Synthesis and properties on some 2-, 4- and 5-aminopyrimidines. Effect of -NH2 and ortho-C methylation on protolytic equilibria and electronic absorption spectra.

A number of new 2-, 4- and 5-aminopyrimidines with sterical hindrance to the amino group rotation were synthesized. The pKa values and u.v. absorption spectra of the aminopyrimidines were measured in order to elucidate the conformation of the amino group depending on the place of substitution.     

Thermochemistry of aqueous solutions of alkylated nucleic acid bases. IV. Enthalpies of hydration of 5-alkyluracils

Enthalpies of sublimation, DeltaH degrees (subl) and of solution in water, DeltaH degrees (sol) were determined for a series of crystalline 1,3-dimethyl-uracil derivatives substituted at the C5-ring carbon atom with alkyl groups (-C(n)H(2n+1), n = 2-4) and some of their C(5.6)-cyclooligomethylene analogues (-(CH2)(n)-, n = 3-5). From these data. enthalpies of hydration DeltaH degrees (hydr)= DeltaH degrees (sol) - DeltaH degrees (subl) were calculated and corrected for energies of cavity formation in pure liquid water in order to obtain enthalpies of interaction, DeltaH degrees (int) of the solutes with their hydration shells. The latter are discussed together with the recalculated DeltaH degrees (int) for variously methylated uracils, obtained previously according to a simplified correction procedure, in terms of perturbations in the energy and scheme of hydration of the diketopyrimidine ring brought about by alkyl substitution. It was found that each -CH2-group added with an alkyl substitution contributes favorably about -20 kJ mol(-1) toDeltaH degrees (int).This contribution is partially cancelled by the unfavorable contribution to DeltaH degrees (int) connected with removal of some water molecules bound in the first and subsequent hydration layers by an alkyl substituent. This is particularly evident on substitution at the polar side of the diketopyrimidine ring on which water molecules are expected to be bound specifically.     

Calorimetric study of 2U:1A three-stranded complexes formed between poly(U) and adenine dinucleotides: ApA and diastereoisomers of nonionic adenine dideoxyribonucleoside methyl phosphonate

Melting parameters of 2U:1A complexes formed by polyuridylic acid [poly(U)] and three adenine dinucleotides, diribonucleoside monophosphonate ApA and diastereoisomers of dideoxyribonucleoside methyl phosphonate [(dApA)₁ and (dApA)₂], in 1M NaCl and at a number of dinucleotide concentrations were obtained from differential scanning microcalorimetric data and interpreted in terms of the theory of helix–coil equilibrium in oligonucleotide–polynucleotide systems. The apparent binding constant, 1/c_m, at 39°C and melting temperatures, T_m, at 1 × 10^?3 M dinucleotide concentration indicate the following order of thermodynamic stability of the complexes: 2 poly(U) · (dApA)₂ (2.27 × 10³M^?1, 44.2°C) > 2 poly(U) · (dApA)₁ (9.9 × 10²M¹, 39.2°C) > 2 poly(U) · (ApA) (5.9 × 10²M^?1, 35.8°C). Corresponding calorimetric enthalpies of melting, ΔH_m: 13.5, 12.7, and 12.8 kcal/mol (UUA base triplets) were found to be considerably lower than the van't Hoff enthalpies, ΔH_app: 29.4, 16.2, and 16.2 kcal/mol, respectively, evaluated from the dependence of the melting temperatures on dinucleotide concentration. Self-association of dinucleotides and their simultaneous binding as monomers, dimers, and higher-order associated species is suggested as the most probable cause of the differences between ΔH_m and ΔH_app values. The differences in thermodynamic properties of the complexes formed by (dApA)₁ and (dApA)₂ diastereoisomers are discussed in connection with their known conformational properties. The higher and essentially enthalpic stability of the 2 poly(U) · (dApA)₂ complex correlates with a lower degree of intramolecular stacking of the (dApA)₂ isomer. The hydrophobically enhanced strong self-association of the latter greatly influences the thermodynamics of its complex formation with poly(U) and results in ΔH_app/ΔH_m = 2.3.     

1H- and 13C-nmr investigations on cis–trans isomerization of proline peptide bonds and conformation of aromatic side chains in H-Trp-(Pro)n-Tyr-OH peptides

¹H and ¹³C high-resolution nmr spectra of cationic, zwitterionic, and anionic forms of the peptides: H-Trp-(Pro)_n-Tyr-OH, n = 0-5, and H-Trp-Pro-OCH₃ were obtained in D₂O solution. Analysis of H^α(Pro₁), H^α(Trp), C^γ(Pro), H^ε(Tyr), and H^δ(Trp) resonances provided evidence for the presence of two predominant backbone isomers: the all-trans one and another with the Trp-Pro peptide bond in cis conformation; the latter constituted about 0.8 molar fraction of the total peptide (n > 1) concentration. Relative content of these isomers varied in a characteristic way with the number of Pro residues and the ionization state of the peptides. The highest content of the cis (Trp-Pro) isomer, 0.74, was found in the anionic form of H-Trp-Pro-Tyr-OH; it decreased in the order of: anion ? zwitterion ≈ cation, and with the number of Pro residues to reach the value of 0.42 in the cationic form of H-Trp- (Pro)₅-Tyr-OH. Isomerization equilibria about Pro-Pro bond(s) were found to be shifted far (?0.9) in favor of the trans conformation. Interpretation of the measured vicinal coupling constants J_α?β′ and J_α?β″ for C^αH-C^βH₂ proton systems of Trp and Tyr side chains in terms of relative populations of g⁺, g^?, and t staggered rotamers around the χ₁ dihedral angle indicated that in all the peptides studied (a) rotation of Trp indole ring in cis (Trp-Pro) isomers is strongly restricted, and (b) rotation of Tyr phenol ring is relatively free. The most preferred χ₁ rotamer of Trp (0.8-0.9 molar fraction) was assigned as the t one on the basis of a large value of the vicinal coupling constant between the high-field H^β and carbonyl carbon atoms of Trp, estimated for the cis (Pro₁) form of H-Trp-Pro-Tyr-OH from a ¹H, ¹³C correlated spectroscopy ¹H detected multiple quantum experiment. This indicates that cis ? trans equilibrium in the Trp-Pro fragment is governed by nonbonding interactions between the pyrrolidine (Pro) and indole (Trp) rings. A molecular model of the terminal cis Trp-Pro dipeptide fragment is proposed, based on the presented nmr data and the results of our molecular mechanics modeling of low-energy conformers of the peptides, reported elsewhere. © 1993 John Wiley & Sons, Inc.     

Interactions of calf spleen purine nucleoside phosphorylase with formycin B and its aglycone - spectroscopic and kinetic studies

Phosphorolysis of 7-methylguanosine by calf spleen purine nucleoside phosphorylase (PNP) is weakly inhibited, uncompetitively, by Formycin B (FB) with Ki = 100 micro M and more effectively by its aglycone (7KPP), IC50 35-100 micro M. In striking contrast, 7KPP inhibits the reverse reaction (synthesis of 8-azaguanosine from 8-azaguanine) competitively, with Ki approximately 2-4 micro M. Formycin B forms only a weakly fluorescent complex with PNP, and 7KPP even less so, indicating that both ligands bind as the neutral, not anionic, forms. 7KPP is a rare example of a PNP non-substrate inhibitor of both the phosphorolytic and reverse synthetic pathways.     

Characterization of the low pH solution structure and dynamics of the region 4 of Escherichia coli RNA polymerase sigma70 subunit

Solution structure of the region 4 of sigma(70) subunit of Escherichia coli RNA polymerase, whose 4.2 subregion is involved in specific recognition of the -35 element of cognate promoters, has not been yet studied. Using multinuclear NMR spectroscopy, we have assigned recently all the backbone and aliphatic side-chain (13)C resonances for a recombinant His(6)-tagged protein containing the whole region 4 and a part of region 3.2 of sigma(70) in aqueous solution at pH 2.8 (Poznański, J., Zhukov, I., Bolewska, K., and Wierzchowski, K. L. (2001) J. Biomol. NMR 20, 181-2). The protein proved to be sufficiently soluble and did not aggregate only in the protonated state. In this paper, the structure and dynamics of this state at pH 2.8 have been extensively examined using CD and NMR spectroscopy. Both analysis of CD spectra and NMR observables (secondary chemical shifts of the (13)Calpha, (13)CO, and (1)Halpha nuclei and of vicinal (3)J(HNH)(alpha) coupling constants) indicated that a significant amount of helical structure remained in the protonated protein. The amount of this structure increased upon deprotonation of carboxylic amino acids, as shown by pH titration CD experiments. 2,2,2-Trifluoroethanol induced an even more extensive build up of this structure. Distribution along the protein sequence of the secondary shifts and (3)J(HNH)(alpha) couplings demonstrated partition of the helical secondary structure into three helices located similarly as in the crystal structures of the homologous region 4 of the sigma(A) subunit of Thermus aquaticus RNA polymerase (Campbell, E. A., Muzzin, O., Chlenov, M., Sun, J. L., Olson, A., Weinman, O., Trester-Zedlitz, M. L., and Darst, S. A. (2002) Mol. Cell 9, 527-39) and sigma(70) of the Thermus thermophilus RNA polymerase (Vassylyev, D. G., Sekine, S., Laptenko, O., Lee, J., Vassylyeva, M. N., Borukhov, S., and Yokoyama, S. (2002) Nature 417, 712-9.). Spectral density analysis of NMR relaxation parameters, R(1) and R(2), and [(1)H]-(15)N heteronuclear NOEs indicated that backbone fluctuations in the whole region embracing the three helices and intervening nonhelical sequences are severely restricted on the nanosecond time scale as compared with the N- and C-terminal protein segments. Inspection of the side-chain contacts stabilizing the crystal structures well explains the observed folding and solution properties of sigma(70)(4) protein in its protonated state.