Pyrazolo[3,4-d]pyrimidine ribonucleosides related to 2-aminoadenosine and isoguanosine: synthesis, deamination and tautomerism

Abstract

The syntheses and properties of 8-aza-7-deazapurine (pyrazolo[3,4-d]pyrimidine) ribonucleosides related to 2-aminoadenosine and isoguanosine are described. Glycosylation of 8-aza-7-deazapurine-2,6-diamine 5 with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose (12) in the presence of BF(3) x Et(2)O as a catalyst gave the N(8) isomer 14 (73%) with a trace amount of the N(9) isomer 13a (4.8%). Under the same reaction conditions, the 7-halogenated 8-aza-7-deazapurine-2,6-diamines 6-8 afforded the thermodynamically more stable N(9) nucleosides 13b-d as the only products (53-70%). Thus, a halogen in position 7 shifts the glycosylation from N(8) to N(9). The 8-aza-7-deazapurine-4,6-diamine ribonucleosides 1a-d were converted to the isoguanosine derivatives 3a-d by diazotization of the 2-amino group. Although compounds 1a,b do not contain a nitrogen at position 7 (the enzyme binding site), they were deaminated by adenosine deaminase; however, their deamination occurred with a much slower velocity than that of the related purines. The pK(a) values indicate that the 7-non-functionalized nucleosides 1a (pK(a) 5.8) and 15 (pK(a) 6.4) are possibly protonated in neutral conditions when incorporated into RNA. The nucleosides 3a-d exist predominantly in the keto (lactam) form with K(TAUT) (keto/enol) values of 400-1200 compared to 10(3)-10(4) for pyrrolo[2,3-d]pyrimidine isoguanosine derivatives 4a-c and 10 for isoguanosine itself, which will reduce RNA mispairing with U.

Structural Requirements for the Activation of Escherichia coli CTP Synthase by the Allosteric Effector GTP Are Stringent, but Requirements for Inhibition Are Lax

“…For example, fitting the curve for GTP-dependent activation and inhibition of Gln-dependent CTP formation gives K i ϭ 0.25 Ϯ 0.01 mM and K i ϭ 0.41 Ϯ 0.01 mM using Equations 3 and 4, respectively. Interestingly, for all GTP/guanosine analogues that activated Gln-dependent CTP formation, the K i values determined by fitting the v i /[E] T data with Equation 3 agree well with the corresponding observed IC 50 values, although this agreement may be fortuitous. It is important to note that both kinetic mechanisms are written such that the observed initial velocity is the rate of CTP production and not Gln production.…”

“…With respect to inhibition of Gln-dependent CTP formation, guanosine, GMP, GDP, and GtetraP all exhibited approximately the same IC 50 values, indicating that inhibition, for the most part, may be attributed to the nucleoside. Consequently, guanosine analogues were employed in many of our inhibition studies rather than the more expensive 5Ј-triphosphates.…”

“…Unfortunately, guanine was not sufficiently soluble under the assay conditions, so its ability to inhibit Gln-dependent CTP formation could not be determined. However, 6-thioguanine was soluble (using 20% Me 2 SO as a co-solvent) and inhibited CTPS with an IC 50 value that was only 1.7-fold greater than that observed for 6-thio-G. Inhibition of Gln-dependent CTP formation, therefore, arises primarily from interactions between CTPS and the purine base.…”

“…The IC 50 values for inosine and ITP were ϳ16-fold greater than the value obtained for guanosine, and the IC 50 value for adenosine was elevated 34-fold relative to guanosine. These results reinforce our conclusion that the 2-amino group plays a major role in binding.…”

“…The effects of varying concentrations of guanosine (0 -0.6 mM), GMP (0 -0.6 mM), GDP (0 -0.6 mM), GTP (0 -1.0 mM), ITP (0 -8.0 mM), and ACV (0 -0.6 mM) on NH 3 -dependent CTP formation were examined using fixed concentrations of NH 4 50 is the concentration of inhibitor that yields 50% inhibition (42,43).…”