Volume 2 number 7 July 1975 Nucleic Acids Research Nucleoside 5'-phosphordiamidates, synthesis and some properties A. Simoncsits and J. Tomasz Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, H-6701, Szeged, Hungary Received 11 June 1975 ABSTRACT A simple way of preparing nucleoside 5'-phosphordiamidate is described. The procedure is based on the ammonolysis of nucleo- side 5'-phosphordichloridates by dilute aqueous ammonium hyd- roxide. The behaviour of nucleoside phosphordiamidates under acidic and alkaline conditions is also reported. Alkaline hyd- rolysis results in the formation of the parent nucleoside, whereas one amide group can be removed selectively by mild acid hydrolysis. This property of nucleoside phosphordiamidates served as a basis for the elaboration of a simple synthesis of nucleoside phosphoramidates starting from nucleosides. INTRODUCTION In contrast to the well-known and widely used nucleoside- phosphoramidates 1, nucleoside phosphordiamidates have been studied to a much lesser extent. To the best of our knowledge, the simplest members of this type of compounds derived from ammonia /I/ have not yet been described. Only some diimidazol- idates3 and dianilidates4 have been prepared from the approp- riate dichloridates by imidazole and from nucleosides by di- anilidophosphorchloridate under anhydrous conditions. 0 H N-P-ON' N1= nucleoside residue, 2 1 H2N I Adenosine 5'-phosphordiimidazolidate was used as starting ma- 5 terial for the synthesis of'1'ATP . Dianilidate has served as 1223 Nucleic Acids Research a phosphate protectinX group in a chemical synthesis of oligo- -deoxyribonucleotides In our investigations on the mechanism of phosphorylation of ribonucleosides by pyrophosphoryl chloride, an easy and gen- eral approach to the preparation of the simplest phosphordi- amidate derivatives of nucleosides has been arrived at. This is based on the ammonolysis of nucleoside phosphordichloridates by dilute aqueous ammonium hydroxide. The structure of the com- pounds prepared in this way has been verified and some of their properties have also been studied. RESULTS AND DISCUSSION The 5'-phosphordiamidates of ribonucleoside 2',3'-cyclic- phosphates, 2',3'-0-isopropylidene-ribonucleosides and 2'-de- oxyribonucleosides were prepared by ammonolyzing the appropri- ate dichloridates with 1-7 N NH4OH below 100 for 10 min. The reaction was tested with several compounds and the following three derivatives were synthesized as representatives on a pre- parative scale: guanosine 2',3'-cyclic phosphate 5'-phosphordi- amidate (/H2N/2pG>p), 2', 3'-0-isopropylidene-guanosine 5'- -phosphordiamidate (/H2N/2pG>Ip) and thymidine 5'-phosphordi- amidate (d/H2N/pT). The compounds were isolated by ion-exchange chromatography in 50-60 % yields. Besides the main products, the reaction mixtures always contained the respective 5'-phos- phoramidates and occasionally, traces of 5'-phosphates, too. In order to prepare the respective dichloridates, pyro- phosphoryl chloride6 (for /H2N/2pG>p and /H2N/2pG>Ip) and phos- phoryl chloride in trimethyl phosphate7 (for d/H2N/2pT) were used. The phosphorylation by pyrophosphoryl chloride resulted in the formation of compounds of type II8,9. The ammonolysis of II gave only guanosine 5'-phosphate derivatives. The absence of higher phosphorylated compounds indicated a much more prefer- able ammonolytical fission of P-0-P bonds compared to P - Cl linkages. On the other hand, the ammonolysis of pyrophosphoryl chloride by liquid ammonia broke the P - Cl linkages giving py- rophosphoryl amidelO. In the synthesis of /H2N/2pG>p the forma- tibn of the uncharged guanosine 2',3'-cyclic phosphoramidate 5'-phosphordiamidate was never demonstrated in agreement with 1224 Nucleic Acids Research literature data C1- 0l[- lO-CH2 G 0 0 G = guanine-9- R n = 0,1,2, R = P IIII -,~~~~1 ~ClC or -%C'CH3~CH3 The compounds synthesized were rather stable and could thus be stored at room temperature for several weeks without detectable decomposition. /H2N/2pG>Ip could be recrystallized from hot water, and d/H2N/2pT remained unchanged in absolute pyridine at 250 for 4 days. The diamidates, however, were high- ly sensitive to both acid and alkali. One of the two amide groups could selectively be removed by mild acid hydrolysis /0.001 N HC1, 24 hr, or 0.1 N HC1, 5 min, 250/ without opening of the markedly acid sensitive 2',3'- -phosphodiester cycle, of /H2N/2pG>p. As expected, under more vigorous conditions /1.0 N HCl, 250 , 1 hr/ a quantitative con- version to monophosphates was attained. Consequently, two atoms of nitrogen per mole diamidate could be measured in the form of ammonia after acid hydrolysis. A similar transformation of di- imidazolidates has been reported12 In alkaline medium the nucleoside phosphordiamidates were quantitatively converted to the parent nucleoside derivatives with the apparent liberation of one mole of inorganic phosphate /Pi/ per mole diamidate /0.1 N NaOH, 250, 5 min/13. The liber- ation of Pi was the consequence of a further breakdown of the orthophosphordiamidate released under the acidity during phos- phate determination. Sodium ethoxide in absolute ethanol pro- duced a similar transformation, but no Pi was liberated. Con- trary to our finding, nucleoside 5'-phosphordiimidazolidates could selectively be transformed to the respective 5'-mono- 1225 Nucleic Acids Research imidazolidates by careful alkaline hydrolysis3. The two processes, i.e. alkaline hydrolysis and ethoxide ion-catalyzed alcoholysis may occur by similar, acylfission mechanisms and can be considered either as bimolecular reactiors in analogy to the alkaline hydrolysis of phosphoric acid tries- ters15 /Fig. la/ or alternatively, as unimolecular transforma- tions, proceeding through a metaphosphorimidate-amide inter- mediate /Fig. lb/. A similar mechanism has been proposed for the alkaline hydrolysis of N.N-disubstituted phosphordiamidic chloride16. The quantitative removal of one amide group of nucleoside phosphordiamidate under mild conditions offers a simple and al- ternative way of synthesis of the important nucleoside phosphor- amidates. These compounds have generally been prepared from nucleotides and ammonia in the presence of N.N-dicyclohexyl- carbodiimide /DCC/17'18, whereas our synthesis starts from nuc- leosides. In order to demonstrate the utility of this approach thymidine 5'-phosphoramidate (dH2NpT)and guanosine 2',3'-cyclic phosphate 5'-phosphoramidate (H2NpG>p) were prepared. The latte was successfully used for the synthesis of guanosine 2',3'-cy- clic phosphate 5'-triphosphate /pppG>p/. H2, NH2 NH 2 NH NH2 RO H-NH-P-ONI -R HN=P + ONI - RO- -NH + HON1 +(OR i -ROH N11 R = H or C2H5 Nt= 5'-nucleoside residue FIG. 1. 1226 Nucleic Acids Research EXPERIMENTAL All chemicals were of reagent grade. Nucleosides were pur- chased from Zellstoffabrik Waldhof, trimethyl phosphate from Fluka, E. coli alkaline phosphatase from Worthington and ribo- nuclease T1 /RNAse T1/ from Sigma. Pyrophosphoryl chloride19 and 2',3'-0-isopropylidene nucleosides 20 were prepared accord- ing to known procedures. Trimethyl phosphate was purified by distillation under reduced pressure. Evaporations were carried out in a flash-evaporator /Buch- ler Instruments/ under reduced pressure at 300. Absorption spectra were recorded in a Cary 15 spectrophotometer at pH 1 or 2, 7 and 11. Phosphorus was determined according to Eibl and 21 Lands . Before microanalysis, the compounds were dried under reduced pressure over phosphorus pentoxide at 250 for 24 hr. Thin-layer chromatography /TLC/ was performed in the fol- lowing systems - A, n-propanol: cc. NH4OH : H20 = 11 : 7 : 2 /v/v/; B, satd. /NH4/2S04 : 0.05 M Na2B407 : 0.1 N Na-acetate i-propanol = 60 : 19 : 19 : 2; C, ethanol : 0.1 M Na-acetate = 5 :2; D, n-butanol : acetic acid : H20 = 4: 1 : 1 on cellulose MN 300 /Macherey-Nagel/ : Silica Gel HF254 /Merck/ = 8 :2 /g/g/ and E, 0.1 M NaCl, F, 0.2 M NaCl, G, 1.0 M KH2PO4, pH 4.2, on pre-coated polyethyleneimine-impregnated cellulose sheets /poly- gram Cel 300 PEI/UV 254' Macherey-Nagel/. In the case of system B, the layers were sprayed with 0.05 M Na2B407 before use. Paper electrophoresis /PE/ was performed on Whatman 3 MM paper using 0.05 M KH2PO4, pH 4.8, and 0.05 M Na2HP04, pH 8.6 buffers in a Markham-Smith type equipment 2 with 70 V/cm for 15 min. Compounds were detected under a short-wave /254 nm/ UV lamp /Desaga Uvis/. Rf values and electrophoretic mobilities are summarized in Table I. Guanosine 2',3'-cyclic phosphate 5'-phosphordiamidate (/H2N/2pG>p) A suspension of anhydrous guanosine (G) /1.42 g, 5 mmoles/ in pyrophosphoryl chloride /7 ml, 50 mmoles/ was stirred at 00 for 3 hr, then the viscous pale-yellow solution was poured into 1227 Nucleic Acids Research Thin-layer Chromatography Paper electrophoresis rela- Compound Rf values in System tive mobilities to pG or dpT A B C D E F G pH 4.8 pH 8.6 ~~~~~~~~~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ G 0.73 0.26 0.38 0.69 0.00 0.00 /HH2NI/ 2G>P 0.56 0.30 0.47 0.74 0.54 0.50 H2NpG>p 0.54 0.34 0.12 1.16 1.05 /H2N/2pGp 0.34 0.48 H2NpGp 0-320.20.53"0.60 1.29 1.32 pG>p 0.38 0.45 0.62 1.29 1.35 pGp 0.28 0.66 0.46 1.37 1.48 H2NpG 0.45 0.28 G>p 0.72 0.28 0.41 0.10.42 * Gp 0-41 0.51 0.08 pppG>p 0.26 0.61 0.26 /H2N/2pG>Ip 0.36 0.80 0.00 /0.70/* 0.00 G>Ip 0.60 0.89 0.00 0.00 H2NpG>Ip 0.24 0.71 0.92 0.54 pG>Ip 0.25 0.68 0.94 0.96 /0.34/* pG 0.38 0.33 0.00 0.07 1.00 1.00 d/H2N/2pT 0.63 0.92 0.00 -0..00 dH2NpT 0.61 0.75 1.00 0.61 dpT 0.53 0.30 1.00 1.00 dT 0.80 0.94 0.00 0 .00 J *Values in parentheses are those observed on Silica Gel HF254 tmThe faster moving compound of the two spots is the 2'isomer.