Synthesis of a modified thymidine monomer for site-specific incorporation of reporter groups into oligonucleotides

Article abstract of DOI:10.1016/S0040-4039(01)00224-6

The efficient incorporation of reporter groups into oligonucleotides at specific sites has been facilitated by the synthesis of a novel modified thymidine monomer with an FMOC-protected hydroxyl group on a linker. The primary hydroxyl group can be deprotected during or after solid-phase oligonucleotide synthesis and reacted with any reporter phosphoramidite.

Full text of DOI:10.1016/S0040-4039(01)00224-6

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 2587–2591
Synthesis of a modified thymidine monomer for site-specific
incorporation of reporter groups into oligonucleotides
Lynda J. Brown,b Jonathan P. Maya and Tom Browna,*
aDepartment of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, UK
bOswel Research Products, Biological and Medical Sciences Building, University of Southampton, Bassett Crescent East,
Southampton SO16 7PX, UK
Received 1 February 2001; accepted 7 February 2001
Abstract—The efficient incorporation of reporter groups into oligonucleotides at specific sites has been facilitated by the synthesis
of a novel modified thymidine monomer with an FMOC-protected hydroxyl group on a linker. The primary hydroxyl group can
be deprotected during or after solid-phase oligonucleotide synthesis and reacted with any reporter phosphoramidite. © 2001
Elsevier Science Ltd. All rights reserved.
Oligonucleotides possessing fluorescent labels attached
to the heterocyclic bases are of increasing importance in
molecular diagnostics.1 There are currently two main
methods for introducing such labels. The first involves
the reaction of functional groups attached to the bases
(e.g. alkylamino groups) with activated dye molecules
(e.g. N-hydroxysuccinimide esters) after oligonucleotide
synthesis.2 However, this post-synthetic method does
not readily allow the incorporation of different dyes at
separate sites, additional purification steps are required
and the yields are often low. The second is to construct
a labelled nucleoside unit that can be incorporated as a
phosphoramidite during automated DNA synthesis.
This method is site-specific but is constrained by the
availability of expensive monomers, each of which has
to be individually prepared.3
The monomer 8 was prepared as follows (Fig. 1):
6-aminohexan-1-ol was protected using BOC anhy-
dride. The alcohol 1 was reacted with FMOC chloride
in pyridine and following prompt acidic deprotection
the amine 3 was obtained in a yield of 65% for steps (i)
to (iii). 5-Iodo-2%-deoxyuridine was protected as its 4,4-
dimethoxytrityl ether to give 4 in good yield (87%). The
nucleoside was then reacted with propargylamine under
Sonagashira conditions, without need for prior protec-
tion of the amine.5 Treatment of the product 5 with
succinic anhydride, and subsequent coupling of amine 3
gave the nucleoside 7. Finally phosphitylation of the 3%
hydroxyl group in an argon atmosphere using 2-cyano-
ethyl-N,N-diisopropyl chlorophosphine afforded the
phosphoramidite 8 in 70% yield.
Monomer 8 was incorporated into a 13-mer oligonucleo-
tide (TCTCAC8ACTATC, Fig. 2) which was assem-
bled using standard phosphoramidite chemistry6 on an
ABI 394 DNA synthesiser (coupling time of 6 minutes
for monomer 8, Fig. 2). Coupling efficiencies of >98%
were observed throughout. The column with the
attached, fully protected oligonucleotide was removed
from the synthesiser and a solution of piperidine in
DMF (20%) was passed through the column by syringe.
A reaction time of 5 minutes was sufficient to remove
the FMOC group; significantly longer reaction times
causing partial cleavage of the oligonucleotide from the
resin (a reaction time of 5 minutes gave a 96% yield of
oligonucleotide when compared to a standard synthe-
sis). The column was washed thoroughly with anhy-
drous DMF and acetonitrile before being returned to
the DNA synthesiser. The support-bound oligonucleo-
In this report we describe the synthesis of a new
thymidine-based monomer that can be used to intro-
duce any phosphoramidite monomer into an oligomer
during solid-phase synthesis. The molecule has a side-
chain on the five-position of thymine which consists of
a triple bond (essential for efficient PCR4), an addi-
tional spacer and a terminal FMOC-protected hydroxyl
group. Once the monomer is incorporated into an
oligonucleotide during routine automated synthesis, the
FMOC protection can be removed and the hydroxyl
group coupled with any chosen phosphoramidite
(monomer). FMOC deprotection can be performed
immediately after incorporation of the monomer, or at
the end of oligonucleotide synthesis.
* Corresponding author. E-mail: tb2@soton.ac.uk
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00224-6

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L. J. Brown et al. / Tetrahedron Letters 42 (2001) 2587–2591
OH
OH
OFMOC
OFMOC
NH2
(i)
(ii)
(iii)
H2N
BOCHN
BOCHN
H2N
O
5
5
5
5
3
1
2
O
N
O
N
I
I
HN
HN
HN
(iv)
(v)
DMTO
O
HO
O
DMTO
O
N
O
O
O
OH
OH
OH
4
5
(vi)
O
O
H
N
OH
O
O
O
OFMOC
N
H
NH
5
O
HN
HN
DMTO
O
N
DMTO
O
N
O
O
(vii)
OH
OH
H2N
OFMOC
4
7
6
3
(viii)
O
H
N
O
OFMOC
N
H
5
O
HN
DMTO
O
N
O
O
P
CN
N
O
8
Figure 1. Reagents and conditions: i, BOC anhydride (1 equiv.), DMF, rt, 97%, ii, FMOC chloride (1.1 equiv.), pyridine, rt; iii,
trifluoroacetic acid, CH2Cl2 (2:1), rt, 67% (two steps); iv, 4,4%-dimethoxytrityl chloride (1.1 equiv.), pyridine, rt, 87%; v,
propargylamine (2 equiv.), Cu(I) iodide (0.25 equiv.), Pd(Ph3)4 (0.1 equiv.), triethylamine (2 equiv.), DMF, rt, dark, 87%; vi,
succinic anhydride (1.05 equiv.), pyridine; vii, EDC (1.2 equiv.), pyridine, rt, 57% (two steps); viii, 2-cyanoethyl-N,N-diisopropyl
chlorophosphine (1.1 equiv.), DIPEA (1.5 equiv.), CH2Cl2, rt, 70%.
tide was treated with tetrachloro-fluorescein phospho-
ramidite (TET, PE Biosystems) and tetrazole using a
standard synthesis cycle, and then cleaved from the
support and deprotected using standard conditions
(conc. aq. ammonia, 5 h, 55°C, Fig. 2). Reversed-phase
HPLC analysis showed the presence of one main peak,
which was shown to be the correct labelled product by
electrospray mass spectrometry.
treatment prior to addition of the dye monomer (R=ii,
Fig. 2). In a third method, after ‘trityl-off’ DNA syn-
thesis, the detritylated 5%-end can be capped with acetic
anhydride using a special synthesis cycle before removal
of the FMOC group and addition of the dye (R=iii,
Fig. 2).
An important application of this monomer is the syn-
thesis of Cyanine Dye™ (CyDye™) labelled oligonucleo-
tides. The commercially available non-nucleosidic Cy5
phosphoramidite (Amersham-Pharmacia Biotech, Fig.
3a) can be incorporated within the oligonucleotide
backbone (Fig. 3b) or at the 5% end.
In order to add the dye phosphoramidite to the
hydroxyl of monomer 8 selectively, the 5%-hydroxyl
group of the oligonucleotide requires protection. In the
previous experiment this 5%-OH was conveniently
masked by addition of a trifluoroacetylaminohexyl
phosphoramidite (R=i, Fig. 2). Alternatively, the
oligonucleotide can be synthesised ‘trityl-on’ and for
the addition of the dye phosphoramidite only, a
modified cycle can be used that does not involve acid
Ultraviolet thermal melting studies using the oligonu-
cleotides O1–O3 and their complementary strand O4
(Fig. 4) showed that an oligonucleotide containing the
Cy5 monomer internally caused significant destabilisa-

L. J. Brown et al. / Tetrahedron Letters 42 (2001) 2587–2591
2589
O
H
N
O
N
H
OFMOC
NC
5
O
HN
O
TcTcac*
RO
RO
RO
O P O
O
8
O
N
Automated
DNA synthesis
O
O
CN
cTaTca
O
P O
O
Piperidine: DMF (1:5)
O
H
N
O
N
H
OH
NC
5
O
HN
O
TcTcac
O P O
O
O
O
N
O
CN
cTaTca
O
P O
O
Dye phosphoramidite
O
CN
H
N
O
O
N
H
O P O DYE
NC
5
O
HN
O
O
TcTcac
O P O
O
O
O
N
O
CN
cTaTca
O
P O
O
R =
i, PO3(CH2)6NHCOCF3
or
i,TCA / CH2Cl2
ii, DMT
or
ii, Conc. aq. ammonia
iii, COCH3
TCTCAC8(DYE)ACTATC
Figure 2. * Bases in lower case are protected, and upper case are unprotected.
N
N
N
N
O
O
O
R1 O P O
OH
O P O R2
OH
MMTO
O
P
N
CN
3a
3b
Figure 3.

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L. J. Brown et al. / Tetrahedron Letters 42 (2001) 2587–2591
Figure 4.
Figure 5.
tion of the duplex (−17.4 K) in comparison to the
normal A.T base pair. However, when the Cy5 phos-
phoramidite was attached to the modified thymidine
monomer 8 the destabilising effect was very small (−2.5
K). This has important implications in the design of
probes containing these dyes.
side-chain of monomer 8 that possess acid-labile pro-
tecting groups (e.g. MMT on Cy5) require detritylation
and capping before synthesis of the oligonucleotide can
continue. This is easily achieved on an automated DNA
synthesiser.
In conclusion, the modified thymidine monomer 8 per-
mits the facile incorporation of phosphoramidite at a
specific location along an oligonucleotide chain. The
ability to synthesise oligonucleotides of this nature is
particularly important in the field of DNA diagnostics.8
Fluorescence resonance energy transfer (FRET) is an
important phenomenon used extensively to allow dyes
of various emission wavelengths to be excited with a
single light source on fluorescence-based genetic analy-
sers.7 We synthesised three oligonucleotides; two
labelled internally, one with fluorescein (FAM, PE
Biosystems), one with Cy5 (both on monomer 8) and a
complementary strand (Fig. 5). Cy5 has an excitation
wavelength of 650 nm and emits at 665 nm, therefore
using a light source of 490 nm the Cy5 labelled oligonu-
cleotide was not excited (Fig. 5, i). The FAM labelled
oligonucleotide was excited and emits at 520 nm, thus
no emission was observed at the wavelength associated
with Cy5 emission (Fig. 5, ii). However, when the two
labelled oligonucleotides hybridise adjacent to each
other on a complement, the FAM is excited at 490 nm
and can transfer its energy to the Cy5 causing it to emit
at 665 nm (Fig. 5, iii). In this example, the ability to
label oligonucleotides internally is crucial as, if the dyes
are placed at the end of the oligonucleotides, they are
too close and fluorescence quenching occurs.
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