Methylene analogues of adenosine 5′‐tetraphosphate

Abstract

Adenosine 5¢-polyphosphates have been identified in vitro, as products of certain enzymatic reactions, and in vivo. Although the biological role of these compounds is not known, there exist highly specific hydrolases that degrade nucleoside 5¢-polyphosphates into the corresponding nucleoside 5¢-triphosphates. One approach to understanding the mechanism and function of these enzymes is through the use of specifically designed phosphonate analogues. We synthesized novel nucleotides: a,b-methylene-adenosine 5¢-tetraphosphate (pppCH 2 pA), b,c-methylene-adenosine 5¢-tetraphosphate (ppCH 2 ppA), c,d-methylene-adenosine 5¢-tetraphosphate (pCH 2 pppA), ab,cd-bismethylene-adenosine 5¢-tetraphosphate (pCH 2 ppCH 2 pA), ab, bc-bismethylene-adenosine 5¢-tetraphosphate (ppCH 2 pCH 2 pA) and bc, cd-bis(dichloro)methylene-adenosine 5¢-tetraphosphate (pCCl 2 pCCl 2 ppA), and tested them as potential substrates and ⁄ or inhibitors of three specific nucleoside tetraphosphatases. In addition, we employed these p 4 A analogues with two asymmetrically and one symmetrically acting dinucleoside tetraphosphatases. Of the six analogues, only pppCH 2 pA is a substrate of the two nucleoside tetraphosphatases (EC 3.6.1.14), from yellow lupin seeds and human placenta, and also of the yeast exopolyphosphatase (EC 3.6.1.11). Surprisingly, none of the six analogues inhibited these p 4 A-hydrolysing enzymes. By contrast, the analogues strongly inhibit the (asymmetrical) dinucleoside tetraphosphatases (EC 3.6.1.17) from human and the narrow-leafed lupin. ppCH 2 ppA and pCH 2 pppA, inhibited the human enzyme with K i values of 1.6 and 2.3 nm, respectively, and the lupin enzyme with K i values of 30 and 34 nm, respectively. They are thereby identified as being the strongest inhibitors ever reported for the (asymmetrical) dinucleoside tetraphosphatases. The three analogues having two halo ⁄ methylene bridges are much less potent inhibitors for these enzymes. These novel nucleotides should prove valuable tools for further studies on the cellular functions of mono-and dinucleoside polyphosphates and on the enzymes involved in their metabolism.Abbreviations Ap 3 A, diadenosine 5¢,5¢¢¢-P 1 ,P 3 -triphosphate; Ap 4 A, diadenosine 5¢,5¢¢¢-P 1 ,P 4 -tetraphosphate; Np n N¢, dinucleoside 5¢,5¢¢¢-P 1 ,P n -polyphosphate; p 4 A, adenosine 5¢-tetraphosphate; p 5 A, adenosine 5¢-pentaphosphate; pCCl 2 pCCl 2 ppA, bc,cd-bis(dichloro)methylene-adenosine 5¢-tetraphosphate; pCH 2 ppCH 2 pA, ab,cd-bismethylene-adenosine 5¢-tetraphosphate; pCH 2 pppA, c,d-methylene-adenosine 5¢-tetraphosphate; p n N, nucleoside 5¢-polyphosphate; ppCH 2 pCH 2 pA, ab,bc-bismethylene-adenosine 5¢-tetraphosphate; pppCH 2 pA, a,b-methylene-adenosine 5¢-tetraphosphate; pppCH 2 ppA, b,c-methylene-adenosine 5¢-tetraphosphate.

Adenosine 5′-Tetraphosphate Is a Highly Potent Purinergic Endothelium-Derived Vasoconstrictor

“…These substances are also potent vasodilators in the rat kidney. 19 We used methylene AP4 analogs ␣,␤-meAP4, ␤,␥-meAP4, and ␥,␦-meAP4, 17,18 which are less susceptible to enzymatic hydrolysis than AP4. In the Ang II preconstricted vessels and after ␣,␤-meATP desensitization, there was no constriction by the stable AP4 analogs and no vasodilation, indicating that the vasodilatory AP4 effects are indeed mediated by degradation products.…”

“…To exclude any vasoactive effects of metabolites arising from degradation of AP4, we investigated the effect of methylene derivatives of AP4, ␣,␤-meAP4, ␤,␥-meAP4, and ␥,␦-meAP4, 17,18 which are less susceptible to hydrolysis than AP4. All these derivatives induced a strong and dose-dependent increase of the perfusion pressure in the isolated perfused rat kidney.…”

Phosphonate analogues of nucleoside polyphosphates

“…Diphenyl chlorophosphate [40][41][42][43] , N,N´-carbonyldiimidazole (CDI) 31,44,45 , imidazole/2,2´-dithiopyridine/Ph3P system [46][47][48][49][50] , dicyclohexylcarbodiimide (DCC) 51,52 , and trifluoracetic anhydride 53,54 are the most widely used activating reagents for the synthesis of methylene-modified nucleoside tri-and polyphosphates. All coupling methods have the same strategy in common: one nucleotide subunit (usually nucleoside monophosphate or bisphosphonate) is converted via an activation process into an electrophilic substrate and then reacted with a second phosphate or phosphonate subunit acting as a nucleophile.…”

“…69 A similar approach has been successfully exploited for the synthesis of mono-and dinucleoside di-, tri-, tetra-and even penta-phosphates and their methylene analogues. [46][47][48][49][50] The coupling reactions occurred efficiently without significant accumulation of by-products which is important because of purification difficulties common for this class of compounds.…”

“…Additionally, nucleoside phosphorimidazolidate can react sluggishly with methylenebisphosphonate, requiring prolonged reaction times or the use of catalysts such as ZnCl2 or CdCl2. 48,69 In some cases, reversal of the commonly used strategy, such that the α,β-methylene NDP is the nucleophile and the phosphorimidazolidate is the electrophile, resulting in a high yield of the desired NTP analogue without the need for catalyst or long reaction time.…”