Oxygen-17 n.m.r. of peptides

Peptide-17O chemical shifts of linear dipeptides with and without protecting groups in H2O, CH3OH, CH2Cl2, CHCl3, CCl4, CH3CN and DMSO were between 256-350 ppm downfield from external water. Increasing solvent H-bond donating ability correlated with shifts to higher field. The 17O resonance of several cyclic dipeptides appeared at higher field relative to comparable linear dipeptides (303-317 p.p.m. vs. 327-337 p.p.m.). Separate signals were simultaneously observed by 13C and 17O n.m.r. for cis and trans N-tert.-butyl-formamide in binary mixtures with H2O, (CH3)2CO, and CCl4. The differences in the 17O nuclear screening of the amide isomers and most probably for cis and trans peptides were independent of contributions from H-bonding at the amide or peptide linkage, apparently reflecting differences between geometric isomers in electron distribution and through space effects. Peptide-17O of Gly-Ala, Gly-Leu and Gly-Glu in aqueous solution experienced upfield shifts of 6-12 p.p.m. and 12-16 p.p.m. upon deprotonation of the C-terminal COOH and of the N-terminal NH3+ groups respectively. These observations were rationalized in terms of the attendant changes in substituent effects, especially on the pi electron donating ability of the N atom at the peptide linkage and increased partial negative charge on the peptide oxygen. Temperature studies of peptide-17O of Gly-Ala between pH 1.5-9.0 revealed a chemical shift coefficient of 0.08 p.p.m./degree K and similar behavior of T1 and T2 relaxation times. Ea for molecular rotation was 5 kcal/mol between 301-331 degrees K. Rotational correlation times, tau c, were within the range expected from the Stokes-Einstein relation.     

Synthesis and oxidation of carboxylate-bridged diiron(II) complexes with substrates tethered to primary alkyl amine ligands

The synthesis and crystallographic characterization of a series of diiron(II) complexes with sterically hindered terphenyl carboxylate ligands and alkyl amine donors are presented. The compounds [Fe(2)(mu-O(2)CAr(Tol))(4)(L)(2)] (L=NH(2)(CH(2))(2)SBn (1); NH(2)(CH(2))(3)SMe (2); NH(2)(CH(2))(3)CCH (3)), where (-)O(2)CAr(Tol) is 2,6-di(p-tolyl)benzoate, and [Fe(2)(mu-O(2)CAr(Xyl))(2)(O(2)CAr(Xyl))(2)(L)(2)] (L=NH(2)(CH(2))(3)SMe (4); NH(2)(CH(2))(3)CCH (5)), where (-)O(2)CAr(Xyl) is 2,6-di(3,5-dimethylphenyl)benzoate, were prepared as small molecule mimics of the catalytic sites of carboxylate-bridged non-heme diiron enzymes. The compounds with the (-)O(2)CAr(Tol) carboxylate form tetrabridged structures, but those containing the more sterically demanding (-)O(2)CAr(Xyl) ligand have only two bridging ligands. The ancillary nitrogen ligands in these carboxylate-rich complexes incorporate potential substrates for the reactive metal centers. Their oxygenation chemistry was studied by product analysis of the organic fragments following decomposition. Compound 1 reacts with dioxygen to afford PhCHO in approximately 30% yield, attributed to oxidative dealkylation of the pendant benzyl group. Compound 3 decomposes to form Fe(II)Fe(III) and Fe(III)Fe(IV) mixed-valence species by established bimolecular pathways upon exposure to dioxygen at low temperatures. Upon decomposition, the alkyne-substituted amine ligand was recovered quantitatively. When the (-)O(2)CAr(Tol) carboxylate was replaced by the (-)O(2)CAr(Xyl) ligand in 5, different behavior was observed. The six-coordinate iron(III) complex with one bidentate and two monodentate carboxylate ligands, [Fe(O(2)CAr(Xyl))(3)(NH(2)(CH(2))(3)CCH)(2)] (6), was isolated from the reaction mixture following oxidation.     

Synthesis of a carboxamide linked T*T dimer and its incorporation in oligonucleotides.

The condensation of 5'-O-protected 3'-O-(2-aminoethyl)thymidine with 1,2-dideoxy-1-thyminyl-beta-D-erythro-pentofuranuronic acid gives a T*T dimer with * representing a 3'-OCH2CH2NHC(O)-4' linkage connecting the two pentofuranosyl moieties. The incorporation of this dimer in oligonucleotide sequences show only moderately lowered Tm values when hybridized with a complementary DNA relative to the unmodified DNA duplex. Consistently, no looped-out or bubble-type structure could be detected in DNA duplexes with an internal T*T module. Moreover, the 5-atom carboxamide linker causes complete stop on DNA polymerization and on exonuclease III degradation.     

Synthesis and fluorescence studies of thiazole orange tethered onto oligonucleotide: development of a self-contained DNA biosensor on a fiber optic surface

Thiazole orange dyes were derivatized with ethylene glycol linkers of various lengths, and were covalently linked to the 5' end of the oligonucleotides after solid-phase synthesis. The labeled oligonucleotides exhibited enhanced fluorescence upon hybridization to complementary DNA sequences at the surfaces of optical fibers, providing for a self-contained labeling strategy. It was determined that the melt temperatures of DNA hybrids using one mixed polypyrimidine base oligonucleotide sequence were dependent on the length of the tethers, and that the melt temperature could be shifted by more than 10 degrees C when tethers were introduced.     

N-(3-Triethoxysilylpropyl)-4-(N'-maleimidylmethyl)cyclohexanamide (TPMC): a heterobifunctional reagent for immobilization of oligonucleotides on glass surface

A new heterobifunctional reagent, namely, N-(3-triethoxysilylpropyl)-4-(N'-maleimidylmethyl)cyclohexanamide (TPMC) was developed and its potentiality for fixing of thiol (-SH) modified oligonucleotides were tested. The covalent attachment of oligonucleotides with the reagent was achieved through its maleimide functionality at one end via stable thioether linkage while the other end bearing triethoxysilyl functionality has been utilized for coupling with the virgin glass surface with simplified methodologies. Immobilization of oligonucleotides was achieved by two alternating ways. The PATH-1 involves formation of conjugate of reagent and SH-modified oligonucleotides through thioether linkage and was subsequently immobilized on unmodified glass surface through triethoxysilyl group and alternatively, PATH-2 involves reaction of reagent first with unmodified glass surface to get maleimide functionality on the surface and then the SH-modified oligonucleotides were immobilized via thioether linkage. The specificity of immobilization was tested by hybridization study with complementary fluorescein labeled oligonucleotide strand.     

Structure of leukotriene C. Identification of the amino acid part.

Leukotriene C, a “Slow Reacting Substance” (SRS) from mouse mast cell tumors, was earlier shown to be a derivative of 5-hydroxy-7,9,11,14-eicosatetraenoic acid with a cysteine containing substituent in thioether linkage at C-6 (Murphy, R.C., Hammarström, S., Samuelsson, B.: Proc. Natl. Acad. Sci. USA, $\text{76}$, 4275–4279 (1979)). The substituent has now been identified as γ-glutamylcysteinylglycine (glutathione).     

Parallel double stranded helices and the tertiary structure of nucleic acids

Thermal denaturation of four oligonucleotides, viz. 3'-d(AT)5pO(CH2)6Opd(AT)5-3'(par(AT], 3'-d(AT)5pO(CH2)6Opd(AT)5-5'(anti(AT],3'-d(A)10pO(CH2) 6Op(T)10-3'(par(A-T], and 3'-d(A)10pO(CH2)6Opd(T)10-5' (anti(A-T], was studied in 0.01 M phosphate buffer, pH 7, in the presence of 0.1, 0.25, 0.5 and 1.0 M NaCl. All the oligomers were found to exist at a lower temperature (0 to 20 degrees C) as complexes composed either of two oligomer molecules (a canonical duplex) or of more oligomer molecules whereas, at a higher temperature (30 to 70 degrees C), they formed hairpins with a parallel (par(AT) and par(A-T] or antiparallel (anti(AT) and anti(A-T) orientation of the chains. Melting curves (A260(T] were used to calculate thermodynamic parameters for the formation of hairpins and "low-temperature" duplexes. Experiments on ethidium bromide binding to the oligonucleotides have shown that the oligomer anti(A-T) exists, at a low ionic strength, as a four stranded complex ("quadruplex") contains two antiparallel helices, d(A).d(T), which have a parallel orientation and are bound to one another owing to the formation of additional hydrogen bonds between nucleic acid bases. The possible biological function of quadruplexes is discussed.     

O6-ethylguanine carcinogenic lesions in DNA: an NMR study of O6etG.T pairing in dodecanucleotide duplexes

High-resolution two-dimensional NMR studies are reported on the self-complementary d-(C1-G2-C3-O6etG4-A5-G6-C7-T8-T9-G10-C11-G12) duplex (designated O6etG.T 12-mer) containing two symmetrically related O6etG.T lesion sites located four base pairs in from either end of the duplex. Parallel studies were undertaken on a related sequence containing O6meG.T lesion sites (designated O6meG.T 12-mer) in order to evaluate the influence of the size of the alkyl substituent on the structure of the duplex and were undertaken on a related sequence containing G.T mismatch sites (designated G.T 12-mer duplex), which served as the control duplex. The exchangeable and nonexchangeable proton and the phosphorus nuclei have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOE) and correlated spectra of the O6etG.T 12-mer, O6meG.T 12-mer, and G.T 12-mer duplexes in H2O and D2O solutions. The distance connectivities observed in the NOESY spectra of the O6alkG.T 12-mer duplexes establish that the helix is right-handed and all of the bases adopt an anti conformation of the glycosidic torsion angle including the O6alkG4 and T9 bases at the lesion site. The imino proton of T9 at the O6alkG.T lesion sites resonates at 8.85 ppm in the O6etG.T 12-mer duplex and at 9.47 ppm in the O6meG.T 12-mer duplex. The large upfield shift of the T9 imino proton resonance at the O6alkG4.T9 lesion site relative to that of the same proton in the G4.T9 wobble pair (11.99 ppm) and the A4.T9 Watson-Crick pair (13.95 ppm) in related sequences establishes that the hydrogen bonding of the imino proton of T9 to O6alkG4 is either very weak or absent. The imino proton of T9 develops NOEs to the CH3 protons of the O6etG and O6meG alkyl groups across the base pair, as well as to the imino and H5 protons of the flanking C3.G10 base pair and the imino and CH3 protons of the flanking A5.T8 base pair in the O6alkG.T 12-mer duplexes. These observations establish that the O6alkG4 and T9 residues are stacked into the duplex and that the O6CH3 and O6CH2CH3 groups of O6alkG4 adopt a syn orientation with respect to the N1 of the alkylated guanine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hybrid characteristics of oligonucleotides consisting of isonucleoside 2',5'-anhydro-3'-deoxy-3'-(thymin-1-yl)-D-mannitol with different linkage modes

Oligonucleotides consisting of the isonucleoside repeating unit 2',5'-anhydro-3'-deoxy-3'-(thymin-1-yl)-D-mannitol (4) were synthesized with the monomeric unit 4 incorporated into oligonucleotides as 1'-->4' linkage 4a (oligomer I) or 6'-->4' linkage 4b (oligomer II). The hybrid properties of the two oligonucleotides I and II with their complementary strands were investigated by thermal denaturation and CD spectra. Oligonucleotide I (4a) formed a stable duplex with d(A)(14) with a slightly reduced T(m) value of 36.6 degrees C, relative to 38.2 degrees C for the control duplex d(T)(14)/d(A)(14), but oligomer II (4b) failed to hybridize with a DNA complementary single strand. The spectrum of the duplex oligomer I/d(A)(14) showed a positive CD band at 217 nm and a negative CD band at 248 nm attributable to a B-like conformation. Molecular modeling showed that in the case of oligomer I: the C6' hydroxy group of each unit could be located in the groove area when hybridized to the DNA single strand, which might contribute additional hydrogen bonding to the stability of duplex formation.     

Internal mobility in a double-stranded B DNA hexamer and undecamer. A time-dependent proton-proton nuclear Overhauser enhancement study

The internal mobility of the deoxyribose H2'-H2" and base C(H5)-C(H6) and T(CH3)-T(H6) vectors has been investigated by means of time-dependent nuclear Overhauser enhancement (NOE) measurements in a B DNA hexamer and undecamer. Cross-relaxation rates between these proton pairs are determined from the initial slopes of the time development of the NOEs, and, as the interproton distances between these proton pairs are fixed, apparent correlation times for the 3 interproton vectors are calculated from the cross-relaxation rate data. It is shown that there is little residue to residue variation in the cross-relaxation rates of the interproton vectors within each oligonucleotide, that the mean apparent correlation times of the C(H5)-C(H6) and T(CH3)-T(H6) vectors are approximately equal and significantly greater than that of the H2'-H2" vectors, and that the data for the H2'-H2" vectors of both oligonucleotides and the C(H5)-C(H6) and T(CH3)-T(H6) vectors of the undecamer cannot be accounted for by isotropic tumbling alone. The data are analysed in terms of a two motion model with isotropic tumbling and a single internal motion. The relaxation time of the internal motion at 23 degrees C is less than or equal to 1 ns for the H2'-H2" vectors of both oligonucleotides and less than or equal to 3 ns for the C(H5)-C(H6) and T(CH3)-T(H6) vectors of the undecamer. In the case of the H2'-H2" vectors, however, the amplitude of the internal motion is found to be too large to be compatible with the known stereochemistry of DNA. This finding can only be explained by invoking additional degrees of internal freedom with a larger number of internal motions of small amplitude of the type deduced from the analysis of crystallographic thermal factors [(1984) J. Mol. Biol. 173, 361-388].     

Chemical and enzymatic properties of bridging 5'-S-phosphorothioester linkages in DNA.

We describe physicochemical and enzymatic properties of 5' bridging phosphorothioester linkages at specific sites in DNA oligonucleotides. The susceptibility to hydrolysis at various pH values is examined and no measurable hydrolysis is observed at pH 5-9 after 4 days at 25 degrees C. The abilities of three 3'- and 5'-exonuclease enzymes to hydrolyze the DNA past this linkage are examined and it is found that the linkage causes significant pauses at the sulfur linkage for T4 DNA polymerase and calf spleen phosphodiesterase, but not for snake venom phosphodiesterase. Restriction endonuclease (Nsi I) cleavage is also attempted at a 5'-thioester junction and strong resistance to cleavage is observed. Also tested is the ability of polymerase enzymes to utilize templates containing single 5'-S-thioester linkages; both Klenow DNA polymerase and T7 RNA polymerase are found to synthesize complementary strands successfully without any apparent pause at the sulfur linkage. Finally, the thermal stabilities of duplexes containing such linkages are measured; results show that T m values are lowered by a small amount (2 degrees C) when one or two thioester linkages are present in an otherwise unmodified duplex. The chemical stability and surprisingly small perturbation by the 5' bridging sulfur make it a good candidate as a physical and mechanistic probe for specific protein or metal interactions involving this position in DNA.     

Parallel double helix and tertiary structure of nucleic acids

The thermal denaturation of four oligonucleotides, viz. 3'-d(AT)5pO(CH2)6Opd(AT)5-3' (parAT), 3'-d(AT)5pO(CH2)5Opd(AT)5-5' (antiAT), 3'-d(A)10pO(CH2)6Op(T)10-3' (parA-T) and 3'd(A)10pOX X (CH2)6Opd(T)10-5' (antiA-T) in 0.01 M phosphate buffer at pH 7 in presence 0.1, 0.25, 0.5 and 1.0 M NaCl have been studied. It was shown that at lower temperature (0-20 degrees C) all oligomeres exist as complexes of two (canonic duplex) or four (eight) molecules of oligonucleotides, but at higher temperature (30-70 degrees C)- as hairpins with parallel (parAT and parA-T) of antiparallel (antiAT and antiA-T) orientation of chains. Thermodinamic parameters of separated strands-hairpins and hairpins--"low temperature complexes" transition were computated from the melting curves [A260 (T)] by nonlinear regression. AntiA-T was shown by ethidium bromide binding to exist at low strength (0.01 M phosphate buffer without NaCl) as four-stranded complex from two antiparallel double stranded helices parallely oriented and bonded by satisfy hydrogen-bond of groups not involved in WC-pairing. At higher ionic strength the two of such tetramers was conjugated by hydrophobic interaction into octamers. We speculate that four-stranded complexes serves to bring together, and zipper up two antiparallel double stranded helices at replication of DNA, cross-over of gomologues chromosomes and other biochemically important processes.     

RNA mimetics: oligoribonucleotide N3'-->P5' phosphoramidates.

The synthesis and properties of novel RNA mimetics, oligoribonucleotide N3'-->P5' phosphoramidates, are described. These oligonucleotides contain 3'-aminoribonucleosides connected via N3'-->P5' phosphoramidate linkages, replacing the native RNA O3'-->P5' phosphodiester counterparts. The key monomers 2'-t-butyldimethylsilyl-3'-(monomethoxytrityl)-amino-5'-phospho ramidi tes were synthesized and used to prepare the oligonucleotide phosphoramidates using a solid phase methodology based on the phosphoramidite transfer reaction. Oligoribophosphoramidates are very resistant to enzymatic hydrolysis by snake venom phosphodiesterase. These compounds form stable duplexes with complementary natural phosphodiester DNA and RNA strands, as well as with 2'-deoxy N3'-->P5' phosphoramidates. The increase in melting temperature, Delta T m, was 5-14 degrees C relative to the 2'-deoxy phosphoramidates for decanucleotides. Also, the thermal stability of the ribophosphoramidatehomoduplex was noticeably higher (Delta T m +9.5 degrees C) than that for the isosequential 2'-deoxy phosphoramidate complex. Furthermore, the oligopyrimidine ribo N3'-->P5' phosphoramidate formed an extremely stable triplex with an oligopurine/oligopyrimidine DNA duplex with Delta T m +14.3 degrees C relative to the 2'-deoxy N3'-->P5' phosphoramidate counterpart. The properties of the oligoribonucleotide N3'-->P5' phosphoramidates indicate that these compounds can be used as hydrolytically stable structural and functional RNA mimetics.     

Sequence specificity modification of double-stranded DNA with an alkylating derivative of oligodeoxyribonucleotide pT(CT)6

It is shown that in slightly acidic solution (pH approximately 5.3) reagent CIRCH2NHpT(CT)6 (RCl = -C6H4-N(CH3)CH2CH2Cl) modifies a double-stranded DNA fragment (120 b. p.) containing A(GA)6.T(CT)6 sequence at a single nucleotide residue, viz. G29 located near to this sequence in the DNA chain. The location of this modification point suggests formation of a triple-stranded reactive complex with parallel orientation of the pyrimidine oligonucleotide moiety of the reagent and pyrine sequence of the target DNA. Analysing the modification extent dependence of the reagent concentration the association constant Kx between the reagent and DNA was calculated (Kx = (0.95 +/- 0.03).10(5) M-1, 25 degrees C, pH = 5.3, [NaCl] = 0.1 M). The modification by the reagent ClRCH2NHpT(m5CT)6 has the same quantitative characteristics as in the case of ClRCH2NHpT(CT)6.     

Comparison of the base pairing properties of a series of nitroazole nucleobase analogs in the oligodeoxyribonucleotide sequence 5'-d(CGCXAATTYGCG)-3'.

The nucleoside analogs 1-(2'-deoxy-beta-D-ribofuranosyl)- 3-nitropyrrole (9), 1-(2'-deoxy-beta-D-ribofuranosyl)-4-nitropyrazole (10), 1-(2'-deoxy-beta-D-ribofuranosyl)-4-nitroimidazole (11) and 1-(2'-deoxy-beta-D-ribofuranosyl)-5-nitroindole (21) were incorporated into the oligonucleotide 5'-d(CGCXAATTYGCG)-3'in the fourth position from the 5'-end. Procedures for synthesis of two of the nitroazole nucleosides, 10 and 11, were developed for this study. Each of the nitroazoles was converted into a 3'-phosphoramidite for oligonucleotide synthesis by conventional automated protocols. Four oligonucleotides were synthesized for each modified nucleoside in order to obtain duplexes in which each of the four natural bases was placed opposite (position 9) the nitroazole. In order to assess the role of the nitro group on base stacking interaction, sequences were also synthesized in which the fourth base was 1-(2'-deoxy-beta-D-ribofuranosyl)pyrazole. Corresponding sequences containing an abasic site, as well as sequences containing inosine, were synthesized for comparison. Thermal melting studies yielded T m values and thermodynamic parameters. Each nucleoside analog displayed a unique pattern of base pairing preferences. The least discriminating analog was 3-nitropyrrole, for which T m values differed by 5 degrees C and Delta G 25 degrees C ranged from -6.1 to -6.5 kcal/mol. 5-Nitroindole gave duplexes with significantly higher thermal stability, with Tm values varying from 35.0 to 46.5 degrees C and -Delta G 25 degrees C ranging from 7.7 to 8.5 kcal/mol. Deoxyinosine (22), a natural analog which has found extensive use as a universal nucleoside, is far less non-discriminating than any of the nitroazole derivatives. Tm values ranged from 35.4 degrees C when paired with G to 62.3 degrees C when paired with C. The significance of the nitro substituent was determined by comparison of the base pairing properties of a simple azole nucleoside, 1-(2'-deoxy-beta-D-ribofuranosyl)pyrazole (12). The pyrazole-containing sequences melt at 10-20 degrees C lower than the corresponding nitropyrazole-containing sequences. On average, the pyrazole-containing sequences were equivalent in stability (average Delta G = -4.8 kcal/mol) to the sequences containing an abasic site (average Delta G = -4.7 kcal/mol).     

Linkage isomerization reaction of intrastrand cross-links in trans-diamminedichloroplatinum(II)-modified single-stranded oligonucleotides.

The stability of trans-(Pt(NH3)2[d(CGAG)-N7-G,N7-G]) adducts, resulting from cross-links between two guanine residues at d(CGAG) sites within single-stranded oligonucleotides by trans-diamminedichloro-platinum(II), has been studied under various conditions of temperature, salt and pH. The trans-(Pt(NH3)2[d(C GAG)-N7-G,N7-G]) cross-links rearrange into trans-(Pt(NH3)2[d(CGAG)-N3-C,N7-G]) cross-links. The rate of rearrangement is independent of pH, in the range 5-9, and of the nature and concentration of the salt (NaCl or NaCIO4) in the range 10-400 mM. The reaction rate depends upon temperature, the t1/2 values for the disappearance of the (G,G) intrastrand cross-link ranging from 120 h at 30 degrees C to 70 min at 80 degrees C. The linkage isomerization reaction occurs in oligonucleotides as short as the platinated tetramer d(CGAG). Replacement of the intervening residue A by T has no major effect on the reaction. The C residue adjacent to the adduct on the 5' side plays a key-role in the reaction; its replacement by a G, A or T residue prevents the reaction occuring. No rearrangement was observed with the C residue adjacent to the adduct on the 3' side. It is proposed that the linkage isomerization reaction results from a direct attack of the base residue on the platinum(II) square complex.     

Peptide nucleic acid-DNA duplexes containing the universal base 3-nitropyrrole

A peptide nucleic acid (PNA) monomer containing the universal base 3-nitropyrrole was synthesized by coupling 1-carboxymethyl-3-nitropyrrole to ethyl N-[2-(tert-butoxycarbonylamino)ethyl]glycinate. The PNA sequence H-TGTACGTXACAACTA-NH2 (X = 3-nitropyrrole and C) and DNA sequence 5'-TGTACGTXACAACTA-3' were synthesized and thermal melting studies with the complementary DNA sequence 5'-TAGTTGTYACGTACA-3' (Y = A,C, G, T) compared. The T(m) data show that 3-nitropyrrole pairs indiscriminately with all four natural nucleobases as a constituent of either DNA or PNA. However, 3-nitropyrrole-containing PNA-DNA (average T(m) value = 51.1 degrees C) is significantly more thermally stable than 3-nitropyrrole-containing DNA-DNA (average T(m) value = 39.6 degrees C). From circular dichroism measurements, it is apparent that 3-nitropyrrole in the PNA strand causes a significant change in duplex structure.     

Recognition of specific DNA sequences by mitomycin C for alkylation.

Synthetic oligodeoxyribonucleotides were reacted with mitomycin C (MC) under conditions which restricted MC to monofunctional alkylating activity. The yields of monofunctional alkylation of oligonucleotides with variable sequence were determined by enzymatic digestion of the reaction mixture to unreacted nucleosides and the product of alkylation, a MC-deoxyguanosine adduct (2), followed by quantitative analysis by HPLC. The relative yields of 2 reflected relative monoalkylation reactivities. They were compared in a series of oligonucleotides having the sequence 5'-NGN' in which the 5'-base was varied while the 3'-base was kept constant as T. Under Na2S2O4 activation conditions a striking enhancement of the yield was observed at the 5'-CG sequence: 36%, compared to 2% at 5'-AG and 4.1% at 5'-TG. The 5'-GG sequence also showed enhanced reactivity although to a lesser extent (14.7%). The enhancements were specific to the duplex state of the oligonucleotides. Using NADPH:cytochrome c reductase as the reducing agent gave similar results. MC activated by acidic pH also displayed 5'-CG alkylation specificity. 10-Decarbamoyl-MC activated by Na2S2O4 showed the same 5'-CG specificity as MC. Replacement of deoxyguanosine by deoxyinosine in the opposite strand at a 5'-CG site abolished the enhancement of alkylation. Such replacement at a 5'-GG site had a similar effect. It was found that the base 3' to the guanine had only a relatively modest modulating effect on the enhanced reactivity of the G at the 5'-CG sequence. This 3'-base effect appeared to be independent of the 5'-base of the 5'-NGN' triplet. The order of reactivity is 3'-(C greater than T greater than A).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of chain-linkage on the structure of phosphatidyl choline bilayers. Hydration studies of 1-hexadecyl 2-palmitoyl-sn-glycero-3-phosphocholine.

While hydrated dipalmitoyl phosphatidylcholine (DPPC) forms tilted chain L beta' bilayers in the gel phase, the ether-linked analogue dihexadecyl phosphatidylcholine (DHPC) exhibits gel phase polymorphism. At low hydration DHPC forms L beta' phases but at greater than 30% H2O a chain-interdigitated gel phase is observed (Ruocco, M. J., D. S. Siminovitch, and R. G. Griffin. 1985. Biochemistry. 24:2406-2411; Kim, J.T., J. Mattai, and G.G. Shipley. 1987. Biochemistry. 26:6599-6603). In this study we report the behavior of a phosphatidylcholine (PC) with both types of chain linkage, 1-hexadecyl-2-palmitoyl-sn-glycero-3-phosphocholine (HPPC). HPPC has been investigated as a function of hydration using differential scanning calorimetry (DSC) and x-ray diffraction. By DSC, over the hydration range 5. 1-70.3 wt% H2O, HPPC exhibits two reversible transitions. The reversible main chain-melting transition decreases from 69 degrees C, reaching a limiting value of 40 degrees C at full hydration. X-ray diffraction patterns of hydrated HPPC have been recorded as a function of hydration at 20 degrees and 50 degrees C. At 50 degrees C, melted-chain L alpha bilayer phases are observed at all hydrations. At 20 degrees C, at low hydrations (less than 34 wt% H2O) HPPC exhibits diffraction patterns characteristic of bilayer gel phases similar to those of the gel phase of DPPC. In contrast, at greater than or equal to 34 wt% H2O, HPPC shows a much reduced bilayer periodicity, d = 47 A, and a single sharp reflection at 4.0 A in the wide angle region. This diffraction pattern is identical to that exhibited by the interdigitated phase of DHPC.(ABSTRACT TRUNCATED AT 250 WORDS)