Volume 2 number 10 October 1975

Nucleic Acids Research

The synthesis of the internucleotide (phosphodiester) bond by a base-catalysed reaction*

Richard von Tigerstrom**, Patricia Jahnke and Michael Smith*** Department of Biochemistry, Faculty of Skdicine, University of British Columbia Vancouver, B.C., Canada

Received 8 July 1975 ABSTRACT

Potassium tert-butoxide in hexamethyl phosphoramide and dimethyl formamide provides an excellent catalyst for the reaction of a 3'-hydroxyl or a 5'-hydroxyl group of a nucleoside with an appropriate nucleoside phosphorofluoridate to yield the dinucleoside phosphate. This paper describes the experiments leading to the development of this reaction together with the synthesis of thymidylyl-(5'>3')-thymidine (dT-dT). INTRODUCTION

The basic procedures for the chemical synthesis of the internucleotide linkage in polynucleotides involve one of two chemical strategies. The most important of these, from a practical standpoint, involves the activation of phosphate in the form of an anhydride. This activated phosphate is used to phosphorylate an unionized sugar hydroxyl group. Either phosphomonoesters or phosphodiesters can be activated, the products being phosphodiesters or phosphotriesters, respectively (1,2,3). In this class of reaction, the phosphorylating agent is non-selective and consequently, other functional groups in the reacting nucleotides and nucleosides require chemical protection. The second strategy, which is more specific, involves attack by phosphate anion on an activated sugar carbon of a nucleoside (4). This class of reaction is particularly effective in the synthesis of thiophosphate esters (5). We have sought an alternate procedure in which there is specific reaction between sugar hydroxide and phosphate in order to eliminate the need for protection of the heterocyclic bases whilst achieving efficient phosphodiester synthesis. A logical approach is to activate the phosphate to a lesser degree than is 1727

Nucleic Acids Research done in the non-specific acid-anhydrides whilst converting the sugar hydroxyl to a more powerful (and, hopefully, dominant) nucleophile by ionization. The basic chemical reaction proposed is that illustrated in equation 1.

o

II RO-P-X 1

0

+ OR'

-

U RO-P-OR'

+

X

Equation 1

We were encouraged in our efforts to develop this reaction by the effective use of the same principle in the synthesis of nucleoside-3',5' cyclic phosphates (6). This communication describes experiments which have led to a successful method for the synthesis of the internucleotide bond together with the application of the method to the synthesis of thymidylyl-(5' + 3')-thymidine (dT-dT). Some of these results have been reported in preliminary form (7). MATERIALS AND METHODS Solvents were redistilled and dried over CaH2 or over Linde 4A molecular sieve. Snake venom phosphodiesterase and E. coli phosphomonoesterase were from Worthington Biochemical Corp. Descending paper chromatography on Whatman 40 paper was performed in the solvent systems; 1, isopropanol: conc. NH40H: H20 (7:1:2); 2, isobutryic acid: M NH40H (5:3); 3, 0.1 M sodium phosphate, pH 6.8: solid (NH4)2S04: n-propanol (100:60:2) using appropriate reference compounds. Elution of nucleotides from paper was done with water or, in the case of protected nucleotides, with 50% ethanol. Thin-layer chromatbgra,phy was carried out on silica

gel (GF-254, Macherey Nagel) ti methanol:chloroform (1:9). Phosphate analysis was carried out as described by Ames (8). Spectrophotometry was carried out using a Unicam SP800 spectrophotometer. M potassium tert-butoxide solutions were prepared by dissolving freshly cut 'potassium (0.4 g) in M tert-butanol in hexamethyl phosphoramide (10 ml), or by dissolving potassium (0.4 g) in tert-butanol (10 ml), or by dissolving freshly sublimed potassium tert-butoxide in dimethyl formamide or dimethyl sul-

phoxide.

Thymidine-3',5' Cyclic Phosphate. Thymidine-5' p-nitrophenyl phosphate (9), thymidine-5' 2,4-dinitrophenyl phosphate (10) or 1728

Nucleic Acids Research thymidine-5' phosphorofluoridate (11), ammonium salt was dried The nucleotide (10 pmoles) was over P205 at 250 for 24 hrs. dissolved in dimethyl sulfoxide or in dimethyl formamide (1 ml) to which was added M potassium tert-butoxide (100 p1) in either tert-butanol, dimethyl formamide, dimethyl sulfoxide or hexamethyl phosphoramide. After an appropriate time at 250, the reaction was quenched with AG-50 x 2 resin (Bio-Rad, pyridinium form) and the products analysed either on a column of Cellex T (Bio-Rad, 12 mm x 250 mm) in the carbonate form by elution with a linear gradient of NH4HCO3 (10) or by paper chromatography in solvent 1 (6). The results are summarized in Table 1. TABLE I

Synthesis of thymidine-3',5' cyclic phosphate from thymidine-5' phosphate derivatives catalysed by potassium tert-butoxide at 250. For details see Materials and Methods.

Compound

Potassium tert-butoxide solvent

Thymidine-5' p-nitrophenyl

Reaction Reaction solvent time (minutes)

Yield of thymidine3',5' cyclic phosphate

t-BuOH DMF t-BuOH DMSO HMP

DMF DMF DMSO DMSO DMF

20 20 20 20 20

80% 80 86 69 92

2,4-dinitrophenyl phosphate

DMF t-BuOH DMSO HMP

DMF DMSO DMSO DMF

20 20 20 20

0 0 0 17

Thymidine-5'

HMP

DMSO

120

80

phosphate

Thymidine-5'

phosphorofluoridate Abbreviations: t-BuOH, tert-butanol; DMF, dimethyl formamide; DMSO, dimethyl sulfoxide; HMP, hexamethyl phosphoramide. In all of the reactions, the substrate was completely used and the only nucleotide product, other than thymidine-3',5' cyclic phosphate, was thymidine-5' phosphate.

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Nucleic Acids Research 3' -0-Monomethoxytritylthymi ine-5' Phosphate. Thymidine-5' phosphate (2.4 mmoles) as the pyridinium salt was allowed to react with monomethoxytrityl chloride (4.8 mmoles) in pyridine (7 ml) for 3 days at 250, in analogy to the reaction of Nacetyldeoxyguanosine-5' phosphate with dimethoxytrityl chloride (11). After removal of solvent in vacuo, the products, in 50% ethanol, were applied to a column of DEAE-cellulose (25 mm x 400 mm), in the carbonate form, packed in 50% ethanol containing 5 x 104 M NH4HCO3. The column was washed successively with 5 x 10- 4M NH4HCO3 in 50% ethanol (500 ml), 0.1 M NH4HCO3 in water (250 ml) and 5 x 10- 4M NH4HCO3 in 50% ethanol (250 ml). The ammonium salt of 3'-0-monomethoxytritylthymidine-5' phosphate was eluted with 0.1 M NH4HCO3 in 50% ethanol (650 ml) and isolated as a solid after rotary evaporation and lyophilization. The yield was 2.2 mmoles and the product was homogeneous on chromatography in solvent 1, Rf 0.70. Calculated; P, 4.78%; found; P, 4.72%. 3'-0-Monomethoxytritylthymidine. 3 '-0-bonomethoxytritylthymidine5' phosphate, ammonium salt (2.2 mmoles) in 0.1 M NH4HCO3 (1 litre) was treated with E. coli alkaline phosphatase (1.5 mg) at 350 for 44 hrs. More enzyme (0.5 mg) was added and the reaction continued for 22 hrs. At this time the reaction was 95% complete as judged by release of inorganic phosphate. The 3'0-monomethoxytritylthymidine, which precipitated during the reaction, was collected by filtration, washed with 2% NH40H at 00 and dried over CaC12 in vacuo. The yield was 1.72 mmoles and the product was >98% pure on thin-layer chromatography. Calculated; N, 5.45%; found; N, 5.23%. 5'-0-Dimethoxytritylthyrmidine. This was prepared by the method of Schaller et al. (12) except that it was isolated by chromatography on silicic acid, being eluted with a gradient of 2 to 6% methanol in chloroform containing a drop of conc NH40H. After removal of solvent the residue was dissolved in methanol and precipitated with 2% NH40H at 0°. The precipitate was dried over CaCl2 in vacuo. 5'-O-Dimethoxytritylthymidine-3' p-Nitrophenyl Phosphate. This was synthesized and isolated as its ammonium salt by the method of Borden and Smith (9). 5'-0-Dimethoxytritylthymidine-3' Phenyl Phosphate. 5'-0-Mono-

methoxytritylthymidine (0.2 mmoles) and phenyl phosphorodichlor1730

Nucleic Acids Research idate (0.4 mmoles) in pyridine (2 mls) were allowed to react at 200 for 21 hrs. Water (6 ml) and pyridine (2 ml) were added and after 1 hr at 250 the solvent was removed in vacuo. The residue was dissolved in 80% acetic acid (5 ml) and kept at 400 for 1 hr to remove the monomethoxytrityl group. The solvent was removed in vacuo and the residue suspended in water (50 ml) and washed with ether (50 ml). Thymidine-3' phenyl phosphate was isolated as its ammonium salt by chromatography on DEAE-cellulose using a linearly increasing gradient of NH4HCO3 for elution. Thymidine-3' phenyl phosphate (0.4 mmoles), pyridinium salt, was treated with dimethoxytrityl chloride (1.6 mmoles) in pyridine (2 ml) for 18 hrs at 250. Water (4 ml) was added and the solvent removed in vacuo. The 5'-O-dimethoxytritylthymidine-3' phenyl phosphate was isolated as its ammonium salt (0.3 mmole) by chromatography on DEAE-cellulose using the protocol described for the isolation of 3'-O-monomethoxytritylthymidine-5' phosphate. Calculated; P, 4.33%; found; P, 4.01%.

5'-O-Dimethoxytritylthymidine-3' Phosphorofluoridate. Thymidine3' phosphorofluoridate (0.25 mmole) prepared by the method of Wittmann (12), as the pyridinium salt, was reacted with dimethoxytrityl chloride (2 mmoles) in pyridine (12 ml) at 200 for 18 hrs. The solvent was removed in vacuo and the desired product isolated as its ammonium salt (0.87 mmole) after chromatography on DEAE-cellulose using the protocol described for 3'-O-monomethoxytritylthymidine-5' phosphate. The product was homogeneous in solvent 1, Rf 0.85. Calculated; P, 4.82%; found; P, 4.64%. 3'-O-Monomethoxytritylthymidine-5' Phosphorofluoridate. Thymidine5' phosphorofluoridate (1 mmole) was reacted with monomethoxytrityl chloride (2 mmoles) in pyridine (10 ml) for 4 days at 250. Isolation of the ammonium salt was as described in the synthesis of 5'-O-dimethoxytritylthymidine-3' phosphorofluoridate (yield, 0.7 mmole). Calculated; P, 5.06%; found; P, 4.94%. 5'-O-Monomethoxytritylthymidine. This was prepared by the method of Schaller et al. (13). Thymidylyl-(5' 3')-thymidine. The appropriate protected nucleotide (20 pmoles) and nucleoside (10 imoles) were dried in vacuo over CaH2 and then placed in a dry box kept anhydrous with P205 under a slight positive pressure of dry nitrogen. The reactants were dissolved in dimethyl formamide on dimethyl sulfoxide (1 ml) 1731

Nucleic Acids Research after which M potassium tert-butoxide in hexamethyl phosphoramide was added in 10-fold excess over all ionizable groups (600 pl). After an-appropriate time at 250, the reaction was terminated by adding 2 to 3 ml of AG50 x 2 resin (pyridinium form). The resin The monomethoxytrityl and dimethoxywas removed by filtration. trityl groups were removed by 80% acetic acid at 250 for 4 hrs. The acetic acid was removed in vacuo, the residue taken up in water, and after an ether extraction, the water-soluble nucleotides were isolated on DEAE-cellulose using a NH4HCO3 gradient. The experiments are summarized in Table 2. All the products were characterized by paper chromatography in three systems using appropriate reference compounds. In addition, thymidylyl-(5' 3')-thymidine was degraded, completely, by snake venom phosphodiesterase (14) and the resultant thymidine and thymidine-5' phosphate characterized by paper chromatography. Yields were estimated spectrophotometrically.

TABLE II Synthesis of thymidylyl-(5' + 3')-thymidine(dT-dT) by reaction of 5'-O-dimethoxytritylthymidine-3' phosphate derivatives with 3'-O-monomethoxytritrylthymidine in the presence of potassium tert-butoxide at 250. For details see Materials and Methods.

Solvent

Reaction time (hours)

Yield of dT-dT

DMF

0.25

14%

p-Nitrophenyl

DMSO

6

ester

DMF

24

Phosphorofluoridate

DMSO DMSO DMF DMF

0.25 72 0.25 72

Nucleotide |Phenyl ester

1732

2 2 17 11 87 40

Nucleic Acids Research RESULTS AND DISCUSSION As a first step in establishing the best potential leaving group (X, in Equation 1) for internucleotide bond synthesis, the series p-nitrophenoxide, 2,4-dinitrophenoxide and fluoride were carefully evaluated in the formation of thymidine-3',5' cyclic phosphate from the appropriate thymidine-5' phosphate derivative (Table I). Under a variety of conditions, thymidine-5' p-nitrophenyl phosphate was converted by potassium tert-butoxide to thymidine-3',5' cyclic phosphate rapidly and in excellent yield. However, thymidine-5' 2,4-dinitrophenyl phosphate was not converted to the cyclic nucleotide at all under three sets of conditions and only in 17% yield under conditions which resulted in a 92% yield from thymidine-5' p-nitrophenyl phosphate. The only nucleotide product from thymidine-5' 2,4-dinitrophenyl phosphate was thymidine-5' phosphate. The production of a number of yellow compounds, separable on DEAE-cellulose, but uncharacterized, suggests that this results from nucleophilic attack on the 2,4dinitrobenzene rather than from base-catalysed hydrolysis. The reaction of thymidine-5' phosphorofluoridate with potassium tert-butoxide, whilst being more sluggish, was very effective in producing thymidine-3',5' cyclic phosphate. The conclusion from this series of experiments was that there was no point in examining 2,4-dinitrophenoxide as a potential leaving group in internucleotide bond synthesis. Derivatives of thymidine-3' phosphate were used for the first investigation of the synthesis of the internucleotide bond because the anion of a 5'-hydroxyl group is a more effective nucleophile in nucleoside-3',5' cyclic phosphate synthesis than is that of a 3'-hydroxyl group (6). As well as the leaving groups p-nitrophenoxide and fluoride, phenoxide was investigated because it would be expected to be more resistant to direct nucleophilic attack than p-nitrophenoxide. As can be seen from Table 2, the most effective method for thymidylyl-(5' -* 3')thymidine synthesis involved limited exposure to tert-butoxide using fluoride as the leaving group. In reactions of longer duration, there was extensive production of thymine, presumably resulting from tert-butoxide catalysed degradation of the dinucleotide. When p-nitrophenoxide was used as the leaving group,

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Nucleic Acids Research the yield of dinucleotide was very low. Analysis of the deprotected products showed that the major nucleotide was thymidine-3' phosphate. Presumably, nucleophilic attack on the nitrobenzene residue was responsible. With phenoxide as leaving group, a moderate yield of dinucleotide was obtained. Examination of the deprotected products showed that the only other nucleotide was thymidine-3' phenyl phosphate. Thus, the lower yield indicates that phenoxide is a less effective leaving group than fluoride, but it is not destroyed by tert-butoxide. The conclusion from this series of experiments is that a nucleoside-3' phosphorofluoridate is a very effectivj intermediate in tert-butoxide catalysed phosphodiester synthesis. It was next of interest to establish whether an internucleotide bond could be synthesized from a nucleoside-5' phosphorofluoridate in base-catalysed reaction with a nucleoside 3hydroxyl function. A reaction of 5'-O-monomethoxytritylthymidine with 3'-O-monomethoxytritylthymidine-5' phosphorofluoridate catalysed by potassium tert-butoxide under the standard conditions in dimethyl formamide was allowed to proceed for 15 minutes at 250. After the usual work-up, thymidylyl-(5' -* 3')-thymidine was isolated in 65% yield. Thus, this route to the internucleotide bond was significantly less efficient. The release of thymine on prolonged exposure of the reaction products to excess potassium tert-butoxide demanded that this type of degradation be minimized. With a 10-fold excess of tertbutoxide, a maximum yield of dinucleotide could be obtained after 10 minutes at 250. Under these conditions, there was two percent degradation of nucleotide to thymine. In order to establish conditions for minimum exposure to tert-butoxide, reactions catalyzed by decreasing amounts of base were carried out (Table 3). Under standard conditions each reaction contains 60 pmoles of ionizable groups (including the thymine residues). Only a small excess of base was required to achieve efficient phosphodiester synthesis. In conclusion, the experiments reported in this paper establish that the tert-butoxide catalysed reaction of a suitably protected deoxyribonucleoside and a deoxyribonucleoside-3' phosphorofluoridate provides an efficient, rapid route to the

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Nucleic Acids Research TABLE III

Effect of the amount of potassium tert-butoxide on the yield of thymidylyl-(5' + 3')-thymidine (dT-dT) produced by reaction of 5'-O-dimethoxytritylthymidine-3' phosphorofluoridate with 3'-Omonoethoxytritylthymidine in dimethyl formamide at 250 for 15 minutes using standard reaction conditions and isolation procedure (Materials and Methods). M potassium tert-butoxide (Pls)

600 360 90 75 60

Yield of dT-dT (%) 81 81 80 46 0

dinucleoside phosphate. In two following papers, a new approach to the synthesis of deoxyribonucleoside phosphorofluoridates is described together with an evaluation of the application of the base-catalyzed reaction to the synthesis of the internucleotide bond involving the other common deoxyribonucleosides and the synthesis of oligodeoxyribonucleotides.

* Research supported by the Medical Research Council of Canada ** Department of Microbiology, University of Alberta, Edmonton, Alberta, Canada * Medical Research Associate of the Medical Research Council of Canada

REFERENCES Chem. 17, 349-381. Khorana, H.G. (1968) Pure-p Agarwal, K.L., Yamazaki, A., CashiTon, P.J. and Khorana, (1972) Angew Chem. Intl. Ed. 11, 451-459. Letsinger, R.L. and Ogilvie, K.K. (1969) J. Am. Chem. Soc. 91, 3350-3355.

1. 2. H.G. 3.

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Nucleic Acids Research 4. Nagyvary, J. and Nagpol, K.L. (1972) Science 177, 272-274. 5. Nagyvary, J., Chladek, S. and Roe, J. (1970) Fiochem. Biophys. Res. Comm. 39, 878-882. 6. Borden, R.K. an Smith, M. (1966) J. Or. Chem. 31, 32473253. von Tigerstrom,. R.G. and Smith, M. (1970) Science 167, 7. 1266-1268. 8. Ames, B.N. (1966) in Methods in Enzymology, Vol. 8, pp. 115-118, Academic Press, New York. 9. Borden, R.K. and Smith, M. (1966) J. Or. Chem. 31, 32413246. 10. von Tigerstrom, R. and Smith, M. (1969) Biochemistry 8, 3067-3070. 11. Ralph, R.K., Connors, W.J., Schaller, H. and Khorana, H.G. (1963) J. Am. Chem. Soc. 85, 1983-1988. 12. Wittmann, R. (1963) Ehem. Ber. 96, 771-779. 13. Schaller, H., Wiemann, G., Lerch, B. and Khorana, H.G. (1963) J. Am. Chem. Soc. 85, 3821-3827. 14. Giiham, P.T. andlKhorana, H.G. (1958) J. Am. Chem. Soc. 80, 6212-6222.

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The synthesis of the internucleotide (phosphodiester) bond by a base-catalysed reaction.

Volume 2 number 10 October 1975 Nucleic Acids Research The synthesis of the internucleotide (phosphodiester) bond by a base-catalysed reaction* Ric...
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