Synthesis of a benzotriazole phosphoramidite for attachment of oligonucleotides to metal surfaces

Article abstract of DOI:10.1016/S0040-4039(01)00108-3

A method for the addition of a benzotriazole moiety to the 5′-terminus of an oligonucleotide via phosphoramidite chemistry has been developed. Use of a monomethoxytrityl protecting group on the benzotriazole allowed fast on column detritylation purification by reverse-phase HPLC. Surface enhanced Raman scattering (SERS) of the modified oligonucleotides was obtained from silver colloid.

Full text of DOI:10.1016/S0040-4039(01)00108-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 2197–2200
Synthesis of a benzotriazole phosphoramidite for attachment of
oligonucleotides to metal surfaces
Rachel Brown, W. Ewen Smith and Duncan Graham*
Department of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow G1 1XL, UK
Received 7 April 2000; revised 10 January 2001; accepted 17 January 2001
Abstract—A method for the addition of a benzotriazole moiety to the 5%-terminus of an oligonucleotide via phosphoramidite
chemistry has been developed. Use of a monomethoxytrityl protecting group on the benzotriazole allowed fast on-column
detritylation purification by reverse-phase HPLC. Surface enhanced Raman scattering (SERS) of the modified oligonucleotides
was obtained from silver colloid. © 2001 Elsevier Science Ltd. All rights reserved.
Oligonucleotides containing a metal complexing group
are of interest for the attachment of oligonucleotides to
metal surfaces. Traditionally oligonucleotides have been
attached to gold surfaces by a thiol linker via the
5%-terminus of the oligonucleotides.1,2 Recently Mirkin
and co-workers have reported extensive use of gold
colloid coated in oligonucleotides for DNA sequence
analysis and also fabrication of nano-structures.3–6 In
this letter we report an alternative linker that allows
attachment of oligonucleotides to other metal surfaces
such as silver and copper. This allows oligonucleotide
modified metal surfaces to be constructed, but is also of
particular interest for surface enhanced Raman scatter-
ing (SERS). For SERS to occur the target molecule
must be adsorbed onto a suitable metal surface.7,8 The
metal surface used in our spectroscopic studies is silver
colloid and as such requires a suitable complexing
agent to form an irreversible complex between the
complexing agent and the metal. A suitable complexing
agent for this purpose is benzotriazole.9 Benzotriazole
is known to form a polymeric layer with silver and
copper metals and is commonly used as an anti-corro-
sion agent to prevent tarnishing.10,11 As part of our
research we have developed a convenient route for the
synthesis of a benzotriazole phosphoramidite and used
the monomer to prepare 5%-benzotriazole modified
oligonucleotides. This allowed oligonucleotides to com-
plex to metal surfaces via the 5%-terminus unlike
unmodified oligonucleotides which do not complex and
hence do not produce SERS.
Previously we have added a benzotriazole moiety to an
amino linker at the 5%-end of an oligonucleotide but this
takes time, requires a manual-coupling step and is not
as high yielding. In our preferred approach we synthe-
sised a benzotriazole phosphoramidite that contained
an alkyl spacer between the benzotriazole and the phos-
phorus. The spacer ensured that the action of the
benzotriazole was not affected by the presence of the
larger oligonucleotide.
The starting material for the synthesis of the phospho-
ramidite was benzotriazole-5-carboxylic acid, which is
commercially available (Fig. 1). In a separate reaction
6-aminohexanol was protected with a tert-butyldi-
phenylsilyl group to yield 6-tert-diphenyl-silanyloxy-
hexylamine (1). The amine was then reacted with the
carboxylic acid to form an amide linkage (2) using
carbonyldiimidazole as the activating agent. Car-
bonyldiimidazole was chosen as it has been used before
with benzotriazole carboxylic acid and allows amide
formation at the acid function without the need for
protection of the triazole ring system.12 However, the
benzotriazole ring proton still required protection prior
to the formation of the phosphoramidite. Thus, the
monomethoxytrityl group was chosen for this purpose
as it is compatible with solid-phase synthesis of
oligonucleotides and allows trityl on purification. The
benzotriazole linker was protected using monomethoxy-
trityl chloride in pyridine with a catalytic amount of
dimethylaminopyridine. This yielded the fully protected
compound N - [4 - methoxytrityl) - benzotriazoyl - 5 - car-
boxylic acid - (6 - tertbutyl - diphenyl - silanyloxy - hexyl)-
amide (3) in 54% yield. A benzoyl protecting group was
also tested but subsequent phosphitylation resulted in
a poor yield hence the use of the more favoured
Keywords: benzotriazole; HPLC; nucleic acids; phosphoramidites;
solid-phase synthesis; SERS.
* Corresponding author. Tel.: (+44) 141 548 4701; fax: (+44) 141 552
0876; e-mail: duncan.graham@strath.ac.uk
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00108-3

2198
R. Brown et al. / Tetrahedron Letters 42 (2001) 2197–2200
Figure 1. Synthesis of the benzotriazole phosphoramidite.
and diisopropylethylamine to yield the product (5) in
86% after 2 hours. The purity of the phosphoramidite
was confirmed by 31P NMR (148.78 ppm) prior to
dissolution in anhydrous THF and used in routine
solid-phase oligonucleotide synthesis.
monomethoxytrityl group. N-Acylated benzotriazole
can act as an acylating agent itself as the benzotriazole
is comparable to a weak halide in leaving group nature
in this case.
Removal of the silyl-protecting group was accom-
plished by tetrabutylammonium fluoride in THF fol-
lowed by treatment with DOWEX H+ in 92% yield to
produce the primary alcohol for phosphitylation (4). A
standard phosphitylation was performed in THF using
2 - cyanoethyl - N,N - diisopropylchlorophosphoramidite
Two 12 mer sequences and one 30 mer were synthesised
using fast deprotection monomers13 and the benzotria-
zole monomer added at the 5%-terminus via an extended
coupling cycle using a double delivery and 15 minute
coupling time. Deprotection by ammonia at room tem-

R. Brown et al. / Tetrahedron Letters 42 (2001) 2197–2200
2199
Figure 2. Analytical HPLC trace of on-column detritylation and purification for the two sequences at 260 nm. 12 mer 5%-BT TCT
ATA TTC ATC and 30 mer 5%-BT GTA TCT ATA TTC ATC ATA GGA AAC ACC ATT.
Figure 3. SERS spectra of benzotriazole modified oligonucleotide (A) and the control spectra of colloid with an unmodified
oligonucleotide (B).
perature for 2 hours was followed by purification by
reverse phase HPLC using a Poros oligo R3™ column
and on column detritylation14 (Fig. 2). The coupling
efficiency was estimated as being between 80 and 90%
by HPLC integration. The identity of both oligonucleo-
tides was confirmed by electrospray MS.15
previously produced excellent SERS from benzotriazole
azo dyes.9 In this case the oligonucleotides were pre-
mixed with spermine (to neutralise the phosphate
backbone16) and added to the silver colloid then left for
15 minutes to allow surface complexation. The colloidal
suspension was concentrated by centrifugation and the
supernatant removed before examination of the residue
by SERS.17 SERS signals were obtained from the ben-
zotriazole oligonucleotides at a level of 5×10−8 mol,
however, nothing could be seen from the control sam-
ple of colloid with DNA and spermine (Fig. 3). The
bands arising at 1373 and 1616 cm−1 were assigned to
the triazole ring and the phenyl ring systems of the
benzotriazole, respectively, with the band at 1270 cm−1
The benzotriazole oligonucleotides were then investi-
gated for their ability to complex to a silver surface by
examination of their SERS activity. SERS can only be
seen for molecules on the surface of the silver colloid
hence the oligonucleotide will only produce SERS if
attached to the metal surface. The metal surface used
was that of citrate reduced silver colloid, which has

2200
R. Brown et al. / Tetrahedron Letters 42 (2001) 2197–2200
arising from the phenyl-triazole ring system. The signals
were similar to those obtained for the benzotriazole
carboxylic acid starting material as expected.
4. Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mirkin, C.
A.; Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 1959–
1964.
5. Mitchell, G. P.; Mirkin, C. A.; Letsinger, C. A. J. Am.
Chem. Soc. 1999, 121, 8122–8123.
In conclusion, we have developed a simple route for the
synthesis of a benzotriazole phosphoramidite and used
it to incorporate benzotriazole into oligonucleotides via
routine solid-phase synthesis. This has produced
oligonucleotides with specific metal complexing proper-
ties. The benzotriazole modified oligonucleotides were
shown to produce SERS from silver colloid thus
providing evidence of surface attachment. This method
of oligonucleotide modification will be of use in
anchoring DNA to a number of metal surfaces for use
in both structural and analytical studies.
6. Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science
2000, 289, 1757–1760.
7. Fleischmann, M.; Hendra, P. J.; McQuillan, A. J. J.
Chem. Phys. Lett. 1974, 26, 163–166.
8. Hildebrandt, P.; Stockburger, M. J. Phys. Chem. 1984,
88, 5935–5944.
9. Graham, D.; McLaughlin, C.; McAnally, G.; Jones, J.
C.; White, P. C.; Smith, W. E. Chem. Commun. 1998, 11,
1187–1188.
10. Altura, D.; Nobe, K. Corrosion 1972, 28, 345.
11. Ling, Y.; Guan, Y.; Han, K. N. Corrosion 1995, 51,
367–375.
Acknowledgements
12. Hirokawa, Y.; Yamazaki, H.; Yoshida, N.; Kato, S.
Bioorg. Med. Chem. Lett. 1998, 8, 1973–1978.
13. Sinha, N. D.; Davis, P.; Usman, N.; Perez, J.; Hodge, R.;
Kremsky, J.; Casale, R. Biochimie 1993, 75, 13–23.
14. Buffer A—50 mM NH4OAc pH 10; Buffer B—50 mM
NH4OAc pH 10+5% MeCN; Buffer C—0.2% TFA;
Buffer D—65% MeOH 35% H2O. Method run on Biocad
Sprint HPLC load, 3 min A, 3 min B, 3 min A, 4 min C,
3 min A, 4 min D at 5 ml/min.
The authors wish to thank the BBSRC for the award of
a David Phillips fellowship to D.G. and Astra Zeneca
for funding R.B.
References
15. Electrospray MS found 3971.0, calculated 3970.8.
16. Graham, D.; Smith, W. E.; Linacre, A. M. T.; Munro, C.
H.; Watson, N. D.; White, P. C. Anal. Chem. 1997, 69,
4703–4707.
17. SERS signals were accumulated using a Renishaw 2000
Raman Microprobe instrument with 514.5 nm excitation.
Spectra were accumulated three times for 30 seconds.
1. Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119,
8916–8920.
2. Steel, A. B.; Levicky, R. L.; Herne, T. M.; Tarlov, M. J.
Biophys. J. 2000, 79, 975–981.
3. Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J.
J. Nature 1996, 382, 607–609.
.