Equilibrium and kinetic studies of the aquation of the dinuclear platinum complex [[trans-PtCl(NH3)2]2(mu-NH2(CH2)6NH2)]2+: pKa determinations of aqua ligands via [1H,15N] NMR spectroscopy

By the use of [1H,15N] heteronuclear single quantum coherence (HSQC) 2D NMR spectroscopy and electrochemical methods we have determined the hydrolysis profile of the bifunctional dinuclear platinum complex [[trans-PtCl(15NH3)2]2(mu-15NH2(CH2)(6)15NH2)]2+ (1,1/t,t (n = 6), 15N-1), the prototype of a novel class of potential antitumor complexes. Reported are estimates for the rate and equilibrium constants for the first and second aquation steps, together with the acid dissociation constant (pKa1 approximately pKa2 approximately pKa3). The equilibrium constants determined by NMR at 25 and 37 degrees C (I = 0.1 M) were similar, pK1 approximately pK2 = 3.9 +/- 0.2, and from a chloride release experiment at 37 degrees C the values were found to be pK1 = 4.11 +/- 0.05 and pK2 = 4.2 +/- 0.5. The forward and reverse rate constants for aquation determined from this chloride release experiment were k1 = (8.5 +/- 0.3) x 10(-5) s-1 and k-1 = 0.91 +/- 0.06 M-1 s-1, where the model assumed that all the liberated chloride came from 1. When the second aquation step was also taken into account, the rate constants were k1 = (7.9 +/- 0.2) x 10(-5) s-1, k-1 = 1.18 +/- 0.06 M-1 s-1, k2 = (10.6 +/- 3.0) x 10(-4) s-1, k-2 = 1.5 +/- 0.6 M-1 s-1. The rate constants compare favorably with other complexes with the [PtCl(am(m)ine)3]+ moiety and indicate that the equilibrium of all these species favors the chloro form. A pKa value of 5.62 was determined for the diaquated species [[trans-Pt(15NH3)2(H2O)]2(mu-15NH2(CH2)(6)15NH2)]4+ (3) using [1H,15N] HSQC NMR spectroscopy. The speciation profile of 1 and its hydrolysis products under physiological conditions is explored.     

Tandem electrostatic effect from the first to the third aglycon in the trimeric RNA owing to the nearest-neighbor interaction

We here show an electrostatic polar-pi interaction from the first to the third aglycon, via the second aglycon, in the ground state in two single stranded trimeric RNAs, 5'-GpA(1)pA(2)-3' (3) and 5'-GpApC-3' (4), as a result of intramolecular nearest neighbor offset-stacking. The experimental evidence in support of this conclusion has been obtained by comparing the pK(a)s of each aglycone in the two trimers with those of guanosine 3'-ethyl phosphate, GpEt (1) and 5'-GpA-3' (2): Thus, the pK(a) of N(1)-H of guanin-9-yl of 5'-GpA(1)pA(2)-3' (3) could be measured by pH titration (pH 7.3-11.6) of its own deltaH8G (pK(a) 9.75 +/- 0.02) as well as from deltaH8A(1) (pK(a) 9.72 +/- 0.02) and deltaH2A(1) (pK(a) 9.83 +/- 0.04) of the neighboring pA(1)p moiety and the deltaH8A(2) (pK(a) 9.83 +/- 0.02) of the terminal pA(2) moiety. Similarly, the pH titration of GpApC (4) shows the pK(a) of N(1)-H of guanin-9-yl from its own deltaH8G (pK(a) 9.88 +/- 0.03) as well as from deltaH8A (pK(a) 9.87 +/- 0.01) of the neighboring pAp moiety, and deltaH5/H6C (pK(a) 9.88 +/- 0.01 and 9.90 +/- 0.01 respectively) of the 3'-terminal cytosin-1-yl. This intramolecular nearest neighbor electrostatic interaction in the single-stranded RNA modulates the pseudoaromaticity of the nearest neighbors by almost total transmission of because they constitute an extended array of offset-stacked coupled aromatic heterocycles within a polyanionic sugar-phosphate backbone at the ground state. The enhanced basicity of Gp residue by ca. 0.6 pK(a) unit in the trimers compared to that of the dimer is a result of the change in the electrostatic microenvironment owing to the nearest neighbors in the former (the nucleobases as well as the phosphates). Thus, the from the 5'-guanylate ion to the 3'-end aglycon via the central adenin-9-yl is 55 to 56 kJ mol(-)(1) in each step through a distance spanning approximately 6.8 A in an unfolded state. As a result, the pK(a) of guanin-9-yl moiety has become 9.25 +/- 0.02 in GpEt (1), 9.17 +/- 0.02 in GpA (2), 9.75 +/- 0.02 in GpApA (3), and 9.88 +/- 0.03 in GpApC (4). This means that guanin-9-yl moiety of trimers 3 and 4 is more basic than in the monomer or the dimer. The net outcome of this electrostatic cross-talk between the two neighboring heterocycles is creation of new hybrid aglycones in an oligo or polynucleotide, whose physicochemical property and the pseudoaromatic character are completely dependent both upon the nearest neighbors, and whether they are stacked or unstacked. Thus, this tunable physicochemical property of an aglycon (an array of the extended genetic code) may have considerable implication in our understanding of the specific ligand binding ability of an aptamer, the pK(a) and the hydrogen bonding ability of nucleic acids in a microenvironment, or in the triplet usage by the anticodon-codon interaction in the protein biosynthesis in the ribosome.     

酰氨基硫脲及相关杂环衍生物的研究XXIX: 2-(3'-溴苯胺基)-5-[5'-氨基-1'-(4"-氯苯基)-1',2',3'-三唑-4'-基]-1,3,4-噻二唑的晶体结构

经1-[5'-氨基-1'-(4"-氯苯基)-1',2',3'-三唑-4'-甲酰基]-4-(3'-溴苯基)-3-氨基硫脲在浓硫酸作用下制得2-(3'-溴苯胺基)-5-[5'-氨基-1'-(4"-氯苯基)-1',2',3'-三唑-4'-基]-1,3,4-噻二唑化合物。该化合物的晶体结构经X射线衍射分析确定, 化合物属三斜晶系, P1空间群, a=1.1784(2), b=1.4455(2),c=1.1353(1)nm; α=100.68(1), β=109.50(1), γ=79.89(1)°; V=1.7779nm^3; 分子式C~1~6H~1~1BrClN~7S, Mr=448.75; Dc=1.673g/cm^3, Z=4,μ=58.16cm^-^1, 最终偏离因子R=0.084, Rw=0.086。分析化合物的键长, 键角数据表明, 该分子具有离域π键结构。     

Trinuclear copper(II) complex showing high selectivity for the hydrolysis of 2'-5' over 3'-5' for UpU and 3'-5' over 2'-5' for ApA ribonucleotides

The cooperative action of multiple Cu(II) nuclear centers is shown to be effective and selective in the hydrolysis of 2'-5' and 3'-5' ribonucleotides. Reported herein is the specific catalysis by two trinuclear Cu(II) complexes of L3A and L3B. Pseudo first-order kinetic studies reveal that the L3A trinuclear Cu(II) complex effects hydrolysis of Up(2'-5')U with a rate constant of 28 x 10(-)(4) min(-)(1) and Up(3'-5')U with a rate constant of 0.5 x 10(-)(4) min(-)(1). The hydrolyses of Ap(3'-5')A and Ap(2'-5')A proceed with rate constants of 24 x 10(-)(4) min(-)(1) and 0.5 x 10(-)(4) min(-)(1) respectively. The L3A trinuclear Cu(II) complex demonstrates high specificity for Up(2'-5')U and Ap(3'-5')A. Similar studies with the more rigid L3B trinuclear Cu(II) complex shows no selectivity and yields lower rate constants for hydrolysis. The selectivity observed with the L3A ligand is attributed to the geometry of the ligand-bound diribonucleotide which ultimately dictates the proximity of the attacking hydroxyl and the phosphoester to a Cu(II) center for activation and subsequent hydrolysis.     

Thermodynamic and kinetic studies on reactions of Pt(II) complexes with biologically relevant nucleophiles

The effect of different N-N spectator ligands on the reactivity of platinum(II) complexes was investigated by studying the water lability of [Pt(diaminocyclohexane)(H2O)2]2+ (Pt(dach)), [Pt(ethylenediamine)(H2O)2]2+ (Pt(en)), [Pt(aminomethylpyridine)(H2O)2]2+ (Pt(amp)), and [Pt(N,N'-bipyridine)(H2O)2]2+ (Pt(bpy)). Some of the selected N-N chelates form part of the coordination sphere of Pt(II) drugs in clinical use, as in Pt(dach) (oxaliplatin), or are models, regarding the nature of the amines, with higher stability in terms of substitution and hydrolysis of the diamine moiety, as in Pt(en) (cisplatin) and Pt(amp) (AMD473). The effect of pi-acceptors on the reactivity was investigated by introducing one (Pt(amp)) or two pyridine rings (Pt(bpy)) in the system. The pK(a) values for the two water molecules (viz., Pt(dach) (pK(a1) = 6.01, pK(a2) = 7.69), Pt(en) (pK(a1) = 5.97, pK(a2) = 7.47), Pt(amp) (pK(a1) = 5.82, pK(a2) = 6.83), Pt(bpy) (pK(a1) = 4.80, pK(a2) = 6.32) show a decrease in the order Pt(dach) > Pt(en) > Pt(amp) > Pt(bpy). The substitution of both coordinated water molecules by a series of nucleophiles (viz., thiourea (tu), L-methionine (L-Met), and guanosine-5'-monophosphate (5'GMP-) was investigated under pseudo-first-order conditions as a function of concentration, temperature, and pressure using UV-vis spectrophotometric and stopped-flow techniques and was found to occur in two subsequent reaction steps. The following k1 values for Pt(dach), Pt(en), Pt(amp), and Pt(bpy) were found: tu (25 degrees C, M(-1) s(-1)) 21 +/- 1, 34.0 +/- 0.4, 233 +/- 5, 5081 +/- 275; L-Met (25 degrees C) 0.85 +/- 0.01, 0.70 +/- 0.03, 2.15 +/- 0.05, 21.8 +/- 0.6; 5'GMP- (40 degrees C) 5.8 +/- 0.2, 3.9 +/- 0.1, 12.5 +/- 0.5, 24.4 +/- 0.3. The results for k2 for Pt(dach), Pt(en), Pt(amp), and Pt(bpy) are as follows: tu (25 degrees C, M(-1) s(-1)) 11.5 +/- 0.5, 10.2 +/- 0.2, 38 +/- 1, 1119 +/- 22; L-Met (25 degrees C, s(-1)) 2.5 +/- 0.1, 2.0 +/- 0.2, 1.2 +/- 0.3, 290 +/- 4; 5'GMP- (40 degrees C, M(-1) s(-1)) 0.21 +/- 0.02, 0.38 +/- 0.02, 0.97 +/- 0.02, 24 +/- 1. The activation parameters for all reactions suggest an associative substitution mechanism. The pK(a) values and substitution rates of the complexes studied can be tuned through the nature of the N-N chelate, which is important in the development of new active compounds for cancer therapy.     

The photochemistry of thymidylyl-(3'-5')-5-methyl-2'-deoxycytidine in aqueous solution

The photochemistry of the dinucleoside monophosphate thymidylyl-(3'-5')-5-methyl-2'-deoxycytidine (Tpm5dC) has been studied in aqueous solution using both 254 nm and UV-B radiation. A variety of dinucleotide photoproducts containing 5-methylcytosine (m5C) have been isolated and characterized. These include two cyclobutane dimers (CBD) (the cis-syn [c,s]and trans-syn forms), a (6-4) adduct and its related Dewar isomer, and two isomers of a product in which the m5C moiety was converted into an acrylamidine. Small amounts of thymidylyl-(3'-5')-thymidine (TpT) were also formed, presumably as a secondary photoreaction product. In addition, a photoproduct was characterized in which the m5C moiety was lost, thus generating 3'-thymidylic acid esterified with 2'-deoxyribose at the 5-hydroxyl on the sugar moiety. The c,s CBD of Tpm5dC readily undergoes deamination to form the corresponding CBD of TpT. The kinetics of this deamination process has been studied; the corresponding enthalpy and entropy of activation for the reaction have been evaluated at pH 7.4 as being, respectively, 73.4 kJ/mol and -103.5 J/K mol. Deamination was not observed for the other characterized photoproducts of Tpm5dC.     

Sugar conformational effects on the photochemistry of thymidylyl(3'-5')thymidine

The synthesis and conformational analysis of 2'-O,5-dimethyluridylyl(3'-5')-2'-O,5-dimethyluridine (1a), the analogue of thymidylyl(3'-5')thymidine (TpT; 1b) in which a methoxy group replaces each 2'-alpha-hydrogen atom, are described. In comparison with TpT, such modification increases the population of the C3'-endo conformer of the sugar ring puckering at the 5'- and 3'-ends from 30 to 75% and from 37 to 66%, respectively. Photolyses of 1a and TpT at 254 nm are qualitatively comparable (the cis-syn cyclobutane pyrimidine dimer and the (6-4) photoproduct are formed), although it is significantly faster in the case of 1a. These results are explained by the increased propensity of the modified dinucleotide to adopt a base-stacked conformation geometry reminiscent of that for TpT.     

Practical synthesis of an open geodesic polyarene with a fullerene-type 6:6-double bond at the center: diindeno[1,2,3,4-defg;1',2',3',4'-mnop]chrysene

Diindeno[1,2,3,4-defg;1',2',3',4'-mnop]chrysene (1), the smallest possible alkene-centered C60 substructure with a curved pi-system, is obtained in 25-35% yield by flash vacuum pyrolysis of the twisted 1,1'-dibromobifluorenylidene (2) on a 100 mg scale at 1050 degrees C. At 1200 degrees C, the bowl-shaped hydrocarbon 1 rearranges to the planar isomer diindeno[5,6,7,1-defg;5',6',7',1'-lmnop]chrysene (14) by a double 5/6 ring-expansion/ring-contraction. X-ray crystallography establishes that the central carbon atoms of 1 are nearly 80% as pyramidalized as the carbon atoms of C60 (POAV angles = 9.0 degrees and 11.6 degrees for 1 and C60, respectively). A four-step synthesis has been developed to prepare the pyrolysis precursor (2) as a mixture of (E)- and (Z)-isomers in 39% overall yield from commercially available 9-fluorenone-1-carboxylic acid (10).     

A time-dependent density functional theory investigation on the nature of the electronic transitions involved in the nonlinear optical response of [Ru(CF3CO2)3T] (T = 4'-(C6H4-p-NBu2)-2,2':6',2'-terpyridine)

We report a theoretical study based on density functional theory (DFT) and time-dependent DFT (TDDFT) calculations on the nature and role of the absorption bands involved in the nonlinear optical response of the complexes [Ru(CF3CO2)3T] (T = T1, T2; T1 = 4'-(C6H4-p-NBu2)-2,2':6',2'-terpyridine, T2 = 4'-(C6H4-p-NMe2)-2,2':6',2'-terpyridine). Geometry optimizations, performed without any symmetry constraints, confirm a twisting of the -C6H4-p-NBu2 moiety with respect to the plane of the chelated terpyridine. Despite this lack of strong pi interaction, TDDFT excited states calculations of the electronic spectrum in solution provide evidence of a relevant role of the NBu2 donor group in the low-energy LMCT band at 911 nm. Calculations also show that the two bands at higher energy (508 and 455 nm) are not attributable only to LMCT and ILCT transitions but to a mixing of ILCT/MLCT and ILCT/pi-pi* transitions, respectively. The 911 nm LMCT band, appearing at lower wavelength of the second harmonic (670 nm) of the EFISH experiment, controls the negative value of the second-order NLO response. This is confirmed by our calculations of the static component beta0(zzz) of the quadratic hyperpolarizability tensor, showing a large positive value. In addition we have found that the increase of the dipole moment upon excitation occurs, in all the characterized transitions, along the dipole moment axis, thus explaining why the EFISH and solvatochromic experimental values of the quadratic hyperpolarizability agree as sign and value.     

Deoxyribozymes with 2'-5' RNA ligase activity

In vitro selection was used to identify deoxyribozymes that ligate two RNA substrates. In the ligation reaction, a 2'-5' RNA phosphodiester linkage is created from a 2',3'-cyclic phosphate and a 5'-hydroxyl group. The new Mg(2+)-dependent deoxyribozymes provide 50-60% yield of ligated RNA in overnight incubations at pH 7.5 and 37 degrees C, and they afford 40-50% yield in 1 h at pH 9.0 and 37 degrees C. Various RNA substrate sequences may be joined by simple Watson-Crick covaration of the DNA binding arms that interact with the two RNA substrates. The current deoxyribozymes have some RNA substrate sequence requirements at the nucleotides immediately surrounding the ligation junction (either UAUA GGAA or UAUN GGAA, where the arrow denotes the ligation site and N equals any nucleotide). One of the new deoxyribozymes was used to prepare by ligation the Tetrahymena group I intron RNA P4-P6 domain, a representative structured RNA. Nondenaturing gel electrophoresis revealed that a 2'-5' linkage between nucleotides A233 and G234 of P4-P6 does not disrupt its Mg(2+)-dependent folding (DeltaDeltaG degrees ' < 0.2 kcal/mol). This demonstrates that a 2'-5' linkage does not necessarily interfere with structure in a folded RNA. Therefore, these non-native linkages may be acceptable in modified RNAs when structure/function relationships are investigated. Deoxyribozymes that ligate RNA should be particularly useful for preparing site-specifically modified RNAs for studies of RNA structure, folding, and catalysis.     

Synthesis of Conformationally Locked 2＇-Deoxy-2＇-nucleobase-5＇- Isonucleosides: 2＇-Deoxy-2＇-nucleobase-5＇ deoxy-1＇, 4＇ :3＇, 6＇-dianhydro-D-mannitol

Novel bicyclic isonucleosides, 2＇ -deoxy- 2＇ -nucleobase- 5＇ -deoxy- 1＇ , 4＇ : 3＇ , 6＇ -dianhydro D-mannitol 10a-10c, were synthesized from D-glucose. The computer-assisted molecular simu lation indicated that the sugar conformations of compounds 10a-10c were restricted to N-con formation.     

Synthesis and structure of novel conformationally constrained 1',2'-azetidine-fused bicyclic pyrimidine nucleosides: their incorporation into oligo-DNAs and thermal stability of the heteroduplexes

[structures: see text] The synthesis of novel 1',2'-aminomethylene bridged (6-aza-2-oxabicyclo[3.2.0]heptane) "azetidine" pyrimidine nucleosides and their transformations to the corresponding phosphoramidite building blocks (20, 39, and 42) for automated solid-phase oligonucleotide synthesis is reported. The novel bicyclonucleoside "azetidine" monomers were synthesized by two different strategies starting from the known sugar intermediate 6-O-benzyl-1,2:3,4-bis-O-isopropylidene-D-psicofuranose. Conformational analysis performed by molecular modeling (ab initio and MD simulations) and NMR showed that the azetidine-fused furanose sugar is locked in a North-East conformation with pseudorotational phase angle (P) in the range of 44.5-53.8 degrees and sugar puckering amplitude (phi(m)) of 29.3-32.6 degrees for the azetidine-modified T, U, C, and 5-Me-C nucleosides. Thermal denaturation studies of azetidine-modified oligo-DNA/RNA heteroduplexes show that the azetidine-fused nucleosides display improved binding affinities when compared to that of previously synthesized North-East sugar constrained oxetane fused analogues.     

Conformational structure of gaseous 3-chloropropanoyl chloride by electron diffraction, normal coordinate analysis, and ab initio molecular orbital, and density functional theory calculations

The molecular and conformational structures of 3-chloropropanoyl chloride (CH(2)Cl-CH(2)-C(=O)Cl) have been studied by using gas-phase electron diffraction (GED) data obtained at 22 degrees C (295 K) and ab initio molecular orbital (MO) and density functional theory (DFT) calculations up to the levels of MP4(SDQ) and B3LYP using larger basis sets. Normal coordinate calculations (NCA) taking into account nonlinear vibrational effects were also used in the analyses. The title compound may have up to four low-energy conformers in the gas phase, labeled according to the position of each of the two chlorine atoms in relation to the CCC propanoyl backbone, labeling the carbonyl chlorine torsion angle first: AA, AG, GG, and GA; where A is anti (ideal C-C-C-Cl torsion angle of approximately 180 degrees) and G is gauche (ideal C-C-C-Cl torsion angle of approximately 60 degrees). It has been judged from the experimental GED data and the theoretical calculations, as well as from previously published infrared (IR) studies on the molecule in both the liquid phase and in argon-trapped matrices at 10 K, that the gas phase consists of a mixture of at least three conformers: AA (most stable), AG, and GG, with the possibility of a smaller contribution (<10%) from the higher-energy GA form. The GA conformer cannot be ruled out by the GED experimental data. Relevant structural parameter values obtained from the GED least-squares refinements, with calculated ab initio MO MP2/6-31+G(2d,p) values used as constraints, were as follows (AA values with estimated 2sigma uncertainties): Bond lengths (r(h1)): r(C-C(=O)) = 1.505(4) A, r(C-CH(2)Cl) = 1.520(4) A, r(C=O) = 1.197(4) A, r(C(=O)-Cl) = 1.789(3) A, and r(C-Cl) = 1.782(3) A. Bond angles (angle(h1)): angle CCC = 111.5(11) degrees , angle CCO = 127.0(5) degrees, angle CC(O)Cl = 112.5(3) degrees, and angle CCCl = 110.3(3) degrees. Torsion angles (phi(C-C) = phi(ClCCC)): for AA, phi(1)(C-C(O)) = phi(2)(C-CH(2)Cl) = 180 degrees (assumed for true C(s) symmetry); for AG, phi(1)(C-C(O)) = -140(5) degrees, phi(2)(C-CH(2)Cl) = 76(13) degrees; for GG, phi(1)(C-C(O)) = 46(8) degrees, phi(2)(C-CH(2)Cl) = 77(14) degrees; for GA, phi(1)(C-C(O)) = 67.9 degrees (assumed), phi(2)(C-CH(2)Cl) = 177.8 degrees (assumed). The non-AA conformers all have chiral C(1) symmetry with twice the statistical weight (multiplicity) of C(s). The MP2/6-31+G(2d,p) calculated composition (%) based on the zero-point energy (ZPE) corrected energy differences, and the statistical weights for conformers: AA/AG/GG/GA = 28/35/28/9 was assumed in the final GED refinement. The more recent literature concerning the title molecule, as well as for several related molecules, has been examined and a survey has been attempted in the present article. The new experimental results for 3-chloropropanoyl chloride are discussed and compared with the previously published findings.     

Computational analysis of aza analogues of [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranose]-3'-spiro-5"-(4"-amino-1",2"-oxathiole-2",2"-dioxide) (TSAO) as HIV-1 reverse transcriptase inhibitors: relevance of conformational properties on the inhibitory activity

We have carried out a theoretical analysis of aza analogues of [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5"-(4"-amino-1",2"-oxathiole-2",2"-dioxide) by a variety of computational tools, aimed to account for the effect of the endocyclic amino moiety N-2" on the inhibitory activity against HIV-1. Docking studies suggest that compounds substituted at the N-3 and N-2' ' positions present the same binding mode to the [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5"-(4"-amino-1",2"-oxathiole-2",2"-dioxide)thymine prototype, where the endocyclic amino group remains mostly exposed to the solvent. A careful conformational analysis performed through different theoretical levels, from molecular mechanics to high-level quantum mechanical calculations, provides a rationalization based on conformational preferences, which appears as strongly determined by the substitution at N-2", and on electrostatic effects from the bulk water.     

Hydrolytic reactions of guanosyl-(3',3')-uridine and guanosyl-(3',3')-(2',5'-di-O-methyluridine)

Hydrolytic reactions of guanosyl-(3',3')-uridine and guanosyl-(3',3')-(2',5'-di-O-methyluridine) have been followed by RP HPLC over a wide pH range at 363.2 K in order to elucidate the role of the 2'-hydroxyl group as a hydrogen-bond donor upon departure of the 3'-uridine moiety. Under neutral and basic conditions, guanosyl-(3',3')-uridine undergoes hydroxide ion-catalyzed cleavage (first order in [OH(-)]) of the P-O3' bonds, giving uridine and guanosine 2',3'-cyclic monophosphates, which are subsequently hydrolyzed to a mixture of 2'- and 3'-monophosphates. This bond rupture is 23 times as fast as the corresponding cleavage of the P-O3' bond of guanosyl-(3',3')-(2',5'-di-O-methyluridine) to yield 2',5'-O-dimethyluridine and guanosine 2',3'-cyclic phosphate. Under acidic conditions, where the reactivity differences are smaller, depurination and isomerization compete with the cleavage. The effect of Zn(2+) on the cleavage of the P-O3' bonds of guanosyl-(3',3')-uridine is modest: about 6-fold acceleration was observed at [Zn(2+)] = 5 mmol L(-)(1) and pH 5.6. With guanosyl-(3',3')-(2',5'-di-O-methyluridine) the rate-acceleration effect is greater: a 37-fold acceleration was observed. The mechanisms of the partial reactions, in particular the effects of the 2'-hydroxyl group on the departure of the 3'-linked nucleoside, are discussed.     

Proton inventory studies of alpha-thrombin-catalyzed reactions of substrates with selected P and P' sites

Deuterium kinetic solvent isotope effects for the human alpha-thrombin-catalyzed hydrolysis of (1) substrates with selected P(1)-P(3) sites, Z-Pro-Arg-7-amido-4-methylcoumarin (7-AMC), N-t-Boc-Val-Pro-Arg-7-AMC, Bz-Phe-Val-Arg-4-nitroanilide (pNA), and H-D-Phe-L-Pip-Arg-pNA, are (DOD)k(cat) = (2.8-3.3) +/- 0.1 and (DOD)(k(cat)/K(m)) = (0.8-2.1) +/- 0.1 and (2) internally fluorescence-quenched substrates (a) (AB)Val-Phe-Pro-Arg-Ser-Phe-Arg-Leu-Lys(DNP)-Asp-OH, an optimal sequence, and (b) (AB)Val-Ser-Pro-Arg-Ser-Phe-Gln-Lys(DNP)-Asp-OH, recognition sequence for factor VIII, are (DOD)k(cat) = 2.2 +/- 0.2 and (DOD)(k(cat)/K(m)) = (0.8-0.9) +/- 0.1, at the pL (L = H, D) maximum, 8.4-9.0, and (25.0-26.0) +/- 0.1 degrees C. The most plausible models fitting the partial isotope effect (proton inventory) data have been selected on the basis of lowest values of the reduced chi squared and consistency of fractionation factors at all substrate concentrations, assuming rate-determining acylation. The data for Z-Pro-Arg-7-AMC are consistent with a single-proton bridge at the transition state phi(TS) = 0.39 +/- 0.05 and components for solvent reorganization phi(S) = 0.8 +/- 0.1 and phi(S) = 1.22 for k(cat) and k(cat)/K(m), respectively. The data for tripeptide amides fit bowl-shaped curves; an example is N-t-Boc-Val-Pro-Arg-7-AMC: phi(TS)(1) = phi(TS)(2) = 0.57 +/- 0.01 and phi(S) = 1 for k(cat) and 1.6 +/- 0.1 for k(cat)/K(m). Proton inventories for the nonapeptide (2b) are linear. The data for k(cat) for H-D-Phe-L-Pip-Arg-pNA and the decapeptide (2a) are most consistent with two identical fractionation factors for catalytic proton bridging, phi(TS)(1) = phi(TS)(2) = 0.68 +/- 0.02 and a large inverse component (phi(S) = 3.1 +/- 0.5) for the latter, indicative of substantial solvent reorganization upon leaving group departure. Proton inventory curves for k(cat)/K(m) for nearly all substrates are dome-shaped with an inverse isotope effect component (phi(S) = 1.2-2.4) originating from solvent reorganization during association of thrombin with substrate. These large contributions from medium effects are in full accord with the conformational adjustments required for the fulfillment of the dual, hemostatic and thrombolytic, functions of thrombin.     

Design,synthesis, and evaluation of matrix metalloprotease inhibitors bearing cyclopropane-derived peptidomimetics as P1' and P2' replacements

We have previously used trisubstituted cyclopropanes as peptide replacements to induce conformational constraints in known pseudopeptide inhibitors of a number of important enzymes. Cyclopropane-derived peptide mimics are novel in that they are among the few replacements that locally orient the peptide backbone and the amino acid side chain in a predefined manner. Although these dipeptide isosteres have been employed to orient amino acid side chains mimicking the gauche(-) conformation of chi(1)-space, their ability to project the side chains into an anti orientation has not been evaluated. As a first step toward this goal, the conformationally constrained pseudopeptides 8 and 10 and their corresponding flexible analogues 9 and 11 were prepared and tested as inhibitors of matrix metalloproteinases (MMPs). These compounds are analogues of 4 and 5, which were known to be potent MMP inhibitors. The anti orientations of the isopropyl side chain in 8 and the aromatic ring in 10 relative to the peptide backbone substituents on the cyclopropane were predicted to correspond to the known orientations of the P1' and P2' side chains of 5 when bound to MMPs. Hence, 8 and 10 were designed explicitly to probe topological features of the S1' or the S2' binding pockets of the MMPs. They were also designed to explore the importance of the P1'-P2' amide group, which is known to form highly conserved hydrogen bonds in several MMP-inhibitor complexes, and the viability of introducing a retro amide linkage between P2' and P3'. Pseudopeptides 8 and 9 were found to be weak competitive inhibitors of a series of MMPs. Any entropically favorable conformational constraints that were induced by the cyclopropane in 8 were thus overwhelmed by the loss of the hydrogen bonding capability associated with the P1'-P2' amide group. On the other hand, compounds 10 and 11, which contain a P2'-P3' retro amide group, were modest competitive inhibitors of a series of MMPs. The results obtained for 10 and 11 suggest that there may be a loss of hydrogen bonding capability associated with introducing the P2'-P3' retro amide group. However, because the conformationally constrained pseudopeptide 10 was significantly more potent than its flexible analogue 11, trisubstituted cyclopropanes related to 3 may serve as useful rigid dipeptide replacements in some biologically active pseudopeptides.     

Five- and six-membered conformationally locked 2',4'-carbocyclic ribo-thymidines: synthesis, structure, and biochemical studies

Two unusual reactions involving the 5-hexenyl or the 6-heptenyl radical cyclization of a distant double bond at C4' and the radical center at C2' of the ribofuranose ring of thymidine have been used as key steps to synthesize North-type conformationally constrained cis-fused bicyclic five-membered and six-membered carbocyclic analogues of LNA (carbocyclic-LNA-T) and ENA (carbocyclic-ENA-T) in high yields. Their structures have been confirmed unambiguously by long range 1H-13C NMR correlation (HMBC), TOCSY, COSY, and NOE experiments. The carbocyclic-LNA-T and carbocyclic-ENA-T were subsequently incorporated into the antisense oligonucleotides (AONs) to show that they enhance the Tm of the modified AON/RNA heteroduplexes by 3.5-5 degrees C and 1.5 degrees C/modification for carbocyclic-LNA-T and carbocyclic-ENA-T, respectively. Whereas the relative RNase H cleavage rates with carbocyclic-LNA-T, carbocyclic-ENA-T, aza-ENA-T, and LNA-T modified AON/RNA duplexes were found to be very similar to that of the native counterpart, irrespective of the type and the site modification in the AON strand, a single incorporation of carbocyclic-LNA and carbocyclic-ENA into AONs leads to very much more enhanced nuclease stability in the blood serum (stable >48 h) as compared to that of the native (fully degraded <3 h) and the LNA-modified AONs (fully degraded <9 h) and aza-ENA ( approximately 85% stable in 48 h). Clearly, remarkably enhanced lifetimes of these carbocyclic-modified AONs in the blood serum may produce the highly desired pharmacokinetic properties because of their unique stability and consequently a net reduction of the required dosage. This unique quality as well as their efficient use as the AON in the RNase H-promoted cleavage of the target RNA makes our carbocyclic-LNA and carbocyclic-ENA modifications excellent candidates as potential antisense therapeutic agents.     

Determination of the intrinsic site pK(a) value and cooperativity of the symmetrical hexadentate chelator N,N',N'-tris[2-(3-hydroxy-2-oxo-1,2-dihydropyridin-1-yl)acetamido]ethylamine

The three overlapping pK(a) values of N,N',N'-tris[2-(3-hydroxy-2-oxo-1,2-dihydropyridin-1-yl)acetamido]ethylamine, a tripodal hexadentate chelator formed from three 3-hydroxy-2(1H)-pyridinone moieties amide linked to tris-(2-aminoethyl)amine, were determined by simultaneous spectrophotometric and potentiometric titration. The data was analysed by non-linear regression with constraints to deal with (a) the highly correlated absorptivities and (b) the highly correlated pK(a) values. The three pK(a) values were optimized first from the spectrophotometric data (absorbance vs. pH) by non-linear regression to a model in which the molar absorptivity of the ith species ((i)) was constrained by the correlation equation (i) = epsilon (0) + (epsilon (3) - epsilon (0))i 3 with i = 0, 1, 2, 3, where (3) and (0) represent the molar absorptivities of the most protonated and least protonated species, respectively. The molar absorbitivity of the four species defined by three pK(a) values is, therefore, linearly related to proton stoichiometry. The pK(a) values were then optimized from the potentiometric data (pH vs. titrant volume) by non-linear regression to a model in which the three pK(a) values were constrained by the correlation equation pK(a(i)) = pK(a(int)) + b(i - 1) + (i - 2)log(3) where i = 1, 2 or 3. This expresses the three pK(a) values in terms of only two optimizable parameters, the intrinsic site pK(a) (pK(a(int))) and the interaction energy between sites (b). The fixed term (i - 2)log(3) accounts for the statistical effect on the pK(a) values of three equivalent ionizable sites. The modified analytical derivatives required for optimization of these parameters by the Gauss-Newton-Marquardt algorithm and the merits of optimizing pK(a) values with these two correlation equations are discussed. The optimized pK(a) values were 9.31 +/- 0.01, 8.75 +/- 0.01 and 8.19 +/- 0.01. The separation between pK(a) values is 0.58 comprising 0.477 for the statistical effect and 0.081 for the interaction energy while the intrinsic site pK(a) is 8.672 +/- 0.005. The tertiary amine at the centre of the tripodal backbone has a pK(a) of 5.88 +/- 0.03.     

2',3'-二脱氧-2',3'-二去氢鸟嘌呤核苷构象稳定性的理论研究

采用密度泛函方法在B3LYP/6-31＋G**水平上研究了2',3'-二脱氧-2',3'-二去氢鸟嘌呤核苷分子(D4G)的构象. 分别研究在气相中的孤立分子和一水合物异构体的相对稳定性和异构体之间的相互转变过程, 分析了水分子的参与对D4G异构体的相对稳定性和几何结构参数以及自然电荷的影响. 结果表明, 孤立的D4G分子在气相中存在8种稳定构象, 其中构象d4g-2是所有构象中最稳定的, 气相中D4G主要以d4g-2存在. 气相中各构象的相对稳定性为: d4g-2＞d4g-1＞d4g-5＞d4g-3＞d4g-6＞d4g-4＞d4g-8＞d4g-7. 计算得到的各构象键长和键角数据与实验值接近. 一个水分子的加入对D4G分子的构型参数有所影响, 基本不改变D4G分子各构象的稳定性顺序, 但构象转变的能垒有所提高. 氢键在分子构象中发挥了重要作用.