Seiichi Uesugi

A linear relationship between electronegativity of 2′-substituents and conformation of adenine nucleosides

Abstract From the 1 H NMR studies of various 2′-substituted 2′-deoxyadenosines, a relationship between electronegativity of substituents and conformation of the sugar moiety was elucidated.

A possible recognition mode of mRNA cap terminal structure by peptide: Cooperative stacking and hydrogen-bond pairing interactions between m7GpppA and Trp-Leu-Glu

1H-NMR and fluorescence spectroscopic studies on the interaction between the Trp-Leu-Glu and m7GpppA have shown a specific binding mode, in which the pi-pi stacking interaction of the Trp indole ring and the hydrogen-bond pairing of Glu carboxyl side group with 7-methylguanine base are simultaneously formed.

Combination of Trp and Glu residues for recognition of mRNA cap structure : analysis of m7G base recognition site of human cap binding protein (IF-4E) by site-directed mutagenesis

Four mutants of the human cap binding protein (hCBP), in which Trp‐102, Glu‐103, Asp‐104 or Glu‐105 was changed to the aliphatic Leu or Ala, were prepared, and their cap binding abilities were examined. Cap binding abilities of two mutants. W102L (Trp‐102→Leu) and E105A (Glu‐105→Ala), were significantly decreased in comparison with the wild‐type hCBP. This result suggest that Trp‐102 and Glu‐105 are both necessary for the cap binding, and the most probable binding mode with the m7G of cap structure is the combination of the stacking by Trp‐102 and the hydrogen‐bond pairing by Glu‐105, as was already proposed from the model studies.

Improved Synthesis of 2′-Fluoro-2′-Deoxyadenosine and Synthesis and Carbon-13 NMR Spectrum of Its 3′,5′-Cyclic Phosphate Derivative1

Abstract Some improvements were made on synthetic method for 2′-fluoro-2′-deoxyadenosine (11). Thus 11 was obtained in an overall yield of 9.3% starting from adenosine. 2′-Fluoro-2′-deoxyadenosine 3′,5′-cyclic phosphate (13), an analogue of cAMP, was synthesized from 11. The carbon-13 NMR spectrum was measured. The sugar carbon signals can be unambiguously assigned since the C1′ C2′ and C3′ have different 13C-19F coupling constants. Comparison of the data with those of other 3′,5′-cyclic phosphate derivatives confirms the assignments of C3′ and C4′ signals previously proposed by us.

Secretion of recombinant ribonuclease T1 into the periplasmic space of Escherichia coli with the aid of the signal peptide of alkaline phosphatase

The ribonuclease T1 (RNase T1) gene was ligated to a synthetic gene for the signal peptide of Escherichia coli alkaline phosphatase. When this fusion gene was expressed in E.Coli under the control of the trp promoter, active RNase T1 having the correct N‐terminal sequence was secreted into the periplasmic space, indicating that the heterologous signal peptide had been cleaved off correctly. The enzyme could be readily purified from the periplasmic fraction with a yield of 1.8 mg from 1 liter culture. Adopting the same strategy, it was possible to produce a labile mutant of RNase T1 (Glu‐58 → Ala mutant) in E. coli, the yield of the purified mutant enzyme being 2.0 mg from 1 liter culture.

Studies of nucleosides and nucleotides— LIII : Purine cyclonucleosides-18. Selective tosylation of adenine nucleotides. Synthesis of 8,2′-anhydro-8-mercapto-9-β-D-arabinofuranosyladenine 5′- and 3′,5-cyclic phosphate

Abstract Adenosine 5′-monophosphate (AMP) and 8-bromo-AMP were tosylated with tosyl chloride in aqupous alkaline solutions to give 2′-tosyl nucleotides selectively. 8-Bromo-2′-tosyl AMP was easily cyclized to 8,2′anhydro-8-mercapto-9-β- d -arabinofuranosyladenine (8.2′-S-cycloadenosine) 5′-MP by treatment with H 2 S in pyridine or NaSH in water-DMF mixture. Desulfurization of the cyclonucleoside MP gave 2′-deoxy-AMP. 3′,5′-Cyclic 8-bromo AMP could also be tosylated and cyclized to give 8,2′-S-cycloadenosine 3′,5′-MP by the same procedure. Snake venom 5′-nucleotidase. E. coli and sheep intestine alkaline phosphatases hydrolyzed 8,2′-S-cycloadenosine 5′-phosphate to give 8,2′-S-cyclonucleoside.

Synthesis and Properties of 2′4′- and 3′-5′-Linked Ribodinucleoside Monophosphates Containing 2-Aminoadenosine and Uridine

Abstract Self complementary diribonucleoside monophosphates containing 2-aminoadenosine (n2A) and uridine (U) residues, (2′-5′) n2ApU (1), (3′-5′) n2ApU (2), (2′-5′) Upn2A (3) and (3′-5′) Upn2A (4), were synthesized by condensation of suitably protected nucleoside and nucleotide units using dicyclohexylcarbodiimide (DCC). The dimers, (3) and (41, were also obtained from uridine 2′,3′-cyclic phosphate and unprotected 2-aminoadenosine using 2,4,6-triisopropylbenzenesulfonyl chloride (TPS-Cl) as the condensing agent. The conformational properties of these dimers were examined by UV, CD and NMR spectroscopy. The results reveal that the 2′-5′ isomers take a stacked conformation, which contains a larger base-base overlap and is more stable against thermal perturbation with respect to the 3′-5′ isomers. The n2ApU isomers have more stacked structure than the Upn2A isomers.

Synthesis and Properties of Dinucleoside Monophosphates Containing 2-Aminoadenine 8,2′-S- and Uracil 6,2′-O-Cyclonucleosides

Abstract Two sequence isomers of dinucleoside monophosphates containing 8,2′-anhydro-2,6-diamino-8-mercapto-9-β-D-arabinofuranosylpurine (2NH2As) and 6,-anhydro-6-hydroxy-1-s-D-arabinofuranosyluracil (Uo), 2NH2As pUo (1) and Uo p2NH2As (2) were synthesized by the phosphodiester method. Examination of the UV, CD and NMR spectra of these dimers led us to the conclusion that, whereas compound (1) did not take a stacked conformation, compound (2) took a well stacked conformation in which the bases were stacked along a left-handed screw axis. Both the dimers formed a complex with ethidium bromide.

NMR Studies of a DNA Containing 8-Methoxydeoxyguanosine

Abstract 1H NMR experiments have been undertaken to elucidate the structural effects of methoxy substitution at the C8 of a deoxyguanosine residue in a self-complementary dodecadeoxyribonucleotide, d(C-G-C-mo8G-A-A-T-T-C-G-C-G), duplex, which has an 8-methoxy-2′-deoxyguanosine (mo8dG) residue at the 4th position. NMR data indicate that the mo8dG residue takes an anti glycosidic conformation in a mo8dG(anti):dC(anti) base-pair structure in a B-form duplex. The thermal stability of the duplex is reduced, but the overall structure is much the same as that of the unmodified d(C-G-C-G-A-A-T-T-C-G-C-G) duplex.

Characterization of recombinant cosmids containing H2-oxidation genes from Alcaligenes hydrogenophilus

Abstract Ten Pseudomonas oxalaticus OX1 harboring recombinant cosmids containing H 2 -oxidation genes (hox) from Alcaligenes hydrogenophilus showed H 2 -dependent autotrophic growth with different growth rates. Some strains grew autotrophically almost as fast as the strain containing the Hox plasmid pHG21-a. Ten strains had high activity of membrane-bound hydrogenase (MH), but the activity of soluble hydrogenase could not be detected. Sizes of DNA inserts in recombinant cosmids isolated from 10 strains were 26 to 29 kilobases (Kb), and there was a 22-Kb overlapped region. It is suggested that the DNA fragment required for conferring the ability of H 2 -dependent autotrophic growth on CO 2 -fixing P. oxalaticus appears to span a region of more than 20 Kb in A. hydrogenophilus . Structural genes of MH were found in the right end of the overlapped region by DNA hybridization analysis using oligonucleotide probes corresponding to the conserved sequences of MH genes in Bradyrhizobium japonicum and Rhodobacter capsulatus . The MH was induced by H 2 and repressed by the addition of fructose, as in A. hydrogenophilus . Therefore, the inserted DNA fragment contained the DNA region which determines the regulatory properties as well as structural genes of MH.