Stabilized Molecular Diphosphorus Pentoxide, P2O5L2 (L = N-Donor Base), in the Synthesis of Condensed Phosphate-Organic Molecule Conjugates.

Commercial phosphorus pentoxide reacts with some N-donor bases to give the adducts P2O5L2 and P4O10L3 (L = DABCO, pyridine, 4-tert-butylpyridine). The DABCO adducts were structurally characterized by single-crystal X-ray diffraction. It is proposed that P2O5L2 and P4O10L3 undergo interconversion through a "phosphate-walk" mechanism, which was evaluated using DFT calculations. P2O5(pyridine)2 (1) efficiently transfers monomeric diphosphorus pentoxide to phosphorus oxyanion nucleophiles, yielding substituted trimetaphosphates and cyclo-phosphonate-diphosphates (P3O8R)2- (R1 = nucleosidyl, phosphoryl, alkyl, aryl, vinyl, alkynyl, H, F). Hydrolytic ring-opening of these compounds forms linear derivatives [R1(PO3)2PO3H]3-, and nucleophilic ring-opening gives linear disubstituted [R1(PO3)2PO2R2]3- compounds.

Beyond Triphosphates: Reagents and Methods for Chemical Oligophosphorylation.

Oligophosphates play essential roles in biochemistry, and considerable research has been directed toward the synthesis of both naturally occurring oligophosphates and their synthetic analogues. Greater attention has been given to mono-, di-, and triphosphates, as these are present in higher concentrations biologically and easier to synthesize. However, extended oligophosphates have potent biochemical roles, ranging from blood coagulation to HIV drug resistance. Sporadic reports have slowly built a niche body of literature related to the synthesis and study of extended oligophosphates, but newfound interests and developments have the potential to rapidly expand this field. Here we report on current methods to synthesize oligophosphates longer than triphosphates and comment on the most important future directions for this area of research. The state of the art has provided fairly robust methods for synthesizing nucleoside 5'-tetra- and pentaphosphates as well as dinucleoside 5',5'-oligophosphates. Future research should endeavor to push such syntheses to longer oligophosphates while developing synthetic methodologies for rarer morphologies such as 3'-nucleoside oligophosphates, polyphosphates, and phosphonate/thiophosphate analogues of these species.

Synthesis of α,δ-Disubstituted Tetraphosphates and Terminally-Functionalized Nucleoside Pentaphosphates.

The anion [P4O11]2-, employed as its bis(triphenylphosphine)iminium (PPN) salt, is shown herein to be a versatile reagent for nucleophile tetraphosphorylation. Treatment under anhydrous conditions with an alkylamine base and a nucleophile (HNuc1), such as an alcohol (neopentanol, cyclohexanol, 4-methylumbelliferone, and Boc-Tyr-OMe), an amine (propargylamine, diethylamine, morpholine, 3,5-dimethylaniline, and isopropylamine), dihydrogen phosphate, phenylphosphonate, azide ion, or methylidene triphenylphosphorane, results in nucleophile substituted tetrametaphosphates ([P4O11Nuc1]3-) as mixed PPN and alkylammonium salts in 59% to 99% yield. Treatment of the resulting functionalized tetrametaphosphates with a second nucleophile (HNuc2), such as hydroxide, a phenol (4-methylumbelliferone), an amine (propargylamine and ethanolamine), fluoride, or a nucleoside monophosphate (uridine monophosphate, deoxyadenosine monophosphate, and adenosine monophosphate), results in ring opening to linear tetraphosphates bearing one nucleophile on each end ([Nuc1(PO3)3PO2Nuc2]4-). When necessary, these linear tetraphosphates are purified by reverse phase or anion exchange HPLC, yielding triethylammonium or ammonium salts in 32% to 92% yield from [PPN]2[P4O11]. Phosphorylation of methylidene triphenylphosphorane as Nuc1 yields a new tetrametaphosphate-based ylide ([Ph3PCHP4O11]3-, 94% yield). Wittig olefination of 2',3'-O-isopropylidene-5'-deoxy-5'-uridylaldehyde using this ylide results in a 3'-deoxy-3',4'-didehydronucleotide derivative, isolated as the triethylammonium salt in 54% yield.

Bacterial Phosphate Granules Contain Cyclic Polyphosphates: Evidence from 31P Solid-State NMR.

Polyphosphates (polyPs) are ubiquitous polymers in living organisms from bacteria to mammals. They serve a wide variety of biological functions, ranging from energy storage to stress response. In the last two decades, polyPs have been primarily viewed as linear polymers with varying chain lengths. However, recent biochemical data show that small metaphosphates, cyclic oligomers of [PO3](-), can bind to the enzymes ribonuclease A and NAD kinase, raising the question of whether metaphosphates can occur naturally as products of biological activity. Before the 1980s, metaphosphates had been reported in polyPs extracted from various organisms, but these results are considered artifactual due to the extraction and purification protocols. Here, we employ nondestructive 31P solid-state NMR spectroscopy to investigate the chemical structure of polyphosphates in whole cells as well as insoluble fractions of the bacterium Xanthobacter autotrophicus. Isotropic and anisotropic 31P chemical shifts of hydrated whole cells indicate the coexistence of linear and cyclic phosphates. Under our cell growth conditions and the concentrated conditions of the solid-state NMR samples, we found substantial amounts of cyclic phosphates in X. autotrophicus, suggesting that in fresh cells metaphosphate concentrations can be significant. The cellular metaphosphates are identified by comparison with the 31P chemical shift anisotropy of synthetic metaphosphates of known structures. In X. autotrophicus, the metaphosphates have a chemical shift anisotropy that is consistent with an average size of 3-8 phosphate units. These metaphosphates are enriched in insoluble and electron-dense granules. Exogenous hexametaphosphate added to X. autotrophicus cell extracts is metabolized to trimetaphosphates, supporting the presence and biological role of metaphosphates in cells. The definitive evidence for the presence of metaphosphates, reported here in whole bacterial cells for the first time, opens the path for future investigations of the biological function of metaphosphates in many organisms.

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

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.

Zirconium complexes supported by a ferrocene-based ligand as redox switches for hydroamination reactions.

The synthesis of (thiolfan*)Zr(NEt2)2 (thiolfan* = 1,1'-bis(2,4-di-tert-butyl-6-thiophenoxy)ferrocene) and its catalytic activity for intramolecular hydroamination are reported. In situ oxidation and reduction of the metal complex results in reactivity towards different substrates. The reduced form of (thiolfan*)Zr(NEt2)2 catalyzes hydroamination reactions of primary aminoalkenes, whereas the oxidized form catalyzes hydroamination reactions of secondary aminoalkenes.

Functionalization of Intact Trimetaphosphate: A Triphosphorylating Reagent for C, N, and O Nucleophiles.

Trimetaphosphate (TriMP, [P3O9]3-) reacts with PyAOP ([(H8C4N)3PON4C5H3][PF6]) to yield an activated TriMP, [P3O9P(NC4H8)3]- (1), incorporating a phosphonium moiety. Anion 1 is isolated as its bis(triphenylphosphine)iminium (PPN) salt in 70% yield and phosphorylates nucleophiles with elimination of phosphoramide OP(NC4H8)3. Treatment of 1 with amines HNR1R2 generates [P3O8NR1R2]2- (2a: R1 = R2 = Et; 2b: R1 = H, R2 = tBu) in greater than 70% yield as mixed PPN and alkyl ammonium salts. Treatment of 1 with primary alcohols in the presence of a tertiary amine base results in salts of intact TriMP alkyl esters [P3O9R]2- (3a: R = Me; 3b: R = Et) in greater than 60% isolated yield. Reaction of 1 with [PPN][H2PO4] provides orthophosphoryl TriMP (4, [P4O12H2]2-) in 40% yield as the PPN salt. Treatment of 1 with Wittig reagent H2CPPh3 (4 equiv) provides phosphorus ylide [P3O8CHPPh3]2- (5) in 61% yield as a mixed salt. Ylide 5 reacts with water to provide [P3O8Me]2- (6) and with aldehydes to give olefins [P3O8CHCHR]2- (7a: R = H; 7b: R = 4-C6H4Br), products in which one TriMP oxygen is replaced by a phosphonate P-C linkage. Treatment of intact TriMP derivatives 2a, 2b, 3a, and 7a with aqueous tetrabutylammonium hydroxide results in ring opening to linear triphosphate derivatives. X-ray crystal structures are provided for salts of 1, 2a, 3a, and 4.