Natsuhisa Oka

Protein-based peptide-bond formation by aminoacyl-tRNA protein transferase

Eubacterial leucyl/phenylalanyl-tRNA protein transferase (LF-transferase) catalyses peptide-bond formation by using Leu-tRNALeu (or Phe-tRNAPhe) and an amino-terminal Arg (or Lys) of a protein, as donor and acceptor substrates, respectively. However, the catalytic mechanism of peptide-bond formation by LF-transferase remained obscure. Here we determine the structures of complexes of LF-transferase and phenylalanyl adenosine, with and without a short peptide bearing an N-terminal Arg. Combining the two separate structures into one structure as well as mutation studies reveal the mechanism for peptide-bond formation by LF-transferase. The electron relay from Asp 186 to Gln 188 helps Gln 188 to attract a proton from the α-amino group of the N-terminal Arg of the acceptor peptide. This generates the attacking nucleophile for the carbonyl carbon of the aminoacyl bond of the aminoacyl-tRNA, thus facilitating peptide-bond formation. The protein-based mechanism for peptide-bond formation by LF-transferase is similar to the reverse reaction of the acylation step observed in the peptide hydrolysis reaction by serine proteases.

Stereocontrolled synthesis of oligonucleotide analogs containing chiral internucleotidic phosphorus atoms

Oligonucleotides, in which one of the two nonbridging oxygen atoms of internucleotidic phosphates is replaced by a different type of atom or a substituent, are useful as therapeutic agents and probes to elucidate mechanisms of enzymatic reactions. The internucleotidic phosphorus atoms of these oligonucleotides are chiral, and the properties of these oligonucleotides are affected by the absolute configuration of the chiral phosphorus atoms. In order to address the issue of chirality, various methods have been developed to synthesize these P-chiral oligonucleotide analogs in a stereocontrolled manner. This critical review focuses on the recent progress in this field (123 references).

Synthesis and biological activity of artificial mRNA prepared with novel phosphorylating reagents

Though medicines that target mRNA are under active investigation, there has been little or no effort to develop mRNA itself as a medicine. Here, we report the synthesis of a 130-nt mRNA sequence encoding a 33-amino-acid peptide that includes the sequence of glucagon-like peptide-1, a peptide that stimulates glucose-dependent insulin secretion from the pancreas. The synthesis method used, which had previously been developed in our laboratory, was based on the use of 2-cyanoethoxymethyl as the 2′-hydroxy protecting group. We also developed novel, highly reactive phosphotriester pyrophosphorylating reagents to pyrophosphorylate the 5′-end of the 130-mer RNA in preparation for capping. We completed the synthesis of the artificial mRNA by the enzymatic addition of a 5′-cap and a 3′-poly(A) tail to the pyrophosphorylated 130-mer and showed that the resulting mRNA supported protein synthesis in a cell-free system and in whole cells. As far as we know, this is the first time that mRNA has been prepared from a chemically synthesized RNA sequence. As well as providing a research tool for the intracellular expression of peptides, the technology described here may be used for the production of mRNA for medical applications.

Stereocontrolled synthesis of oligoribonucleoside phosphorothioates by an oxazaphospholidine approach.

Oligoribonucleoside phosphorothioates (PS-ORNs) stereodefined at the phosphorus atoms were synthesized on solid support. Thermal denaturating experiments of the resultant PS-ORNs showed that a backbone consisting of (Sp)-PS-linkages as well as stereorandom PS-linkages had an unexpectedly large destabilizing effect on a PS-ORN-ORN duplex, whereas a backbone consisting of (Rp)-PS-linkages slightly stabilized a duplex.

A new boranophosphorylation reaction for the synthesis of deoxyribonucleoside boranophosphates

Abstract Deoxyribonucloside 3′-boranophosphate derivatives including adenine, cytosine, guanine, and thymine bases were synthesized in good yields by the use of a new boranophosphorylating reagent. The reaction was successfully applied to the formation of internucleotidic boranophosphate linkages.

Synthesis of glycosyl boranophosphates and their applications as precursors of glycosyl phosphate analogues.

Glycosyl boranophosphate triesters were synthesized via a boranophosphorylation of reducing sugars. The usefulness of the resultant glycosyl boranophosphates as versatile chemically stable precursors of various glycosyl phosphate derivatives is demonstrated.

1,2-Trans-selective synthesis of glycosyl boranophosphates and their utility as building blocks for the synthesis of phosphodiester-linked disaccharides.

A highly 1,2-trans-selective synthesis of glycosyl boranophosphate derivatives by glycosylation of dimethyl boranophosphate with glycosyl iodides was developed. A study on the reaction mechanism indicated that the stereoselectivity of the reactions is controlled by neighboring group participation. The resultant glycosyl boranophosphate triesters were converted into the corresponding boranophosphate diesters and condensed with appropriately protected monosaccharides to give disaccharides linked with an anomeric boranophosphate linkage. Furthermore, the disaccharides worked as precursors of the corresponding phosphodiester-linked disaccharides. The whole synthesis of boranophosphate-linked disaccharides and their conversion to the phosphodiester-linked disaccharides were accomplished in good yields without loss of stereopurity at the anomeric position, indicating that the method is useful to synthesize diastereopure glycosyl phosphate-containing biomolecules.

Synthesis of Oligo(α-d-glycosyl phosphate) Derivatives by a Phosphoramidite Method via Boranophosphate Intermediates

An efficient method for the synthesis of short oligo(α-D-glycosyl boranophosphate) derivatives by using an α-D-glycosyl phosphoramidite as a monomer unit was developed. The synthesis of oligomers was carried out by repeating a cycle consisting of the condensation of the monomer unit with a terminal hydroxy group of carbohydrates, boronation of the resultant phosphite intermediates, and terminal deprotection. The phosphoramidite monomer unit was synthesized from the corresponding glycosyl iodide and methyl N,N-diisopropylphosphonamidate in a highly α-selective manner. Di- and tri(α-D-glycosyl boranophosphate) derivatives obtained by the synthetic cycle were converted into the corresponding H-phosphonate diester derivatives, which were then used to synthesize di- and tri(α-D-glycosyl phosphate) derivatives including a fragment of Leishmania glycocalyx lipophosphoglycans.

α-Selective ribofuranosylation of alcohols with ribofuranosyl iodides and triphenylphosphine oxide.

Ribofuranosylation of a variety of alcohols with ribofuranosyl iodides in the presence of a base and triphenylphosphine oxide afforded the corresponding α-ribofuranosides with diastereoselectivities ≥ 99:1. This reaction can be carried out under mildly basic conditions and is thus compatible with acid-sensitive functional groups.

Stereoselective synthesis of alpha-glycosyl phosphites and phosphoramidites via O-selective glycosylation of H-phosphonate derivatives.

A highly stereo- and chemoselective glycosylation of H-phosphonate derivatives with glycosyl iodides was discovered as a reverse reaction of the formation of a glycosyl iodide from a glycosyl phosphite and I- under mild acidic conditions. Further study on the unique reaction showed that the reaction provided various alpha-glycosyl phosphites and phosphoramidites in a highly stereoselective manner with complete O-selectivity.