39 Some Aspects of the Chemical Synthesis of Oligodeoxyribonucleotides Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 29, 2018 | https://pubs.acs.org Publication Date: November 11, 1981 | doi: 10.1021/bk-1981-0171.ch039
COLIN B. REESE and LYDIA VALENTE Department of Chemistry, King's College, Strand, London WC2R 2LS, England
We report some recent studies on the synthesis of oligodeoxy ribonucleotides. We have prepared three dodecamer restriction site adaptors which were required by Dr. Peter Rigby of the Department of Biochemistry, Imperial College of Science and Technology, University of London, in connection with his studies on the cloning of cDNA (complementary deoxyribonucleic acids). The nucleotide sequences are d[AATTCGGTACCG], d[AATTCGAGCTCG] and d[AATTCGTCGACG] which are Eco RI -> Κpn I, Eco RI->Sst I and Eco RI->Pst I adap tors, respectively. These three dodecamers were prepared in satis factory yields from four fully-protected hexamers (d[Dbmb -ApApTpTpCpG-Px], d[Dbmb-GpTpApCpCpG-Px], d[Dbmb-ApCpCpTpCpG-Px] and d[Dbmb-TpCpGpApCpG-Px]. The system of abbreviations for pro tected oligodeoxynucleotides has been described previously (1). [o-Dibromomethylbenzoyl (2) and 9-phenylxanthen-9-yl (3) are abbre viated to Dbmb and Px, respectively. 6-N-(p-t-Butylbenzoyl)-2' -deoxyadenosine, 4-N-benzoyl-2'-deoxycytidine and 2-N-(p-t-butylphenylacetyl)-2'-deoxyguanosine residues are represented by A, C and G, respectively; phosphate residues which are protected by o -chlorophenyl groups are represented by p.]
0097-6156/81/0171-0191$05.00/ 0 © 1981 American Chemical Society Quin and Verkade; Phosphorus Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
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PHOSPHORUS CHEMISTRY
Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 29, 2018 | https://pubs.acs.org Publication Date: November 11, 1981 | doi: 10.1021/bk-1981-0171.ch039
Scheme 1
Scheme 2 (a)
d[Obmb-ApApTpTpCpG-Vx] [Obmb-GpTpApCpCpG-T?x]
(b) d (c)
(12)
+ (23)
d[Obmb-ApApTpTpCpGp]
(12)
d[HO-GpTpApCpCpG-Px]
(13)
d[Dbmb-ApApTpTpCpGpGpTpApCpCpG-Px]
(14)
(d) (14)
d[AATTCGGTACCG] (15)
ReagentsÎ
( i ) ( a ) ( 2 ) / a c e t o n i t r i l e - p y r i d i n e , (b) E t N - H 0 ; (ii)(3)/ pyridine; (iii)(4)/pyridine; (iv)(a) s i l v e r perchlorate2,4,6-collidine/acetone-water (98:2 v / v ) , (b) morpholine; (ν) t o l u e n e - p - s u l f o n i c acid/CHCl -MeOH (95:5 v / v ) ; (vi) Ν ,Ν ,N_^,N -tetramethylguanidinium s y n - 4 - n i t r o benzaldoximate i n dioxan-water (1:1 v / v ) , 20°C, 24 h r ; ( v i i ) aqueous ammonia (d 0.88), 20°C, 48 h r ; ( v i i i ) a c e t i c acid-water (4:1 v / v ) , 20°C, 15 min. 3
2
3
1
1
3
Quin and Verkade; Phosphorus Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 29, 2018 | https://pubs.acs.org Publication Date: November 11, 1981 | doi: 10.1021/bk-1981-0171.ch039
39.
REESE A N D VALENTE
Synthesis of
Oligodeoxyribonucleotides
193
The procedure used for the preparation of the fully-protected hexamers is indicated in Scheme 1. The nucleoside building blocks required (5) are prepared (1,2) by treating thymidine or appropriate N-acyl derivatives of 2'-deoxyribonucleosides (7) with o-dibromomethylbenzoyl chloride (1) in acetonitrile-pyridine solution. The resulting Dbmb-derivatives (5), which are obtained as crystalline solids in good isolated yields, are first treated with a two- to three-fold excess of o-chlorophenyl phosphorodi-(1,2,4triazolide) (2) (1^,4) in acetonitrile-pyridine and the products hydrolyzed with aqueous triethylamine to give the corresponding 3 (o-chlorophenyl) phosphates (6) in very high (usually { 90%) yields. The latter mononucleotide building blocks (6) are isolated as pure solid triethylammonium salts Q,3' -dinucleoside phosphates may be detected in the products. When the mononucleotide blocks (6) are allowed to react (ca. 20 min, room temperature) with thymidine or the appropriate N-acyl2 -deoxyribonucleoside (7) in the presence of an excess (ca. 3 molecular equivalents) of 1-(mesitylene-2-sulfonyl)-3-nitro-l,2,4triazole (3, MSNT) (5,6) in pyridine solution, the corresponding 3 ->5 -partially-protected dinucleoside phosphates (8) are obtained in good yields (usually 60-70%). Phosphorylation occurs on the 5'-hydroxy function of (7) with a very high degree of regioselectivity and the small quantities of the isomeric 3'->3 -dinucleoside phosphates sometimes obtained are less polar and may readily be removed from the desired products (8) by short column chromatography (7) on silica gel. A partially-protected dinucleoside phosphate (d[HO-CpG-Px]) with a free 5'-hydroxy and a protected 3'-hydroxy function is also required. Treatment of the 5*-protected dinucleoside phosphates (8) with 9-phenyl-9-xanthenyl chloride (4, pixyl chloride) (3) in pyridine solution gives their 5'-0-pixyl derivatives in virtually quantitative yields. When the latter are treated first with silver perchlorate and 2,4,6-collidine in acetone-water (98:2 v/v) for ca. 1 hr at room temperature and the products then treated with morpholine, the 5'-Dbmb group (2) is removed and the desired dinucleoside phosphates (9) with protected 3 -terminal hydroxy functions are obtained in good yields. The appropriate 5'protected dinucleoside phosphates (8) are then phosphorylated with o-chlorophenyl phosphorodi-(1,2,4-triazolide) (2) and thereby converted into their 3'-(o-chlorophenyl) phosphates (10) in yields of ca. 90%. The latter (10) are then condensed, in the presence of an excess of MSNT (3) with the 3 -protected dinucleoside phosphates (9) to give fully-protected tetranucleoside triphosphates usually in good yields (ca. 70%). The corresponding partially-protected tetranucleoside triphosphates (11) are then obtained by removing the 5'-Dbmb protecting groups by the procedure described above. The desired fully-protected hexanucleoside pentaphosphates are then prepared in the same way by condensing dinucleotide blocks (10) with the appropriate partially-protected tetranucleoside triphos1
1
1
1
1
1
1
Quin and Verkade; Phosphorus Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 29, 2018 | https://pubs.acs.org Publication Date: November 11, 1981 | doi: 10.1021/bk-1981-0171.ch039
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PHOSPHORUS
CHEMISTRY
phates (11). MSNT (3) i s again used as the condensing agent and s a t i s f a c t o r y y i e l d s (ca. 60%) are u s u a l l y obtained. The p r e p a r a t i o n o f one o f the completely unblocked dodecamers ( 1 5 ; the Eco RI -> Kpn I adaptor) i s i l l u s t r a t e d i n Scheme 2. The f i n a l steps i n the p r e p a r a t i o n and unblocking o f the other two do decamers correspond e x a c t l y . In the f i r s t p l a c e , the hexamer block (d [Dbmb-ApApTpTpCpG-'Px] ) i s t r e a t e d (Scheme 2a) with toluene£-sulfonic a c i d i n chloroform-methanol (95:5 v/v) and the r e s u l t i n g hexamer with a f r e e 3 -hydroxy f u n c t i o n i s phosphorylated with ochlorophenyl p h o s p h o r o d i - ( 1 , 2 , 4 - t r i a z o l i d e ) t o give the correspond ing hexanucleotide block (12). The l a t t e r i s then condensed, i n the presence o f MSNT (3), with the product (13, Scheme 2b) obtained by removing the 5'-Dbmb p r o t e c t i n g group from the hexamer block (diDbmb-GpTpApCpCpG-Vx]), t o g i v e the f u l l y - p r o t e c t e d dodecamer (14, Scheme 2c). The y i e l d i n t h i s f i n a l condensation step i s good but the p u r i f i c a t i o n o f the dodecamers by short column chroma tography on s i l i c a g e l proved sometimes t o be d i f f i c u l t . The removal o f the p r o t e c t i n g groups from (14) i s e f f e c t e d i n a t h r e e step process. The f u l l y - p r o t e c t e d o l i g o n u c l e o t i d e i s f i r s t t r e a t e d with a l a r g e excess (ca. 10 molecular e q u i v a l e n t s p e r phosp h o t r i e s t e r group) o f a 0.3 M-solution o f the Ν , Ν , N , N - t e t r a methylguanidinium s a l t o f syn-4-nitrobenzaldoxime (_5) i n dioxanwater (1:1 v/v) a t 20°C f o r 24 h r to unblock the i n t e r n u c l e o t i d e linkages. We now b e l i e v e (8) t h a t t h i s process i s complete i n a much s h o r t e r time. An ammonolysis step completes the removal o f the N-acyl and 5 -O-Dbmb p r o t e c t i n g groups. The f u l l y - u n b l o c k e d o l i g o n u c l e o t i d e i s obtained f o l l o w i n g the removal o f the p i x y l group by a c i d i c h y d r o l y s i s . 1
1
1
3
3
1
A l l three o f the f u l l y - u n b l o c k e d dodecamers underwent complete d i g e s t i o n t o give t h e i r monomeric components when they were t r e a t e d with C r o t a l u s adamanteus snake venom and spleen phosphodiesterases. T h e i r s t r u c t u r e s were f u r t h e r confirmed i n the usual way. We thank the Science Research C o u n c i l f o r generous f i n a n c i a l support.
Literature Cited 1. Chattopadhyaya, J. B.; Reese, C. B. Nucleic Acids Res. 1980, 8, 2039. 2. Chattopadhyaya, J. B.; Reese, C. B.; Todd, A. H. J. Chem. Soc. Chem., Commun. 1979, 987. 3. Chattopadhyaya, J. B.; Reese, C. B. J. Chem. Soc., Chem. Commun. 1978, 639. 4. Chattopadhyaya, J. B.; Reese, C. B. Tetrahedron Lett. 1979, 5059. 5. Reese, C. B.; Titmas, R. C.; Yau, L. Tetrahedron Lett. 1978, 2727. 6. Jones, S. S.; Rayner, B.; Reese, C. B.; Ubasawa, A.; Ubasawa, M. Tetrahedron 1980, 36, 3075. 7. Hunt, B. J.; Rigby, W. Chem. Ind. (London) 1967, 1868. 8. Reese, C. B.; Valente, L . , unpublished observations. RECEIVED
July 7, 1981. Quin and Verkade; Phosphorus Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
