.) 1992 Oxford University Press

4100 Nucleic Acids Research, Vol. 20, No. 15

One pot general method for the derivatisation of polymer support for oligonucleotide synthesis P.Sharma, Ashwani K.Sharma, V.P.Malhotra1 and K.C.Gupta* Nucleic Acids Research Laboratory, CSIR Centre for Biochemicals, University Campus, Mall Road, Delhi- 10 007 and 1Chemistry Department, D.J. College, Baraut (UP), India Submitted June 18, 1992 The last decade has seen a tremendous improvement in solid phase oligonucleotide synthesis since the introduction of phosphoramidite chemistry and silica-based solid supports. The current methodology for oligonucleotide synthesis utilizes nucleoside anchored on controlled pore glass (CPG) by a succinyl linkage. A number of other linkages such as ester (1), urethane (2) and those cleaved under mild basic conditions (3, 4) are known for synthesising oligonucleotides and modified oligonucleotides. However, succinate linkage remains the most commonly used linkage for solid phase oligonucleotide synthesis. A number of methods (5 - 8) have been reported for the functionalisation of polymer supports with succinate linkage. Most of them involve the time consuming preparation of nucleoside-3'-0-succinates and their anchoring on polymer supports via active ester method (5) or directly by using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (DEC) as coupling reagent (6). However, the time taken for the functionalisation of polymer supports is considerably longer (30 h). Damha et al. (7) have proposed an alternative derivatisation procedure in which 2'-deoxyribo-, ribo- and arabinonucleosides are coupled directly to long chain alkylamine controlled pore glass (LCAA-CPG) via 2 '- or 3'-hydroxyl groups using DEC/ dimethylaminopyridine (DMAP) as a coupling reagent, but the time taken for the derivatisation is very long (3-5 days). In this work we describe a versatile, rapid and economical method in which 2'-deoxyribonucleosides are directly coupled to the amino groups of the polymer supports using a bifunctional reagent, succinyl chloride, in 2 h with excellent nucleoside loadings. Thus, the derivatisation of polymer supports is no longer a time consuming process. 0

/

C

2

TONoOMTrO

OHT~

H

CH3CN/Py

2

8- A,C,G or T

0=

CPG, FRACTOSL,

C=O ~

CH-2 CONH

LCAA

Scheme-I

The functionalisation of polymer supports was carried out as given in Scheme 1 Succinyl chloride (6.3 tl, 0.05 mmol) was added to a solution of triazole (19.8 mg, 0.28 mmol) and pyridine (0.5 ml) in dry acetonitrile (1.0 ml) taken in a septum sealed vial under argon atmosphere. To the clear solution so formed, appropriately protected 2'-deoxyribonucleoside (0.05 mmol) in dry acetonitrile was added using a syringe. The reaction mixture was kept at room temperature for 1 h with occasional shaking. Septum was opened under argon atmosphere and appropriate polymer support (-NH2 = 10 umol) was added, the reaction vial was further shaken for 15 min at room temperature. The .

ACKNOWLEDGEMENT The financial assistance from the DBT is gratefully

acknowledged. REFERENCES 1. Dobrynin,V.N., Filippova,S.A., Bystrove, N.S., Severtsova,I.V. and Kolosov, M.N. (1983) Bioorgn. Khim. 9, 706-710. 2. Sproat,B.S. and Brown,D.M. (1985) Nucleic Acids Res. 13, 2979-2987. 3. Alul,R.H., Singman,C.N., Zhang,G. and Letsinger,R.L. (1991) Nucleic Acids Res. 19, 1527 -1532. 4. Eritja,R., Robles,J., Fernandez,D., Albericio,F., Giralt,E. and Pedroso,E.

(1991) Tet. Lett. 32, 1511-1514.

o

C=__O CH 2- CH 2 C°l

reaction mixture was transferred to a sintered funnel under argon atmosphere and washed successively with dry acetonitrile (2 x 5 ml) and dry methanol (1 x 5 ml) (to cap residual succinyl triazolide groups) followed by washing with dry acetonitrile (2 x 5 ml). The residual amino groups on the support were capped following standard procedure (5). The nucleoside loadings obtained on different polymer supports are given in Table 1. All the supports were tested and compared with the supports functionalised following standard protocols (5) in a PharmaciaLKB Gene Assembler Plus using phosphoramidite chemistry. The coupling efficiency per cycle based on the released 4,4'-dimethoxytrityl cation exceeded 98 % and the isolated yields of the purified oligomers were found to be in full agreement with the oligomers synthesised using the standard supports. This is the fastest method of derivatisation of polymer supports with succinyl linkage known to date. The method obviates the need for the time consuming preparation of four nucleoside-3'-0succinates. The derivatisation of polymer supports is simple and amenable for the functionalisation of large quantities of polymer supports.

5. Atkinson,T. and Smith,M. (1984) In Gait,M.J. (ed.) Oligonucleotide Synthesis: A Practical Approach. IRL Press, Oxford, pp. 35-81. 6. Pon,R.T., Usman,N. and Ogilvie,K.K. (1988) BioTechniques, 6, 768-775. 7. Damha,M.J., Ginnaris,P.A. and Zabarylo,S.V. (1990) Nucleic Acids Res. 18, 3813-3821. 8. Gupta,K.C. and Kumar,P. (1992) Biomed. Chem. Lett. (in press). 9. Reddy,M.P., Rampal,J.B. and Beaucage,S.L. (1987) Tet. Lett. 28, 23-26. Table 1. Loading of the nucleosides on different polymer supports

Loading in zmol/g

Nucleoside

DMTrdAbz DMTrdCb7 DMTrdT

DMTrdG'hU

LCAA (47)a

CPG(52)"

Fractosil(93)a

29.0 32.0 30.0 28.0

43.0 39.0 41.0 43.0

57.0 56.0 55.0 56.0

'Amino groups loading as determined by using

(9).

DMTrCl/silver

nitrate method