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. 2019 Nov 20;141(46):18400-18404.
doi: 10.1021/jacs.9b09760. Epub 2019 Nov 11.

Nucleoside Tetra- and Pentaphosphates Prepared Using a Tetraphosphorylation Reagent Are Potent Inhibitors of Ribonuclease A

Scott M Shepard  1 , Ian W Windsor  1 , Ronald T Raines  1 , Christopher C Cummins  1
Affiliations

Nucleoside Tetra- and Pentaphosphates Prepared Using a Tetraphosphorylation Reagent Are Potent Inhibitors of Ribonuclease A

Scott M Shepard et al. J Am Chem Soc. .

Abstract

Adenosine and uridine 5'-tetra- and 5'-pentaphosphates were synthesized from an activated tetrametaphosphate ([PPN]2[P4O11], [PPN]2[1], PPN = bis(triphenylphosphine)iminium) and subsequently tested for inhibition of the enzymatic activity of ribonuclease A (RNase A). Reagent [PPN]2[1] reacts with unprotected uridine and adenosine in the presence of a base under anhydrous conditions to give nucleoside tetrametaphosphates. Ring opening of these intermediates with tetrabutylammonium hydroxide ([TBA][OH]) yields adenosine and uridine tetraphosphates (p4A, p4U) in 92% and 85% yields, respectively, from the starting nucleoside. Treatment of ([PPN]2[1]) with AMP or UMP yields nucleoside-monophosphate tetrametaphosphates (cp4pA, cp4pU) having limited aqueous stability. Ring opening of these ultraphosphates with [TBA][OH] yields p5A and p5U in 58% and 70% yield from AMP and UMP, respectively. We characterized inorganic and nucleoside-conjugated linear and cyclic oligophosphates as competitive inhibitors of RNase A. Increasing the chain length in both linear and cyclic inorganic oligophosphates resulted in improved binding affinity. Increasing the length of oligophosphates on the 5' position of adenosine beyond three had a deleterious effect on binding. Conversely, uridine nucleotides bearing 5' oligophosphates saw progressive increases in binding with chain length. We solved X-ray cocrystal structures of the highest affinity binders from several classes. The terminal phosphate of p5A binds in the P1 enzymic subsite and forces the oligophosphate to adopt a convoluted conformation, while the oligophosphate of p5U binds in several extended conformations, targeting multiple cationic regions of the active-site cleft.

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Conflict of interest statement

The authors declare the following competing financial interest(s): The tetraphosphorylation reagent is covered in patent US10017388B2.

Figures

Figure 1.
Figure 1.
Cocrystal structure of RNase A bound to an AUAA DNA tetramer revealed the subsites that recognize nucleobases and phosphoryl groups (1rcn, top). The mainchain of RNase A is traced with a cartoon, key active-site and cysteine residues are shown as sticks, and ligands are shown as balls-and-sticks. Residues in subsites are colored blue (P2), red (P1), and green (P0). A cartoon representation of the RNase A active site showing the preferred binder for each subsite (bottom). For simplicity, the P−1 subsite is not shown.
Figure 2.
Figure 2.
Synthesis of nucleoside tetraphosphates by Kowalska and Taylor compared to this synthesis of nucleoside tetra- and pentaphosphates.
Figure 3.
Figure 3.
(A) i. [PPN]2[1] (1.5 equiv) and triethylamine (2 equiv) (DMF, N2 atmosphere, 48 h); ii. [TBA][OH] (7.5 equiv) (DMF/H2O, 2 h) followed by HPLC (50 mM triethylammonium acetate (TEAA)). (B) i. [PPN]2[1] (1.1 equiv) (DMF, N2 atmosphere, 30 min); ii. [TBA][OH] (4.5 equiv) (DMF/H2O, 24 h) followed by HPLC (50 mM TEAA).
Figure 4.
Figure 4.
Interactions between nucleotides and the active site of RNase A. The structures are depicted as described in Figure 1. (A) pA and P3A bind the active site by positioning the terminal phosphate group in the P1 subsite with the adenosine base in the B2 subsite (1z6s, 2w5g). (B) p5A binds similarly to shorter adenosine nucleotides (6pvv). (C) cp4pA binds similarly to p5A, but more efficiently targets Lys7 of the P2 subsite (6pvw). (D) Two molecules of p2U bind RNase A; however, only the B1 subsite is efficiently targeted (3dxh). (E) Chain A of the RNase A·p5U complex is similar to p2U (6pvx). (F) In chain B, p5U only binds in the B1 subsite and alternatively targets the P1 and P2 subsites (6pvx).
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