Palladium(0)-catalyzed modification of oligonucleotides during automated solid-phase synthesis

Full text of DOI:10.1021/ja9836794

4704
J. Am. Chem. Soc. 1999, 121, 4704-4705
Communications to the Editor
Palladium(0)-Catalyzed Modification of
Oligonucleotides during Automated Solid-Phase
Synthesis
for new and alternative procedures that offer greater flexibility
and synthetic ease to modify oligonucleotides. Herein, we report
the site-specific modification of oligonucleotides on solid support
using a novel procedure that combines the advantages of solid-
phase DNA and Pd(0) cross-coupling chemistries.
To demonstrate the generality of this new oligonucleotide
modification procedure, three structurally and electronically
different functionalities were directly incorporated into an
Shoeb I. Khan and Mark W. Grinstaff*
Department of Chemistry
Paul M. Gross Chemical Laboratory
Duke UniVersity, Durham, North Carolina 27708
oligonucleotide during standard automated DNA solid-phase
synthesis.2
0-22
Alkynyl-derivatized amines, biotin, and a transition-
ReceiVed October 22, 1998
metal complex, each of which possesses unique properties and
synthetic requirements, were attached to an oligonucleotide using
Oligonucleotides modified with functional groups have diverse
and important research and clinical applications, including primers
for DNA sequencing, hybridization probes for detecting DNA,
antisense and antigene oligonucleotides for therapy, and spec-
troscopic probes for DNA structure and function studies.1-19
Synthetic strategies toward these complex oligonucleotides focus
primarily on either synthesizing the labeled phosphoramidite for
subsequent incorporation into the nucleic acid strand or modifying
the synthesized nucleic acid single strand after solid-phase
23-26
the Sonogashira Pd(0) cross-coupling reaction.
These alkynyl
precursors (2, 3, 5, 8) were synthesized as shown in Scheme 1.
The protected amines, N-trifluoroacetyl propargylamine (2) and
N-tert-butyloxycarbonyl propargylamine (3) were prepared by
reacting propargylamine with ethyl trifluoroacetate in MeOH and
27,28
di-tert-butyl dicarbonate in water/CHCl
3
, respectively.
The
biotin analogue, 6-((6-biotinoylamino hexanoyl)aminohexanoyl)-
propargylamine (5), was synthesized from the biotin aminohex-
anoylaminohexanoic acid succinimidyl ester (biotin-NHS ester)
1
synthesis. The major hurdles with the first procedure are the
29
and propargylamine in dry DMF. Reaction of Ru(bpy)
Cl
2 2
with
multiple synthetic and purification steps to the desired phos-
phoramidite, which is moisture-sensitive and often possesses
limited solubility in common solvents, followed by the low
coupling yield on the DNA synthesizer and chromatographic
purifications of the oligonucleotide derivative. The second
procedure, oligonucleotide postmodification, does require fewer
synthetic steps. This solution-phase coupling, however, of the
functional group to the oligonucleotide is often hampered by a
low coupling yield, side reactions, and chromatographic purifica-
tions to isolate the final product. Consequently, there is a need
4
′-methyl-2,2′-bipyridine-4-carbonylpropargylamine afforded bis-
(
2,2′-bipyridine)(4′-methyl-2,2′-bipyridine-4-carbonylpropargy-
lamine) ruthenium(II)bis-(hexafluorophosphate), 8. Incorporation
of these groups into the oligonucleotide also required the
preparation of a 5-iodo-substituted pyrimidine nucleoside for
subsequent Pd(0) cross-coupling reaction. Specifically, 5′-DMT-
3′-cyanoethyl-N,N′-diisopropylphosphoramidite-2′-deoxy-5-iodou-
ridine was synthesized in two steps. In the first step, 2′-deoxy-
5
5
-iodouridine was treated with DMT-Cl in dry pyridine to afford
′-DMT-2′-deoxy-5-iodouridine.
22,27
This DMT-protected nucleo-
(
1) (a) Goodchild, J. Bioconjugate Chem. 1990, 1, 165-186. (b) Beaucage,
side was then reacted with 2-cyanoethylchloro-N,N′-diisopropy-
lphosphoramidite, in the presence of diisopropylethylamine, to
yield 5′-DMT-3′-cyanoethyl-N,N′-diisopropylphosphoramidite-2′-
deoxy-5-iodouridine, 9.30
The oligonucleotide syntheses were performed on a commercial
ABI 395 DNA/RNA synthesizer from the 3′ to 5′ end using
standard automated DNA synthesis protocols as shown in Scheme
S. L.; Iyer, R. P. Tetrahedron 1993, 49, 1925-1963. (c) Beaucage, S. L.;
Iyer, R. P. Tetrahedron 1993, 49, 6123-6194. (d)Verma, S.; Eckstein, F.
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(
2) Truffert, J. C.; Asseline, U.; Brack, A.; Thuong, N. T. Tetrahedron
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(
1
14, 5509-5517.
2
(0.2 and 1.0 µmol scale). A 0.1 M solution of 5′-DMT-3′-
(
(
4) MacMillin, A. W.; Verdine, G. L. Tetrahedron 1991, 47, 2603-2616.
5) Kahl, J. D.; McMinn, D. L.; Greenberg, M. M. J. Org. Chem. 1998,
cyanoethyl-N,N′-diisopropyl phosphoramidite-2′-deoxy-5-iodou-
ridine in dry acetonitrile was prepared and installed on the DNA
synthesizer in a standard reagent bottle. All solid-phase syntheses
were performed in such a manner that the sequence was stopped
after incorporation of the 5′-DMT-3′-cyanoethyl-N,N′-diisopropyl
phosphoramidite-2′-deoxy-5-iodouridine, and without deprotecting
the 5′-hydroxyl or cleaving the oligonucleotide from the resin.
The column was subsequently removed from the synthesizer and
6
3, 4870-4871.
(
6) Hunkapillar, M.; Kent, S.; Caruthers, M.; Dreyer, W.; Firca, J.; Giffin,
C.; Horvath, S.; Hunkapiller, T.; Tempst, P.; Hood, L. Nature 1984, 310,
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05-111.
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7) Sanger, F.; Nicklen, S.; Coulson, A. R. Proc. Natl. Acad. Sci. U.S.A.
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977, 74, 5463-5467.
(
(
8) Fodor, S. P. A. Science 1997, 277, 393-395.
9) Wallraff, G.; Labadie, J.; Brock, P.; DiPietro, R.; Nguyen, T.; Nuynh,
T.; Hinsberg, W.; McGall, G. CHEMTECH 1997, February, 22-32.
(
10) Kremsky, J. N.; Pluskal, M.; Casey, S.; Perry-O’Keefe, H.; Kates, S.
A.; Sinha, N. D. Tetrahedron Lett. 1996, 37, 4313-4316.
11) Langer, P. R.; Waldrop, A. W.; Ward, D. C. Proc. Natl. Acad. Sci.
(20) Caruthers, M. H. Acc. Chem. Res. 1991, 24, 278-284.
(21) Caruthers, M. H. Science 1985, 230, 281-285.
(22) Gait, M. J. Oligonucleotide Synthesis: A Pratical Approach; IRL
Press: Washington, DC, 1984; p 217.
(
U.S.A. 1981, 78, 6633-6637.
(
(
12) Cook, P. D. Anti-Cancer Drug 1991, 6, 585-607.
13) Jones, D. S.; Hachmann, J. P.; Osgood, S. A.; Hayag, M. S.; Barstad,
(23) Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett. 1975, 50,
4467-4470.
P. A.; Iverson, G. M.; Coutts, S. M. Bioconjugate Chem. 1994, 5, 390-399.
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14) Chrisey, L. A.; Lee, G. U.; O’Ferrall, C. E. Nucleic Acids Res. 1996,
4, 3031-3039.
15) Silver, G. C.; Sun, J. S.; Nguyen, C. H.; Boutorine, A. S.; Bisagni,
E.; Helene, C. J. Am. Chem. Soc. 1997, 119, 263-268.
16) Perkins, T. A.; Wolf, D. E.; Goodchild, J. Biochemistry 1996, 35,
6370-16377.
17) Holmlin, R. E.; Dandliker, P. J.; Barton, J. K. Angew. Chem., Int. Ed.
Engl. 1997, 36, 2714-2730.
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2623-2624.
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1
(
1
0.1021/ja9836794 CCC: $18.00 © 1999 American Chemical Society
Published on Web 04/28/1999

Communications to the Editor
J. Am. Chem. Soc., Vol. 121, No. 19, 1999 4705
Scheme 1a
Scheme 2a
a
Key: (a) ethyl trifluoroacetate, MeOH, 25 °C, 3 h, yield 82%; (b)
3
water, NaHCO , di-tert-butyl dicarbonate, 25 °C, 3 h, yield 87%; (c)
propargyl amine, DMF, 25 °C, 24 h, yield 83%; (d) 70% ethanol/water,
reflux, 10 h, yield 82%.
Table 1. Site-Specifically Modified Oligonucleotides Synthesized
DNA
coupling
Pd(0)
coupling
functional group
no. sequence 5′-3′
(alkynyl group, no.) (% yield) (% yield)
1
0, CTU*AGCA
amine, 2
Boc-amine, 3
biotin, 8
98%
98%
98%
98%
85%
89%
92%
89%
1
1
1
1, DMT-TU*GCA
2, GTTU*GA
3, TAC ATC CTA U*CT
a
Key: (a) DNA synthesis; (b) off-synthesizer, Pd(Ph
C(O), (CH COC(O), biotin-C(O), and Ru(bpy)
)-C(O))2 ; (c) resume DNA synthesis; (d) 30% NH
, 55 °C,16 h.
ODN ) oligodeoxynucleotide. Prot. ) protected.
3
P)
4
CuI, TEA,
biotin, 8
DMF, 3 h, R ) CF
CH
3
)
3 3
2
(bpy-
1
4, GGT CTT ATT CAC CAC AAT AAC CTC AGT U*CT
+
(
3
3
biotin, 8
98%
96%
92%
89%
2+, 5
Ru(bpy)
3
1
5, DMT-TU*CA
synthesized. Importantly, the type, size, three-dimensional shape,
or charge of the functional group did not significantly affect the
Pd(0) cross-coupling reaction or the ability to synthesize both
short and long modified oligonucleotides.
sparged with N
column was attached to a syringe and the alkyne-derivatized
functional group, Pd(Ph P) , CuI, and 150 µL of dry solvent DMF:
Et N (3.5:1.5) were added. After 3 h, the column was washed
with DMF:Et N (9:1; 10 mL) and acetonitrile (40 mL), dried with
for 30 min, and then reinstalled on the synthesizer. Solid-
2
to maintain the anhydrous condition. Next, the
3
4
3
In conclusion, a simple and convenient procedure for the
derivatization of oligonucleotides using solid-phase nucleic acid
and Pd(0) cross-coupling chemistries is described. The advantages
of this on-column deriVatization method include: (1) fewer overall
synthetic steps, (2) efficient Pd (0) cross-coupling reactions, (3)
practical solid-phase reaction conditions, (4) ease of oligonucle-
otide purification, and (5) wide functional group tolerance. This
new protocol is an attractive alternative to the synthesis and use
of highly functionalized and specialized phosphoramidites for the
preparation of modified oligonucleotides at the nucleobase. These
favorable reaction conditions will facilitate the development and
design of new oligonucleotide derivatives which are of research
and clinical interest.
3
N
2
phase synthesis was resumed, and additional DNA bases were
added.
Collection and analysis of the trityl fractions during automated
synthesis showed efficient phosphoramidite coupling throughout
the procedure, with both the standard pyrimidine and purine
nucleosides as well as with 5-iodouridine (>95%; Table 1).
Finally, the modified oligonucleotide was collected and incubated
3
at 55 °C in NH overnight, to completely deprotect the oligo-
nucleotide. Analysis of the HPLC traces of the crude oligonucle-
otide products showed efficient Pd(0) cross-coupling reactions
for each functional group (yields ranged from 85 to 92%; Table
1
). Enzymatic digestion of the modified oligonucleotides showed
Acknowledgment. This work was supported by Duke University.
selective coupling to 5-iodouridine with no side reactions observed
with the other bases. The free amine, 10, or Boc-protected amine,
Supporting Information Available: Detailed experimental informa-
tion and characterization data (PDF). This material is available free of
charge via the Internet at http://pubs.acs.org.
11, modified oligonucleotides were synthesized from groups 2
and 3, respectively. Three oligonucleotides derivitized with biotin,
5
, were synthesized (12, 13, and 14). An oligonucleotide, 15,
2
+
modified with a transition-metal complex, Ru(bpy)
3
, was also
JA9836794