donors 2 from the corresponding nucleosides 1 using the
fulfill the requirements with respect to insolubility, inertness
to solvents/reagents, polarity, and sufficient surface area.
Tentagel is based on a composite of cross-linked polystyrene
with poly(ethylene glycol) (MW 3000-4000) and is termi-
nally functionalized with NH2 groups. The beads swell in
water, facilitating biological assays in aqueous systems.
Because of their large loading capacity, they have been used
for chemical oligonucleotide syntheses.14 The kieselguhr-
derived support consists of fabricated kieselguhr (SiO2)
containing large pores in which cross-linked polydimethyl-
acrylamide resin resides. Because of the macroporous
structure of the pores, they are freely permeable for very
large molecules and should be suitable for enzymatic
synthesis. Our major efforts were therefore directed toward
the use of this resin. Several 5′-phosphorylated primers
(mainly commercially available) were coupled to the resins
at the free amino group by performing the reaction in 0.1 M
N-methylimidazole buffer (pH 6) in the presence of water-
soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),
affording the resin-primer conjugate 5 having a loading of
about 2 µmol/g as estimated from the UV analysis of the
relevant solution following standard cleavage with NH2OH/
H2O (pH 4.5)14 and from analysis of the products by capillary
electrophoresis (CE). Higher loading was not striven for, but
there is no reason to assume that this is not possible.
procedure of Barrio.8
In the next step, adenylated 3′,5′-deoxycytidine phosphate
(4), although accessible by the method of Hoffmann and
McLaughlin,9 was prepared more conveniently by reacting
the (n-Oct)3NH+ salt of 2a with adenosine 5′-monophos-
phomorpholidate 3. Compound 4 was formed in 22% yield
in addition to some 3′-adenylated deoxycytidine phosphate.
Separation of this isomer was not necessary since it is not a
substrate for the enzyme.
With these compounds and commercially available10
primers pdA5, 5′-pdA(pdA)3prC-3′, and 5′-pdA(pdA)9prC-
3′ in hand, we turned our attention to the solid phase,
surmising that the choice of the solid support and of the linker
could be crucial to the success of the enzymatic oligonucle-
otide synthesis. Indeed, only a limited number of successful
cases of biocatalytic processes on solid supports are known
based on the use of other enzymes and substrates.11 The
commercial resins tentagel12 and kieselguhr/polydimethyl-
acrylamide (PDMA)13 are available in forms having terminal
NH2 groups, ideal for primer attachment. They appeared to
The principle of our solid-phase enzymatic oligonucleotide
synthesis is summarized in Scheme 1.
Scheme 1. Principle of Solid-Phase Enzymatic
Oligonucleotide Synthesis
(8) Barrio, J. R.; Barrio, M. del C.; Leonard, N. J.; England, T. E.;
Uhlenbeck, O. C. Biochemistry 1978, 17, 2077-2081.
(9) Hoffmann, P. U.; McLaughlin, L. W. Nucleic Acids Res. 1987, 15,
5289-5303.
(10) EUROGENTEC, 4102-Seraing, Belgium.
(11) Examples: (a) Elmore, D. T.; Guthrie, D. J. S.; Wallace, A. D.;
Bates, S. R. E. J. Chem. Soc., Chem. Commun. 1992, 1033-1034. (b)
Schuster, M.; Wang, P.; Paulson, J. C.; Wong, C.-H. J. Am. Chem. Soc.
1994, 116, 1135-1136. (c) Yamada, K.; Nishimura, I. Tetrahedron Lett.
1995, 36, 9493-9496. (d) Halcomb, R. L.; Huang, H.; Wong, C.-H. J.
Am. Chem. Soc. 1994, 116, 11315-11322. (e) Ko¨pper, S. Carbohydr. Res.
1994, 265, 161-166. (f) Meldal, M.; Auzanneau, F.-I.; Hindsgaul, O.; Palcic,
M. M. J. Chem. Soc., Chem. Commun. 1994, 1849-1850. (g) Waldmann,
H.; Reidel: A. Angew. Chem. 1997, 109, 642-644; Angew. Chem., Int.
Ed. Engl. 1997, 36, 647-649. (h) Sauerbrei, B.; Jungmann, V.; Waldmann,
H. Angew. Chem. 1998, 110, 1187-1190; Angew. Chem., Int. Ed. 1998,
37, 1143-1146.
The first three steps are repeated as often as desired, the
final step being the cleavage from the resin. Specifically,
the primer attached with its 5′-end to the solid support has
a free 3′-OH group that binds the nucleoside 3′,5′-biphos-
phate in the presence of T4 RNA ligase. The terminal
phosphate blocking group is removed enzymatically by
alkaline phosphatase, and all excess reagents are simply
washed off from the resin. The next nucleoside 3′,5′-
(12) (a) Bayer, E. Angew. Chem. 1991, 103, 117-133; Angew. Chem.,
Int. Ed. Engl. 1991, 30, 113-129. (b) Rapp W. In Combinatorial Peptide
and Nonpeptide Libraries; Jung, G., Ed.; VCH: Weinheim, 1996; pp 425-
464.
(14) Ryabova, T. S.; Shabarova, Z. A.; Prokof’ev, M. A. Dokl. Akad.
Nauk SSSR 1965, 162, 1068-1070.
(13) Dryland, A.; Sheppard, R. C. Tetrahedron 1988, 44, 859-876.
1730
Org. Lett., Vol. 1, No. 11, 1999