Adenylyl imidodiphosphate, an adenosine triphosphate analog containing a P--N--P linkage.

An analog of adenosine triphosphate, adenylyl imidodiphosphate (AMP-PNP), in which a "-grouping replaces the terminal bridge oxygen of the triphosphate chain has been synthesized by the reaction of imidodiphosphate with PI-adenosine P2-diphenyl pyrophosphate (Michelson, A. (1964), Biochim. Biophys. Acta 9/, 1). AMP-PNP is stable (<3z hydrolysis) for months at -20" as the tetrasodium salt and in solution a t 25" a t neutral pH for a t least 16 hr. The principal nonenzymic breakdown products are inorganic phosphate and adenylyl phosphoramidate. Alkaline phosphatase (Escherichia coli) cleaves the terminal phosphate of the analog, a finding which extends the substrate specificity of this enzyme to include P-N-type bonds. Snake venom A T P is perhaps the most important small molecular weight compound in biological systems. Yet our understanding of its interactions with enzymes as both a substrate and as a n allosteric effector is incomplete and unsatisfactory. A similar situation exists with our understanding of the interaction of ATP with most biological transducers, such as the contractile system in muscle and the membranes of nerves and other cells. To better understand these interactions we have been synthesizing analogs of ATP in which the triphosphate chain has been modified (Yount et al., 1966a,b). Recently a n increasing number of such analogs have been synthesized (see Cook, 1970, and references therein for a partial list). One of the earliest and most useful of such compounds was AMP-PCPl (Myers et a/., 1963) a n analog in which a CH, group replaced the terminal bridge oxygen of the triphosphate chain of ATP. Because of the inherent stability of the P-C-P bonds in this compound, AMP-PCP offered the opportunity to study the interaction of a n ATP-like molecule with various enzymes without the possibility of cleavage between the P,y-phosphates. In many cases, however, AMP-PCP has had little effect on the system under study. For example, AMP-PCP was shown by Moos et ul. (1960) not to support contraction of glycinerated muscle fibers, not to dissociate actomyosin at high ionic strength, or even to inhibit the ATPase activity * From the Departments of Agricultural Chemistry and Chemistry, Washington State University, Pullman, Washington 99163. Receiced J U ~ M U J , 12, 1971. This research was supported in part by U. S. Public Health Service Grant AM-05195 and by Muscular Dystrophy Associations of America; College of Agriculture, scientific paper no. 3600, project 1614. Part of this work was communicated to the Proc . r n f . Congr. Biochern., 7th, 926 (1967). t T o whom inquiries concerning this work should be addressed. The follov,ing abbreviations have been used: AMP-PCP, adenylyl methylenediphosphate; AMP-PNP, adenylyl imidodiphosphate; PNPi, inorganic imidodiphosphate; PCPi, methylenediphosphoiiate; A D P . NHr, adenylyl phosphoramidate. 2484 B I O C H E M I S T R Y , V O L . IO, N O . 1 3 , 1 9 7 1 phosphodiesterase cleaves AMP-PNP to adenosine 5 'monophosphate and imidodiphosphate. AMP-PNP is not a substrate for hexokinase (glucose phosphorylation) or for myokinase (phosphorylation of adenosine monophosphate). AMP-PNP binds Ca2+, Mg2+, and Mn2+ at pH 8.5 more tightly than ATP. Similar results were found with the comparable methylene analog, adenylyl methylenediphosphonate, a t p H 7.4 and 9.2 with Ca2+ and Mg2+. An improved method of synthesis of the methylene analog is described and the structural properties of the P-C-P and P-N-P analogs are compared to adenosine triphosphate based on recent X-ray structural data of imidodiphosphate, pyrophosphate, and methylenediphosphonic acid. if excess Mg2+ was present. Since we were particularly interested in such interactions, we reasoned that a n ATP analog with P-N-P bond, if sufficiently stable, would be structurally and chemically more similar to ATP and hence would mimic ATP more effectively. Such a compound, AMP-PNP, has been synthesized (Figure 1). This paper reports on the synthesis and properties of AMP-PNP in comparison to those of AMP-PCP and ATP. During the course of this work the crystal structure of the tetrasodium salt of imidodiphosphate was solved (Larsen et a/ . , 1969) and it was shown to be remarkably similar to the previously known structure of sodium pyrophosphate. This close structural similarity rationalizes the overall similarity of ATP and AMP-PNP in their interactions with a number of enzyme systems. The more detailed studies of the interaction of AMP-PNP and AMP-PCP with myosin, heavy meromyosin, and actomyosin is given in the following paper (Yount et a/., 1971). Experimental Section Materials. Sodium imidodiphosphate was prepared by the hydrolysis of diphenylimidodiphosphoric acid (Kirsanov and Zhmurova, 1958) with the following modification of method B of Neilsen et al. (1961). Diphenylimidodiphosphoric acid (14.5 g, 0.045 mole) was mixed with 40 g (1 mole) of sodium hydroxide, 39 g (0.45 mole) of phenol, and 108 ml of water in a 1-1. flask and heated as rapidly as possible with magnetic stirring to 140" in an oil bath preheated to 170-180". After 10 min at 140-145", the hot solution was transferred to a beaker where on cooling, crystals of sodium imidodiphosphate formed. After filtering the product was dissolved in 250 ml of water and ethanol added to incipient cloudiness and the solution cooled a t 4" overnight. The crystals were filtered and tested for the presence of phenols (Feigl and Jungreis, 1959) and recrystallized as before until a negative phenol test was obtained. Phosphate analysis gave D ow nl oa de d by U O F C A L IF O R N IA D A V IS o n Se pt em be r 15 , 2 00 9 | h ttp :// pu bs .a cs .o rg P ub lic at io n D at e: J un e 1, 1 97 1 | d oi : 1 0. 10 21 /b i0 07 89 a0 09 A D E N Y L Y L I M I D O D I P H O S P H A T E , A P-N-P A N A L O G O F ATP a formula weight of 444 corresponding to sodium imidodiphosphate. decahydrate. Lower degrees of hydration were obtained depending on the mode of drying the crystals and molecular weights were determined for each preparation before using them in a synthetic reaction. Crystals were stored in capped vials a t -20". Mcthylenediphosphonic acid was prepared by the hydrolysis of tetraethyl methylenediphosphonate (Cade, 1959) with 6 N HCI under reflux for 72 hr. After removing HCI and ethanol under reduced pressure, the methylenediphosphonic acid was recrystallized twice from hot glacial acetic acid containing small amounts of water. Phosphorus analysis (Ames and Dubin, 1960) for CIHeOGP2 gave a molecular weight of 173 ; theory 176. Pyridine was refluxed over calcium hydride or sodium hydroxide pellets, distilled through a short column, and stored over calcium hydride or molecular sieves (Linde Division, Union Carbide Corp., Tonawanda, N. Y . , Type 4A, l/,,;-in. pellets). Dimethylformamide and chloroform were commercial preparations dried over molecular sieves for at least 10 days. Trioctylamine (Eastnian) and tributylamine (Aldrich) were redistilled under vacuum and stored in dark bottles at 4". Triethylamine (Aldrich) was refluxed with a few grams of /I-toluenesulfonyl chloride added to react with any secondarq amines, distilled at atmospheric pressure, and stored in the dark at 4". Triethylamnionium bicarbonate solutions (1 M) were prepared by bubbling CO? through a sintered glass diffuser into triethylamine solutions in an ice bath until the pH fell to 7 . 5 . Diphenyl chlorophosphate (Aldrich) was used from freshly opened bottles or from freshly distilled preparations. Chromatographic columns used were either from Pharniacia (Piscataway , N . J . ) or Chromatronix Inc. (Berkeley, Calif.). Anu!)*/icci/ Merliods. Acid-labile phosphate and acidlabile ammonia were determined on P-NP compounds after heating 30 min at 100" in 1 N HCI. P, was measured as the reduced phosphomolybdate complex (Fiske-s subbarow, 1925) and ammonia with Nessler's reagent (Umhreit et u / , , 1962). Total phosphate was determined as P , after ashing with Mg(NOa), and subsequent hydrolysis with I N HCI (Ames and Dubin, 1960). When triethylamnionium bicarbonate was present, it was removed by taking the solution to dryness before doing phosphate analyses since phosphomolybdate precipitates with trialkylamines. Compounds were detected on paper chromatograms and electrophoretograms by ultraviolet quenching using a Transilluminator and Chromato-Vue cabinet (Ultra-violet Products, San Gabriel, Calif.) and by a phosphate spray reagent (KolIolT, 1Y61) after acid hydrolysis. This latter step was accomplished by suspending thc dried, developed chromatograni or electrophoretogram with thin Teflon tubing in a jar over 4 N HCI. The jar was covered with Saran Wrap and heated 30-45 min in an 85 YO" oven. If the paper was not noticeably weakened, an additional 10 min of heating was used. The above spray reagents will detect less than 0.01 pmole of P, . Thc per cent composition (based on adenine) of various nucleotide preparations was determined by measuring the absorbance at 260 mp after eluting the appropriate ultraviolet-absorbing compounds from paper chromatograms with 0.05 N HCI. This method routinely gave values within .f 1 % for duplicate samples if 0.5-1 pmole of total adenine compounds were initially chromatographed. Elemental analyses for the methylene analog were by A. Bcrnhardt (Miilheim, West Germany). OH Oh. Adenyly l imidodiphosphote