Internal labeling of oligonucleotide probes by Diels-Alder cycloaddition

Article abstract of DOI:10.1016/S0040-4039(02)00930-9

A new method of adding fluorescent labels to the middle of oligonucleotides is reported. Diels-Alder cycloaddition was used to add five fluorescent maleimides to an oligonucleotide containing a 2′-deoxyuridine modified at the 5-position with a spaced fura

Full text of DOI:10.1016/S0040-4039(02)00930-9

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 4785–4788
Internal labeling of oligonucleotide probes by Diels–Alder
cycloaddition
Duncan Graham,* Antonio Grondin, Callum McHugh, Ljiljana Fruk and W. Ewen Smith
Department of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow G1 1XL, UK
Received 4 March 2002; revised 30 April 2002; accepted 13 May 2002
Abstract—A new method of adding fluorescent labels to the middle of oligonucleotides is reported. Diels–Alder cycloaddition was
used to add five fluorescent maleimides to an oligonucleotide containing a 2%-deoxyuridine modified at the 5-position with a spaced
furan. This is a new approach to internal oligonucleotide chemistry that opens up a large range of possibilities for further
conjugation. © 2002 Elsevier Science Ltd. All rights reserved.
Addition of external labels to oligonucleotides is neces-
sary to satisfy the need of current DNA assays and
allow the development of new approaches. The most
common current approach for DNA analysis is the use
of fluorescence spectroscopy.1–3 Most fluorophores are
added to the 5% or 3% terminus by a number of different
methods. Internal labeling is more complex to achieve
and a number of approaches have been employed.
These include modification of the backbone, sugar or
base. Addition of fluorophores to the base are most
popular. Current approaches include the use of func-
tional groups such as alkyl amines that react with
activated dyes after oligonucleotide synthesis4 or the
use of a pre-labeled nucleoside phosphoramidite.3 The
post synthesis approach suffers from low yields and
side reactions and the use of dye nucleoside phospho-
ramidites requires complex chemistry and expensive
monomers. Brown et al.5 recently reported the use of an
Fmoc protected alkyl alcohol modified thymidine phos-
phoramidite that could be used to add commercially
available dye phosphoramidites in high yield to the
middle of an oligonucleotide. The aim of the research
reported here was to produce a nucleoside phospho-
ramidite that could be used to add a label to the middle
of an oligonucleotide after solid phase synthesis in a
highly efficient manner.
cycloadditions as a method of adding fluorophores to
the 5%-terminus of oligonucleotides. This demonstrated
the advantages of using Diels–Alder reactions for con-
jugations to oligonucleotides, namely, a fast, quantita-
tive reaction occurring in an aqueous buffer system.
Additionally, as Diels–Alder reactions occur between a
diene and a dienophile there is no need for use of
selective protecting groups to prevent reaction with
common nucleophiles and hence do not suffer from any
side reactions. In this approach one phosphoramidite
can be used to add any number of different dyes.
In this study we report the addition of a diene to the 5
position of 2%-deoxyuridine. The diene was chosen to be
added to the nucleoside, as a number of commercially
available dienophiles in the form of fluorescent
maleimides can be used without further chemical mod-
ification. A range of dienes was considered for addition
to a nucleoside and their reactions with a maleimide
examined.7 Furan was chosen due to the number of
readily available derivatives, the speed and efficiency of
the reaction with maleimides and stability to the condi-
tions used for oligonucleotide synthesis.
The starting material for the synthesis was 5-iodo-2%-
deoxyuridine which was initially protected on the 5%-
hydroxyl with dimethoxytrityl 1. A palladium catalyzed
Sonogashira coupling was used to add an acetylenic
furan derivative 2 prepared by the condensation of
pentynoic acid and furfuryl amine. This protected furan
2%-deoxyuridine 3 was then phosphitylated using stan-
dard procedures to give the monomer 4 ready for solid
phase oligonucleotide synthesis (Scheme 1).
We report the use of Diels–Alder cycloaddition for the
addition of fluorescent dyes to
a modified 2%-
deoxyuridine residue after oligonucleotide synthesis.
Hill et al.6 recently reported the use of Diels–Alder
* Corresponding author. E-mail: duncan.graham@strath.ac.uk
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)00930-9

4786
D. Graham et al. / Tetrahedron Letters 43 (2002) 4785–4788
Scheme 1. (i) Pyridine, 4,4%-dimethoxytrityl chloride (1.2 equiv.), DMAP (0.1 equiv.), 80%; (ii) DMF, carbonyl diimidazole (1.1
equiv.), 40°C, 95%; (iii) DMF, triethylamine (5 equiv.), CuI (0.2 equiv.), 2 (1.5 equiv.), Pd (PPh3)4 (0.1 equiv.), 79%; (iv) THF,
2-cyanoethyl-N,N-diisopropylchlorophosphine (1.1 equiv.), DIPEA (3 equiv.), 98%.
The monomer was then used as a 50 mg/ml solution
(THF/MeCN 1:1) on an Expedite 8909 oligonucleotide
synthesizer with a 15-minute coupling cycle. The HPLC
trace indicated excellent incorporation of the modified
monomer (Fig. 1). A 20mer that has been used as an
antisense oligonucleotide to inhibit MDR1 gene expres-
Figure 1. HPLC trace obtained for the crude modified 20mer after deprotection using a Chromolith reverse phase column running
at 4 ml/min and a buffer system of A=0.1 M ammonium acetate pH 7.0, B=MeCN. (CV=column volume 1.662 ml) 5 CV 5%
B, 25 CV 5–30% B, 5 CV 30% B, 5 CV 30–100% B, 3 CV 100% B.

D. Graham et al. / Tetrahedron Letters 43 (2002) 4785–4788
4787
sion was synthesized.8 The position marked with X
represents the furan modified uridine.
The labeled oligonucleotides were then examined for
fluorescence. The emission wavelengths observed for
the oligonucleotides differed slightly from those quoted.
This is presumed to be due to the use of an aqueous
buffer as opposed to methanol apart from the value
quoted for FAM,9 which is in aqueous buffer and is
identical (Table 1). Interestingly we found that the two
5%CCT CGC GCX CCA GCC CTA CC
Diels–Alder cycloadditions were then attempted using
this oligonucleotide and five fluorophore maleimides.
The following dye maleimides were purchased from
Molecular Probes and used without further manipula-
tion, FAM, Texas red, TAMRA-5, TAMRA-6 and
Alexa Fluor 532 (Fig. 2). The two isomers of TAMRA
i.e. 5 and 6 were examined to see if the different isomers
affected the cycloaddition. The oligonucleotide was dis-
solved in a phosphate buffer (25 mM, pH 5.5) and the
fluorophore maleimides added in an acetonitrile buffer
mixture that gave a final acetonitrile concentration of
30% v/v. The cycloadditions were left in the dark at
40°C for 3 h prior to analysis by reverse phase HPLC.
The TAMRA dyes were not heated due to the thermal
instability of the dye but were left to react for 4 h
instead of 3 h. In each case 3 equiv. of dye maleimide
were used to ensure complete reaction. The HPLC
traces of the cycloadditions showed clear differences
between the furan oligonucleotide peak and the
cycloadduct peak indicating that the cycloadduct had
formed in excellent yield. Fig. 3 shows the HPLC trace
of the oligonucleotides produced by cycloaddition of
three of the maleimides. All of the cycloadducts dis-
played a reduced retention time due to the increased
polarity of the conjugated oligonucleotides. Only three
are shown in Fig. 3 as the Alexa Fluor 532 and FAM
conjugates had the same retention time as did the
TAMRA 6 isomer and Texas Red conjugates.
Figure 3. HPLC of the cycloadducts using a Chromolith
reverse phase column at 4 ml/min and a buffer system of:
A=0.1 M ammonium acetate pH 7.0, B=MeCN. (CV=
column volume 1.662 ml) 0–5 CV 5% B, 5–15 CV 5–10% B,
15–35 CV 10–25% B, 35–45 CV 25–100% B, 45–48 CV 100%
B. Peak 1=TAMRA 5, Peak 2=Texas Red, Peak 3=FAM,
Peak 4=furan oligo. (The peaks have been scaled for clarity.)
Figure 2. The fluorophore maleimides used in the cycloadditions.

4788
D. Graham et al. / Tetrahedron Letters 43 (2002) 4785–4788
Table 1. Comparison of the emission wavelengths of the
labeled oligonucleotides with the literature values
Acknowledgements
Fluorophore
Emission (aq.) (nm)
Lit. value9 (nm)
The authors would like to thank the BBSRC for the
award of a David Phillips fellowship to D.G. and for
funding the work through grant number E11015.
FAM
515
571
558
533
606
515 (aq.)
TAMRA-5
TAMRA-6
Alexa Fluor 532
Texas Red
567 (MeOH)
567 (MeOH)
552 (MeOH)
600 (MeOH)
References
1. Wojczewski, C.; Stolze, K.; Engels, J. W. Synlett 1999,
1667.
2. Christopoulos, T. K. Anal. Chem. 1999, 71, 425R.
3. Davies, M. J.; Shah, A.; Bruce, I. J. Chem. Soc. Rev. 2000,
29, 97.
4. Randolph, J. B.; Waggoner, A. S. Nucleic Acids Res. 1997,
25, 2923.
TAMRA isomers gave emission wavelengths that dif-
fered by 13 nm where the literature values are identical.
This proved that the oligonucleotides were fluorescent
and that the cycloaddition does not interfere with the
fluorescence emission.
5. Brown, L. J.; May, J. P.; Brown, T. Tetrahedron Lett.
2001, 42, 2587.
6. Hill, K. W.; Taunton-Rigby, J.; Carter, J. D.; Kropp, E.;
Vagle, K.; Pieken, W.; McGee, D. P. C.; Husar, G. M.;
Leuck, M.; Anziano, D. J.; Sebesta, D. P. J. Org. Chem.
2001, 66, 5352.
7. Grondin, A.; Robson, D. C.; Smith, W. E.; Graham, D. J.
Chem. Soc., Perkin Trans. 2 2001, 2136.
8. Astriab-Fisher, A.; Sergueev, D. S.; Fisher, M.; Shaw, B.
R.; Juliano, R. L. Biochem. Pharmacol. 2000, 60, 83.
9. http://www.probes.com/
In conclusion, this letter demonstrates a new method of
internally labeling oligonucleotides that is fast, simple
and high yielding. Different types of fluorophore were
used to demonstrate the versatility of this approach and
demonstrate the selectivity of the chemistry. Addition-
ally the one modified phosphoramidite can be used with
any fluorescent maleimide and is therefore a generic
modification.