)和慢(约10 ns)两个衰减过程, 它们分别是由激基复合体和环境极性对激发态TMPyP4分子的影响所致. 单链DNA能诱导TMPyP4产生诱导圆二色信号, 而单分子(碱基、核苷、核苷酸)则无此功能.     

pH‐Independent Recognition of the dG ⋅ dC Base Pair in Triplex DNA: 9‐Deazaguanine N7‐(2′‐Deoxyribonucleoside) and Halogenated Derivatives Replacing Protonated dC

Triplex-forming oligonucleotides (TFOs) containing 9-deazaguanine N⁷-(2′-deoxyribonucleoside) 1a and halogenated derivatives 1b,c were synthesized employing solid-phase oligonucleotide synthesis. For that purpose, the phosphoramidite building blocks 5a – c and 8a – c were synthesized. Multiple incorporations of 1a – c in place of dC were performed within TFOs, which involved the sequence of five consecutive 1a – c ⋅ dG ⋅ dC triplets as well as of three alternating 1a – c ⋅ dG ⋅ dC and dT ⋅ dA ⋅ dT triplets. These TFOs were designed to bind in a parallel orientation to the target duplex. Triplex forming properties of these oligonucleotides containing 1a – c in the presence of Na⁺ and Mg²⁺ were studied by UV/melting-curve analysis and confirmed by circular-dichroism (CD) spectroscopy. The oligonucleotides containing 1a in the place of dC formed stable triplexes at physiological pH in the case of sequence of five consecutive 1a ⋅ dG ⋅ dC triplets as well as three alternating 1a – c ⋅ dG ⋅ dC and dT ⋅ dA ⋅ dT triplets. The replacement of 1a by 9-halogenated derivatives 1b,c further enhanced the stability of DNA triplexes. Nucleosides 1a – c also stabilized duplex DNA.     

Sequence-specific control of azobenzene assemblies by molecular recognition of DNA

We investigated the molecular recognition between the amphiphile AzoAde, which is composed of azobenzene in the hydrophobic and adenine in the hydrophilic portion of the molecule, and oligonucleotides having a homogeneous base (dA30, dT30, dG30, and dC30) at the air-water interface. On the basis of the complementary base-pairing of DNA in the duplex, orderly arrangement of AzoAde on templated dT30 was examined using pi-A isotherm, UV-vis RAS, FT-IR RAS, and XPS measurements. Although there was little interaction between AzoAde and mismatched oligonucleotides (dA30, dG30, and dC30), AzoAde prepared on a dT30 subphase stoichiometrically assembled and interacted with dT30, subsequently forming a J-form assembly at the air-water interface. AFM observation of the LB films revealed the nanostructure of the J-formed AzoAde monolayer on the dT30 subphase as well as the domain structures of the H-formed monolayers on the other oligonucleotide subphases. Therefore, dT30 has a potential application as a template for assembling AzoAde at the air-water interface.     

DNA nucleosides and their radical anions: molecular structures and electron affinities

The deoxyribonucleosides have been studied to determine the properties of combinations of 2-deoxyribose with each of the isolated DNA bases for both neutral and anionic species. We have used a carefully calibrated theoretical method [Chem. Rev. 2002, 102, 231], employing the B3LYP hybrid Hartree-Fock/DFT functional with the DZP++ basis set. Predictions are made of the geometric parameters, adiabatic electron affinities, charge distributions based on natural population analysis, and decomposition enthalpy for the neutral and anionic forms of the four 2'-deoxyribonucleosides in DNA: 2'-deoxyriboadenosine (dA), 2'-deoxyribocytidine (dC), 2'-deoxyriboguanosine (dG), and 2'-deoxyribothymidine (dT). Geometric changes in the anions show that the glycosidic bond exhibits little change with excess charge for the guanosine but significant shortening for the adenosine and for the pyrimidines. The zero-point corrected adiabatic electron affinities in eV for each of the 2'-deoxyribonucleosides are as follows: 0.06, dA; 0.09, dG; 0.33, dC; and 0.44, dT. These values are uniformly greater than those of the corresponding isolated bases (-0.28, A; -0.07, G; 0.03, C; 0.20, T) and the isolated 2-deoxyribose (-0.38) at the same level of theory. The vertical detachment energies of dT and dC are substantial, 0.72 and 0.94 eV, and these anions should be observable. A high VDE, 0.91 eV, is also found for dA but its anion is unlikely to be stable due to the small AEA of 0.06 eV. The high VDE reflects the fact that the molecular structures of the anions and the corresponding neutral species are quite different. Valence character is displayed for the SOMOs of dA, dC, and dT, while some component of diffuse character is visible in the SOMO of dG. Further analysis of electronic changes upon electron attachment include an examination of the NPA charges, which show that in the neutral 2'-deoxyribonucleosides the sum of NPA charges for every base is the same, -0.28 with the sum of 2-deoxyribose charges being positive, +0.28. In the anions, the trend in charge division varies based on the nature of the excess electron in the anions. Thermodynamically, the overall enthalpy change for the reaction of water with the neutral nucleosides to give bases and ribose is approximately zero. The analogous decomposition is exothermic by 8 to 11 kcal mol-1 for the anions, indicating possible challenges for anionic gas-phase nucleoside exploration in the presence of water.     

MECHANISMS OF QUENCHING OF THE FLUORESCENCE OF A BENZO[a]PYRENE TETRAOL METABOLITE MODEL COMPOUND BY 2'-DEOXYNUCLEOSIDES

Abstract— The hydrophobic interactions of bulky polycyclic aromatic hydrocarbons with nucleic acid bases and the formation of noncovalent complexes with DNA are important in the expressions of the mutagenic and carcinogenic potentials of this class of compounds. The fluorescence of the polycyclic aromatic residues can be employed as a probe of these interactions. In this work, the interactions of the (+)-trans stereoisomer of the tetraol 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene (BPT), a hydrolysis product of a highly mutagenic and carcinogenic diol epoxide derivative of benzo[a]pyrene, were studied with 2′-deoxynucleosides in aqueous solution by fluorescence and UV spectroscopic techniques. Ground-state complexes between BPT and the purine derivatives 2′-deoxyguanosine (dG), 2′-deoxyadenosine (dA), and 2′-deoxyinosine (dI) are formed with association constants in the range of ～40–130 M^?1 Complex formation with the pyrimidine derivatives 2′-deoxythymidine (dT), 2′-deoxycytidine (dC), and 2′-deoxyuridine (dU) is significantly weaker. Whereas dG is a strong quencher of the fluorescence of BPT by both static and dynamic mechanisms (dynamic quenching rate constant k_dyn= [2.5 ± 0.41 × 10⁹M¹ s ¹, which is close to the estimated diffusion-controlled value of ～ 5 × 10⁹M^{? 1} s^?1), both dA and dI are weak quenchers and form fluorescenceemitting complexes with BPT. The pyrimidine derivatives dC, dU, and dT are efficient dynamic fluorescence quenchers (K_dyn～ [1.5–3.0] × 10⁹M^?1 s^?1), with a small static quenching component due to complex formation evident only in the case of dT. None of the four nucleosidcs dG, dA, dC and dT are dynamic quenchers of BPT in the triplet excited state; the observed lower yields of triplets are attributed to the quenching of single excited states of BPT by 2′-deoxynucleosides without passing through the triplet manifold of BPT. Possible fluorescence quenching mechanisms involving photoinduced electron transfer are discussed. The strong quenching of the fluorescence of BPT by dG, dC and dT accounts for the low fluorescence yields of BPT-native DNA and of pyrene-DNA complexes.     

Synthesis and nucleic acid-binding properties of water-soluble porphyrins appending platinum(II) complexes.

We synthesized two water-soluble porphyrins appending platinum(II) complexes [alpha,beta-(4a) and alpha,alpha-(4b) 5,15-bis(2-trans-[PtCl(NH3)2]N-2-aminoethylaminocarbonylphenyl) 2,3,7,8,12,13,17,18-octamethylporphyrin] and studied their reactions with a variety of nucleic acids [disodium adenosine-5'-monophosphate (AMP), disodium guanosine-5'-monophosphate (GMP), disodium thymidine-5'-monophosphate (TMP), disodium cytidine-5'-monophosphate (CMP), synthetic polymer poly(dG)-poly(dC), poly(dA)-poly(dT)] by 1H-NMR, UV-vis and FAB-MS spectroscopies. Based on the denaturation experiments of synthetic nucleic acid polymers, we conclude that the presence of the porphyrins (5.6 microM) does not cause significant changes in the melting temperature of poly(dA)-poly(dT) (28 microM) (deltaT=1 degrees C) and shows reannealing. On the other hand, gradual melting of poly(dG)-poly(dC) (28 microM) occurs at a low temperature (deltaT= -27 degrees C) in the presence of the porphyrins (5.6 microM), and the solutions do not show reannealing phenomena. The results of UV-vis and 1H-NMR experiments revealed that the porphyrins bind to guanine bases and that the porphyrins bind to GMP more strongly than to the other nucleotides. The binding modes between the porphyrins and synthetic nucleic acids are affected more by the coordination of the nucleobase [poly(dG)-poly(dC)] to the Pt(II) in the porphyrins than by Coulomb and hydrophobic interactions.     

Silver Ions in Non‐canonical DNA Base Pairs: Metal‐Mediated Mismatch Stabilization of 2′‐Deoxyadenosine and 7‐Deazapurine Derivatives with 2′‐Deoxycytidine and 2′‐Deoxyguanosine

Novel silver‐mediated dA?dC, dA*?dC, and dA*?dG base pairs were formed in a natural DNA double helix environment (dA* denotes 7‐deaza‐dA, 7‐deaza‐7‐iodo‐dA, and 7‐cyclopropyl‐7‐deaza‐dA). 7‐Deazapurine nucleosides enforce silver ion binding and direct metal‐mediated base pair formation to their Watson–Crick face. New phosphoramidites were prepared from 7‐deaza‐dA, 7‐deaza‐7‐iodo‐dA, and 7‐cyclopropyl‐7‐deaza‐dA, which contain labile isobutyryl protecting groups. Solid‐phase synthesis furnished oligonucleotides that contain mismatches in near central positions. Increased thermal stabilities (higher T_m values) were observed for oligonucleotide duplexes with non‐canonical dA*?dC and dA?dC pairs in the presence of silver ions. The stability of the silver‐mediated base pairs was pH dependent. Silver ion binding was not observed for the dA?dG mismatch but took place when mismatches were formed between 7‐deazaadenine and guanine. The specific binding of silver ions was confirmed by stoichiometric UV titration experiments, which proved that one silver ion is captured by one mismatch. The stability increase of canonical DNA mismatches might have an impact on cellular DNA repair.     

5'-O-(2-Isopropoxyprop-2-yl)-protected Phosphoramidite Building Blocks in the Liquid Phase Oligonucleotide Synthesis

5'-O-(2-isopropoxyprop-2-yl) (IIP)-protection was introduced to 5’-OH function of nucleosides in high yields by an acid-catalysed transacetalization with 2,2-diisopropoxypropane. The applicability of this temporal 5’-O-protecting group was demonstrated in the liquid phase oligonucleotide synthesis (LPOS) using the corresponding phosphoramidite building blocks (dA, dG, dC and dT) and a tetrapodal precipitative soluble support. Standard protecting groups were used on nucleobases. Tetrazole as an activator, followed by oxidation using m-chloroperbenzoic acid, was used for the coupling. The IIP was shown to be a capable choice to the 5’-O protection in solution phase synthesis. It could be readily removed with formic acid (t_1/2<10 s in 6 % HCOOH in dichloromethane/methanol (2/1) at RT), resulting in volatile byproducts (acetone and isopropanol).     

Synthesis of Nucleobase‐Functionalized Carbon Nanotubes and Their Hybridization with Single‐Stranded DNA

For the first time ssDNA (25‐aptamer of mixed dA, dT, dG, and dC) was wrapped around functionalized single‐walled carbon nanotubes (SWCNTs), whose external surfaces were attached to multiple triazole‐(ethylene glycol)‐dA ligands. This method of hybridization involved the formation of hydrogen bonds between dT of ssDNA and dA of functionalized SWCNTs. It deviates from the reported π–π stacking between the nucleobases of DNA and the external sidewalls of nanotubes. The structural properties of the functionalized SWCNTs and its ssDNA complex were characterized by spectroscopic (including CD and Raman), thermogravimetric, and microscopic (TEM) methods. The results thus obtained establish a new platform of DNA delivery by use of nanotubes as a new vehicle with great potential in biomedical applications and drug development.     

Theoretical study of the interaction of tetramethylammonium with double-stranded oligonucleotides

Computations are performed on the interaction specificities of tetramethylammonium (TMA) for double-stranded oligonucleotides held in the B conformation. The effects of base sequence and chain length are investigated. In the short oligomers (helices formed from dinucleoside monophosphates and trinucleoside diphosphates), the interaction energies of TMA are larger in the major groove of (dG)_n · (dC)_n than in the minor groove of either (dA)_n · (dT)_n or (dA—dT)_n. Upon lengthening the oligomers, and owing to the gradual shaping of the grooves of the helix and cumulative effect of the phosphates, TMA is shown to increasingly favor the minor groove of (dA)_n · (dT)_n with respect to the major groove of (dG)_n · (dC)_n, with a sizeable energy difference computed at the pentanucleoside hexaphosphate level. The binding of TMA in the minor groove of (dA)_n · (dT)_n involves stabilizing contacts with several sites, on the bases and on the deoxyriboses. Configurations locating the cation closer to the thymine strand are slightly preferred over configurations locating it closer to the adenine strand.     

SUBSTRATE DEPENDENCE OF THE ACTION SPECTRUM FOR PHOTOENZYMATIC REPAIR OF DNA

Abstract— The absolute action spectrum has been determined for photoenzymatic splitting of cyclobutadipyrimidines ("pyrimidine dimers") from natural DNA, and from the synthetic polydeoxyribonucleotides poly(dA)·poly(dT) (forming only cyclobutadithymine) and poly(dG)·poly(dC) (forming only cyclobutadicytosine). These action spectra differ strikingly from each other, even when using the same enzyme preparations. On the other hand, the action spectrum for splitting cyclobutadithymine in natural DNA containing "dimers" of only this one type closely resembles the action spectrum for splitting the total mixture of "dimer" types in natural DNA, and is entirely different from the spectrum for splitting of the same photoproduct from poly(dA)·poly(dT). These results mean that the action spectrum is not simply the absorption spectrum of a chromophore carried by the photoreactivating enzyme, nor is it solely determined by the nature of the substrate photoproduct. It is at least partly determined by the over-all polynueleotide structure (viz. exact helical dimensions, pattern of neighboring bases to the "dimers," or both), affecting a ground state interaction between the enzyme and substrate in the enzyme-substrate complex.     

Oligonucleotides incorporating 8-aza-7-deazapurines: synthesis and base pairing of nucleosides with nitrogen-8 as a glycosylation position

Oligonucleotides incorporating the unusually linked 8-aza-7-deazapurine N8-(2'-deoxyribonucleosides) 3a,b (purine and 6-amino-2-chloropurine analogues) were used as chemical probes to investigate the base pairing motifs of the universal nucleoside 8-aza-7-deazapurin-6-amine N8-(2'-deoxyribofuranoside) 2 (adenine analogue) and that of the 2,6-diamino compound 1. Owing to the absence of an amino group on the nucleoside 3a the low stability of oligonucleotide duplexes incorporating this compound opposite to the four canonical DNA-constituents indicate hydrogen bonding and base pairing for the universal nucleosides 1 and 2 which form much more stable duplexes. When the 6-amino-2-chloro-8-aza-7-deazapurine nucleoside 3b replaces 1 and is located at the same positions, two sets of duplexes are formed (i) high Tm duplexes with 3b located opposite to dA or dC and (ii) low Tm duplexes with 3b located opposite to dG or dT. These results are due to the steric clash of the 2-chloro substituent of 3b with the 2-oxo group of dT or the 6-oxo group of dG while the 2-halogeno substituents are well accommodated in the base pairs formed with dA or dC. For comparison duplexes incorporating the regularly linked nucleosides 4-6a,b containing the same nucleobases as those of 1-3a,b were studied.     

Synthesis and properties of DNA oligomers containing 2'-deoxynucleoside N-oxide derivatives

Cytosine and adenine N-oxide derivatives have long been known as products resulting from the oxidative damage of DNA by peroxides such as hydrogen peroxide. Although the synthesis and properties of 2'-deoxynucleoside N-oxide derivatives have been well described, little has been reported about the chemical and biochemical behavior of initially formed DNA oligomers containing these N-oxide bases. In this study, we established a convenient method for the solid-phase synthesis of oligodeoxynucleotides incorporating 2'-deoxycytidine N-oxide (dC O) or 2'-deoxyadenosine N-oxide (dA O) by using the postsynthetic oxidation of N-protected DNA oligomers except for the target dC or dA site with m-CPBA in MeOH in a highly selective manner. In this strategy, the benzoyl, phthaloyl, and (4-isopropylphenoxy)acetyl groups proved to serve as base protecting groups to avoid oxidation of adenine, cytosine, and guanine, respectively, at the unmodified sites.     

Naphthalene Imide Conjugates: Formation of Supramolecular Assemblies,and the Encapsulation and Release of Dyes through DNA‐Mediated Disassembly

We report the synthesis of two new amphiphilic conjugates 1 and 2 based on naphthalene di‐ and monoimide chromophores and the investigation of their photophysical, self‐assembly and DNA‐binding properties. These conjugates showed aqueous good solubility and exhibited strong interactions with DNA and polynucleotides such as poly(dG?dC)–poly(dG?dC) and poly(dA?dT)–poly(dA?dT). The interaction of these conjugates with DNA was evaluated by photo‐ and biophysical techniques. These studies revealed that the conjugates interact with DNA through intercalation with association constants in the order of 5–8×10⁴ M ^?1. Of these two conjugates, bolaamphiphile 1 exhibited a supramolecular assembly that formed vesicles with an approximate diameter of 220 nm in the aqueous medium at a critical aggregation concentration of 0.4 mM , which was confirmed by SEM and TEM. These vesicular structures showed a strong affinity for hydrophobic molecules such as Nile red through encapsulation. Uniquely, when exposed to DNA the vesicles disassembled, and therefore this transformation could be utilised for the encapsulation and release of hydrophobic molecules by employing DNA as a stimulus.     

Rapid Location of the Preferred Interaction Sites between Small Polar Molecules and Macromolecules. II. Binding of Water to a Model Segment of B-DNA

The procedure developed in Part I of this series is applied to the homopolymeric sequences poly(dA) · poly(dT) and poly(dG) · poly(dC) on the double helical structure of B-DNA. Some aspects of the base sequence influence on the polymer's attraction for water molecule are described. The results are used to discuss the general hydration features of those systems in relation to recent experimental studies of DNA single crystals.     

Synthesis and incorporation of a furan-modified adenosine building block for DNA interstrand crosslinking

2'-O-(3-(Furan-2-yl)propyl)adenosine was synthesized and evaluated for interstrand crosslink (ICL) formation in DNA duplexes. In situ oxidation of the furan moiety with NIS showed rapid crosslink formation to dA and dC, while dT and dG were inactive.     

Photoswitchable Formation of a DNA Interstrand Cross‐Link by a Coumarin‐Modified Nucleotide

A coumarin‐modified pyrimidine nucleoside ( 1 ) has been synthesized using a Cu^I‐catalyzed click reaction and incorporated into oligodeoxynucleotides (ODNs). Interstrand cross‐links are produced upon irradiation of ODNs containing 1 at 350 nm. Cross‐linking occurs through a [2+2] cycloaddition reaction with the opposing thymidine, 2′‐deoxycytidine, or 2′‐deoxyadenosine. A much higher reactivity was observed with dT than dC or dA. Irradiation of the dT‐ 1 and dC‐ 1 cross‐linked products at 254 nm leads to a reversible ring‐opening reaction, while such phenomena were not observed with dA‐ 1 adducts. The reversible reaction is ultrafast and complete within 50–90 s. Consistent photoswitching behavior was observed over 6 cycles of irradiation at 350 nm and 254 nm. To the best of our knowledge, this is the first example of photoswitchable interstrand cross‐linking formation induced by a modified pyrimidine nucleoside.     

Parallel-stranded DNA: enhancing duplex stability by the 'G-clamp' and a pyrrolo-dC derivative

The new pyrrolo-dC derivative 4 tethered with an alkylamino side chain via a triazole linker was synthesized. Oligonucleotides containing the G-clamp 3 or the pyrrolo-dC derivative 4 were prepared. Oligonucleotide synthesis and deprotection under standard conditions led to unwanted side product formation. The side product was identified as an acrylonitrile adduct of the aminoalkyl side chain. Changing the synthesis and work-up conditions to fast-deprotection chemistry and performing β-elimination of the cyanoethyl group on the solid support yielded pure oligonucleotides. Oligonucleotide duplexes with parallel chain orientation were constructed incorporating dA·dT and isoG(d)·dC base pairs. Replacement of dC-residues by the G-clamp 3 led to extraordinarily stable duplexes (ΔT(m) = +11 °C for two incorporations) in ps DNA, while the pyrrolo-dC derivative 4 behaved like dC. Surprisingly, the G-clamp 3 forms an even more stable base pair with 2'-deoxyisoguanosine in DNA with parallel chain orientation than with 2'-deoxyguanosine in aps DNA.