Nucleoside Tetra- and Pentaphosphates Prepared Using a Tetraphosphorylation Reagent Are Potent Inhibitors of Ribonuclease A
Authors: Ian W Windsor
Scott M Shepard
Ronald T Raines
Christopher C Cummins
Journal: Journal of the American Chemical Society (2019)
Discipline: CHEMISTRY
Keywords: Adenosine; Uridine; Tetrametaphosphates; Enzymatic activity; Ribonuclease A
Affiliations: Department of Chemistry, Massachusetts Institute of Technology, Cambridge Massachusetts 02139, United States

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. 

Retracted: No

PubPeer comments:
 Regards to compound [HNEt3]5[Hp5A], I m interested in the actual yield of this compound. The authors state that all the compounds in this paper have had water removed under vacuum at 40C on a Schlenk line. This compound specifically has had this process done 4 times resulting in apparently a colorless solid. The NMR, however, suggests  eight equivalents of residual buffer which apparently has not been removed, which the authors state in the SI. Using lyophilization which is the typical process for drying these compounds, water and buffer is almost always completely removed with maybe 0.5-1 eq remaining at worst. Considering the triethylammonium acetate buffer is an aqueous solution, one might reasonably expect  eight equivalents of residual buffer to also include a significant amount of water that can be hidden behind the HDO residual peak of the D2O solvent. And with the milligram scale that the authors are working with, a decent amount of water in the sample is going to change the yield quite a lot when compared to a product that has been thoroughly dried through lyophilization. This is further supported by the fact that compared to the HDO solvent peak, the rest of the peaks corresponding to the product are extremely miniscule, maybe reflecting that there is indeed a lot of water buffer remaining in this sample. Considering that the other compounds (perhaps with the exception of Hp4A, which has three equivalents of excess buffer) have been purified and dried well, I m wondering why Hp5A in particular was not dried to this extent and whether the 58% yield the authors report is a reproducible one. This is important to know, since for researchers without dry-boxes, we can apparently only get 31% yield for Hp5A, and if buffer is still remaining the  true yield might be even lower for those who wish to reproduce this technique on a Schlenk line. I would be interested to hear the authors response, thank you. ,