Phosphorothioate analogs of 5-phosphoribosyl diphosphate: synthesis, purification, and partial characterization

Abstract

The 1-phosphorothioate analogues of 5-phosphoribosyl 1-diphosphate (P-Rib-PP) have been prepared enzymatically, in reactions catalyzed by P-Rib-PP synthetase from Salmonella typhimurium. 5-Phosphoribosyl 1-O-(2-thiodiphosphate) (P-Rib-PP beta S) was synthesized from ribose 5-phosphate (Rib-5-P) and the Mg2+ complex of adenosine 5'-O-(3-thiotriphosphate). The SP and RP diastereomers of 5-phosphoribosyl 1-O-(1-thiodiphosphate) (P-Rib-PP alpha S) were synthesized from Rib-5-P and the Mg2+ complex of adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) (SP diastereomer, delta-configuration) and the Cd2+ complex of ATP beta S (RP diastereomer, delta-configuration), respectively. The strategy for the synthesis and stereochemical assignment of the P-Rib-PP alpha S diastereomers was based on the specificity of P-Rib-PP synthetase for the (delta)-beta, gamma-bidentate metal-nucleotide substrate and the stereochemical course of the synthetase reaction, leading to inversion of configuration at the P beta atom of the nucleotide [Li, T. M., Mildvan, A. S., & Switzer, R. L. (1978) J. Biol. Chem. 253, 3918-3923], and the known configurations of the Mg2+ and Cd2+ beta, gamma-bidentate complexes of the ATP beta S diastereomers [Jaffe, E. K., & Cohn, M. (1979) J. Biol. Chem. 254, 10839-10845]. The P-Rib-PP analogues were purified by gradient elution from DEAE-Sephadex and characterized by chemical analysis and 31P nuclear magnetic resonance [Smithers, G. W., & O'Sullivan, W. J. (1984) Biochemistry (following paper in this issue)]. A preliminary account of their interaction with human brain hypoxanthine phosphoribosyltransferase and yeast orotate phosphoribosyltransferase (OPRTase) is described.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorothioate analogs of 5-phosphoribosyl diphosphate: phosphorus-31 nuclear magnetic resonance study

“…Thus, in the spectral region associated with the lPa doublet ( = 40.7 ppm) of the SP diasteromer of P-Rib-PPaS (Figure 2), and on the basis of the limitations imposed by the resonance signal amplitudes and noise level of the spectrum, other resonances (arising from possible contamination with the RP diastereomer) could not be observed. This was as expected, on the basis of the strict stereospecificity of the P-Rib-PP synthetase reaction and resolution of the two isomers by DEAE-Sephadex chromatography (Smithers & O'Sullivan, 1984).…”

“…], were evident in the 31P NMR spectrum of this analogue (Figure 2). On the basis of the time-dependent increase in the peak height of the Rib-5-P signal, compared with the 5P resonance, the estimated half-life of P-Rib-PPaS at 28 °C was approximately 4 h. The increased lability of this analogue, compared with both P-Rib-PP and P-Rib-PP/8S, and the limited quantities available (Smithers & O'Sullivan, 1984) prevented a more detailed 31P NMR study.…”

“…Chemicals. The ammonium salts of P-Rib-PP/SS and the SP diastereomer of P-Rib-PPaS were synthesized and purified as described in the preceding paper (Smithers & O'Sullivan, 1984). Other chemicals were analytical reagent grade obtained from local suppliers or the sources described in Smithers & O'Sullivan (1982).…”

“…In the preceding paper (Smithers & O'Sullivan, 1984), we described the preparation and partial characterization of the 1-phosphorothioate analogues of P-Rib-PP. We present here the results of a 31P NMR study of P-Rib-PP/JS and the SP diastereomer of P-Rib-PPaS.…”

“…[Unfortunately, low yields in 1 Abbreviations: NMR, nuclear magnetic resonance; P-Rib-PP, 5phosphoribosyl 1-diphosphate (the designations 5P and 1PP are used to denote the 5-phosphate and 1-diphosphate groups, respectively; IP, indicates the phosphorus atom of the diphosphate group closest to the anomeric carbon of the ribose ring and 1P9 is the successively remote phosphorus atom); P-Rib-PP/3S, 5-phosphoribosyl l-0-(2-thiodiphosphate); P-Rib-PPaS, 5-phosphoribosyl 1 -0-( 1 -thiodiphosphate); J^, spin-spin coupling constant between the Pa and P6 nuclei; Rib-5-P, ribose 5-phosphate; SPP¡, thiopyrophosphate; <5, chemical shift (parts per million from 85% H3PG4); R, [Mg]T/[phosphorylated ligand]x; ADP/5S, adenosine 5'-0-(2-thiodiphosphate); P¡, inorganic phosphate. the synthesis of the RP diastereomer of P-Rib-PPaS prevented a similar study of this analogue (Smithers & O'Sullivan, 1984)].…”