Studies on transfer ribonucleic acids and related compounds. XXXII. Synthesis of ribonucleotides corresponding to residues 1-5 and 6-10 of tRNAfMet from E. coli and their base conversion analogs.

E. Coli tRNAfMet fragments, C-G-C-G-Gp (bases 1-5), U-G-C-G-Gp (base 1 transition, analog) pG-G-C-G-Gp (base 1 transversion analog) and pG-G-s4U-G-Gp (bases 6-10) were synthesized by triester methods using 2'-O-(o-nitrobenzyl) nucleotides including a 3',5'-bisphosphorylated guanosine derivative. The s4U containing pentanucleotide was derived from the pG-G-C-G-Gp by treatment with liquid hydrogen sulfide.

Influence of terminal 3' phosphates or 2',3'-cyclic phosphates on the conformations of oligoriboadenylates, oligoribocytidylates, and the corresponding monomers.

The circular dichroism spectra of chemically synthesized adenylate and cytidylate dinucleotides and trinucleotides bearing terminal 3' phosphates have been compared under a variety of conditions with the spectra obtained from the corresponding oligomers with 2',3'-terminal cyclic phosphate groups. Similar comparisons for the mononucleotides are also presented. Although the base stacking of an oligomer with a terminal cyclic phosphate might be expected to be greater than that of the corresponding oligomer with a 3' phosphate from charge repulsion considerations, the magnitudes of the Cotton effects in the former class are always considerably smaller than those in the latter class.

Inhibitory effect of methylated derivatives of guanylic acid for protein synthesis with reference to the functional structure of the 5'-'cap' in viral messenger RNA.

Guanylic acid modified variously with methyl groups on base or sugar moieties were synthesized chemically and their inhibitory effects on protein synthesis were tesetd in a wheat germ cell-free system using mRNAs from cytoplasmic polyhedrosis virus and tobacco mosaic virus. The confronting dinucleotide m7G5' pppA that corresponds to the most simple 'cap' structure of an eukaryotic mRNA is a strong inhibitor of protein synthesis, but non-methylated G5' pppA or G5' ppA is not inhibitory. The strong inhibitory effect is observed only by 7-methylguanylic acid (pm7G).

Polynucleotides. LVII. Synthesis and properties of poly (2'-chloro-2'-deoxyinosinic acid).

Poly (2'-chloro-2'-deoxyinosinic acid) [poly(Icl)] was synthesized from Icl 5'-DP by polymerization with polynucleotide phosphorylase. UV absorption properties of poly(Icl) are very similar to those of poly(I). Poly(Icl) adopted a multi-stranded ordered form in the presence of 0.

Influence of various 2- and 2'-substituted polyadenylic acids on murine leukemia virus reverse transcriptase.

Several newly synthesized polyadenylic acid [(A)n] analogues, including poly(2-methyladenylic acid) [(m2A)n], poly(2-ethyladenylic acid) [(e2A)n], poly(2-isopropyladenylic acid) [(i-pro2A)n], poly(2-methylthioadenylic acid) [(ms2A)n], poly(2-ethylthioadenylic acid) [(e2A)n], poly(2'-fluoro-2'-deoxyadenylic acid) [(dAfl)n] and poly(2'-azido-2'-deoxyadenylic acid) [(dAz)n] have been evaluated for their effects on the RNA-directed DNA polymerase (reverse transcriptase) activity of Moloney murine leukemia virus; (m2A)n and (e2A)n did not markedly affect reverse transcriptase activity, (dAfl)n served as an efficient template for the reverse transcriptase reaction, and (i-pro2A)n, (ms2A)n, (es2A)n and (dAz)n strongly inhibited reverse transcriptase activity. (dAfl)n also served as an efficient template (Km : 0.025 micron) for the reverse transcriptase of avian myeloblastosis virus.

Polynucleotides XLVII. Synthesis and properties of poly(2-methylthio- and 2-ethylthioadenylic acid). Formation of non-Watson-Crick type complexes.

Poly(2-methyl- and 2-ethylthioadenylic acid) were prepared by polymerization of corresponding diphosphates with Escherichia coli polynucleotide phosphorylase. These polynucleotides have relatively large hypochromicity of 30-35%. Acid titration of these polymers showed abrupt transition at pH 5.

A new method for 3'-labelling of polyribonucleotides by phosphorylation with RNA ligase and its application to the 3'-modification for joining reactions.

P1-Adenosine 5'-P2-o-nitrobenzyl pyrophosphate (nbzlppA) has been synthesized as a substrate for T4 RNA ligase catalyzed 3'-phosphorylation. Incubation of oligoribonucleotides and nbzlppA with RNA ligase yielded oligoribonucleotides having a 3'-o-(o-nitrobenzyl) phosphate. Photochemical removal of the o-nitrobenzyl group provided the free 3'-phosphate.

The crystal and molecular structure of 8-methyladenosine 3'-monophosphate dihydrate.

8-Methyladenosine 3'-monophosphate dihydrate was synthesized and crystallized in the monoclinic space group P21 with the unit cell dimensions: a = 9.095(2) A, b = 16.750(3) A, c = 5.

Dinucleoside monophosphate having a high anti conformation: the crystal structure of 8,2'-S-cycloadenylyl-(3'-5')-8,2'-S-cycloadenosine hydrochloride.

The crystal and molecular structure of 8,2'-S-cycloadenylyl-(3'-5')-8,2'-S-cycloadenosine (AspAs) hydrochloride has been determined by X-ray method. The conformation of two independent AspAs molecules found in an asymmetric unit are almost identical to each other. The torsion angles concerning the sugar-phosphate backbone are different from those in crystalline dinucleoside monophosphates so far determined by X-rays.

Synthesis of the nascent strand of tRNAfMet from E coli.

Oligonucleotides corresponding to the total tRNAfMET FROM E. coli have been synthesized. These fragments were joined by using RNA ligase to yield quarter molecules.

Joining of 3'-modified oligonucleotides by T4 RNA ligase. Synthesis of a heptadecanucleotide corresponding to the bases 61--77 from Escherichia coli tRNAfMet.

Chemically synthesized fragments corresponding to the 3' end of tRNAfMet from Escherichia coli were joined by T4-induced RNA ligase to yield a heptadecanucleotide (bases 61--77). The 3' terminus of C-C-A was modified by introduction of the ethoxymethylidene group to prevent intra- and intermolecular self-joining reactions at the 3' end. The terminal trimer was phosphorylated using polynucleotide kinase and joined to C-A-A with RNA ligase.

Polynucleotides. XLIV. Synthesis and properties of poly (2-azaadenylic acid) and poly(2-azainosinic acid).

Chemically synthesized 2-azaadenosine 5'-diphosphate (n2ADP) and 2-azainosine 5'-diphosphate (n2IDP) were polymerized to yield poly(2-azaadenylic acid), poly(n2A), and poly(2-azainosinic acid), poly(n2I), using Escherichia coli polynucleotide phosphorylase. In neutral solution, poly(n2A) and poly(n2I) had hypochromicities of 32 and 5.5%, respectively.

Carbon-13 nuclear-magnetic-resonance spectra of adenine cyclonucleosides and their phosphates. Effects of neighboring groups for elucidation of fine structure of nucleosides and nucleotides.

Carbon-13 nuclear magnetic resonance spectra of adenine cyclonucleosides, which have a fixed glycosidic conformation in an anti range, and their isopropylidene and phosphate esters are reported; those of 9-beta-D-arabinofuranosyladenine and its 5'-phosphate are also presented. The chemical shifts of the base carbons are affected not only by the bridging atom but also by the position of the bridged sugar carbon which determine the planarity of the third ring formed by cyclization between the base and the sugar. The effects of glycosidic conformation on the sugar-carbon chemical shifts are discussed by comparison of the data for 8:5'-cycloadenosines with the data for adenosine and its 8-substituted derivatives.

Polynucleotides. LVI. Synthesis and properties of poly(2-deoxy-2'-fluoroinosinic acid).

Poly (2'-deoxy-2'-fluoroinosinic acid) [ poly(If)] was synthesized by polymerization of 2'-deoxy-2'-fluoroinosine 5'-diphosphate catalyzed by Escherichia coli polynucleotide phosphorylase. Although the UV absorption properties of poly(If) closely resembled those of poly(I), thermal melting curves at Na+ concentrations of 0.15M and 0.

Interaction between actomyosin and 8-substituted ATP analogs.

Various 8-substituted ATP analogs were synthesized, and their reactions with myosin and actomyosin were studied. The nucleoside triphosphates (NTPs) with an amino group at the 6 position and hydrogen at the 8 position, and formycin 5'-triphosphate (FTP) were hydrolyzed by myosin very slowly in the presence of Mg2+ and rapidly in the presence of EDTA and K+. In contrast, NTPs with substitution of the 8 position, other than FTP, were readily hydrolyzed by myosin in the presence of Mg2+ but were hardly hydolyzed in the presence of EDTA and K+.

Conformationally restricted adenine nucleotide analogs in mitochondrial adenine nucleotide transport.

The conformationally restricted adenine nucleotide analogs 8,3'anhydro-8-oxy-9-(beta-D-xylofuranosyl)adenine-5'-O-tri(di)-phosphate (I), and 8,2'-anhydro-8-oxy-9-(beta-D-arabinofuranosyl) adenine-5'-O-tri(di)-phosphate (II), were prepared chemically as their alpha-32P-labelled compounds and compared with syn-structured 8-bromo AT(D)P in mitochondrial adenine nucleotide translocation. The experimental findings demonstrate that the heterocycle-ribose orientation affects the carrier mediated adenine nucleotide transport very strongly, i.e.

Polynucleotides. LII. Synthesis and properties of poly(2'-deoxy-2'-fluoroadenylic acid).

2'-Deoxy-2'-fluoroadenosine was chemically transformed to its 5'-diphosphate and polymerized with polynucleotide phosphorylase to give poly(2'-deoxy-2'-fluoroadenylic acid) [poly(Af)]. Polymerization proceeded smoothly as in the case of poly(A) and the yield of the polymerization was 55%. The UV absorption spectra of poly(Af) closely resembled those of poly(A) and the hypochromicity was 32% at pH 7.

Polynucleotides. L. Synthesis and properties of poly (2'-chloro-2'-deoxyadenylic acid) and poly (2'-bromo-2'-deoxyadenylic acid).

Poly (2'-chloro-2'-deoxyadenylic acid) and poly (2'-bromo-2'-deoxyadenylic acid) were synthesized from the corresponding diphosphates with the aid of polynucleotide phosphorylase from E. coli. UV, CD, acid titration and mixing with poly (U) were investigated.

Biologic activities of poly (2-azaadenylic acid) and poly (2-azainosinic acid).

Poly (2-azaadenylic acid) [(aza2A)n] and poly(2-azainosinic acid [(aza2I)n], two newly synthesized analogues of (A)n and (I)n, in which CH-2 of the purine ring is replaced by a nitrogen atom, have been evaluated in various biological assay systems. (Aza2A) n formed a complex with (U)n and (br5U)n, and (aza2I)n formed a complex with (C)n and (br5C)n, but these complexes were markedly destabilized relative to the corresponding (A)n or (I)n complexes. The (aza2A)n-and (aza2I)n-derived complexes failed to stimulate the production of interferon in primary rabbit kidney cells and human diploid fibroblasts, under conditions (A)n.

Polynucleotides. XLV Synthesis and properties of poly(2'-azido-2'-deoxyinosinic acid).

Poly (2'-azido-2'-deoxyinosinic acid), [poly (Iz)], was synthesized from 2'-azido-2'-deoxyinosine diphosphate by the action of polynucleotide phosphorylase. Poly (Iz) has UV absorption properties similar to poly (I) and hypochromicity of 11% at 0.15M Na+ and neutrality.