Chemistry Letters Vol.36, No.6 (2007)
769
120
100
80
60
40
20
0
d
c
b
a
295
345
395
445
495
Wavelength/nm
Figure 3. The relative fluorescence spectra of 1) 50-d(CplT)-30 shown
by (a); 2) 50-d(CplTTT)-30 shown by (b); 3) 50-d(CplAAT GGA GCC
AGT)-30 shown by (c) and 50-d(CplG TCA TGT CAG TTC CCC TTG
GTC CTC)-30 shown by (d) in 10 mM phosphate buffer (pH 7.0), 20%
v/v DMF and 0.2 M NaCl by exciting at 346 nm. pl ¼ TBMPS.
Figure 2. Detection limits: Gel electropherograms; lower panel ON4
(26-mer), volume in each well is 200 mL, lane 1: (2.5 mM); lane 2:
(1.5 mM); lane 3: (1.0 mM); lane 4: (0.75 mM); lane 5: (0.50 mM), of
50-d(CplG TCA TGT CAG TTC CCC TTG GTC CTC)-30 (26-mer)
and lane 6: 26-mer without TBMPS group. Sample were illuminated
was used for studies to visualize the effect of length on the
fluorescent properties when covalently attached to sequences
(Figure 1). At the dimer and tetramer level the excitation inten-
sity and the emission intensity were more or less same. But at
13-mer and 26-mer levels the excitation peaks were found to
be too diminished whereas the emission peaks were found to
be having significant intensity (Figure 3). This shows that pyrene
at 50-terminus position is sterically unhindered which gives
a better emission intensity, which makes it detectable through
fluorescence even at as much low concentration. This would
enable to identify easily the desired product in a complex
mixture, where some of the sequences may get truncated in
the synthesis cycle.
pl
using a standard laboratory UV-lamp, ꢀmax ¼ 365 nm. ¼ TBMPS.
absorbance as well as fluorescence were collected and pooled.
The fluorescence clearly indicates that the whole peak complex
represents products of the correct length. Confirmation of the
correct mass has been done by MALDI-TOF mass spectrometry.
The TBMPS group was successfully cleaved by treatment of
2–3 equiv. of tetra-n-butylammonium fluoride (TBAF) in THF
for 3 min at r.t. The purified oligonucleotides after deprotection
did not show any absorbance peak at 346 nm and any fluores-
cence emission at 390 nm.
The 13-mer and 26-mer both were purified on 8 M urea
polyacrylamide gel electrophoresis. As observed with DMTr-
protected oligomers, the TBMPS-protected oligomers are
well separated from non-fluorescent failure sequences. The
fluorescent bands were visible up to at 0.5 OD concentrations
by fluorescence on a wet gel at 350 nm and, because of the
retarding effect of the TBMPS group; it is well separated from
n ꢂ 1 and other failure sequences. The fluorescence studies of
all these pyren-labeled oligonucleotides have been carried out
(Figure 2).
In this paper, the synthesis of oligonucleotide sequences
covalently attached to a silyl fluorescent moiety as purification
handle has been carried out. The synthesis is a simple one-
step procedure and the in situ product formed i.e. di(tert-
butyl)[(pyren-1-yl)methoxy]silyl chloride (1) is subsequently
used for 50-OH protection of nucleosides. Thus, a one-pot
synthesis is possible for protection of all of the nucleosides
(2a–2c). The nucleosides protected with 50-TBPMS group tend
to crystallize easily therefore product can be purified without
expensive column chromatography. The monomer is obtained
in fairly good yield. The acid stability and the base stability
of the monomer show its usefulness that it can withstand the
basic condition while cleaving the sequence from the support.
All the fluorescence studies were carried out at 0.04 OD
concentrations. Since the oligomers were readily soluble in
DMF buffer, therefore, all the spectra were recorded in it
(10 mM phosphate buffer (pH 7.0), 20% v/v dimethylformamide
(DMF) and 0.2 M NaCl). DMF is reported to be an effective
solvent for controlling hybridization stringency as well as having
a similar effect as that of formamide and for enhancing the
fluorescence of pyren labels.13,14 The fluorescence studies
showed a peculiar phenomenon. Four different lengths of
sequences i) dimer; ii) tetramer; iii) 13-mer, and iv) 25-mer
In conclusion we found that TBPMS–Cl react selectively
with the hydroxy groups of nucleosides and preferentially with
the 50-hydroxy group. Because of the compatibility of TBPMS
group with other acid and base labile groups, almost complete
manipulation of protecting group is now possible. The TBMPS
group is stable to phosphorylation. The nucleosides protected
with 50-TBPMS group tend to crystallize easily therefore product
can be purified without expensive column chromatography.
Purification of 50-TBMPS-labeled oligonucleotides of different
length viz. 2-mer, 4-mer, 13-mer, and 26-mer is easily achieved
using this approach. Thus, the desired sequences may be purified
from the rest of (n ꢂ 1) failure sequences at 0.5 OD unit on
denaturing polyacrylamide gel aids in quantitative recovery of
the full length product (Figure 2).
References and Notes
1
2
3
M. Kwiatkowski, J. Chattopadhya, Acta Chem. Scand., Ser. B 1984, 38,
657.
4
C. J. Welch, X. Zhon, J. Chattopadhyay, Acta Chem. Scand., Ser. B 1986,
40, 817.
5
6
7
8
H. Tanimura, T. Imada, Chem. Lett. 1990, 1715.
M. Kawai, P. Neogi, P. S. Khattri, Y. Butsugan, Chem. Lett. 1990, 577.
J. L. Fourrey, J. Varenne, C. Blonski, P. Dousset, D. Shire, Tetrahedron
9
M. H. Caruthers, 8th International Conference on Polymer-based
Technology, Ma’ale Hachamisha, Israel, 1998.
12 S. L. Beaucage, in Protocols for Oligonucleotide and Analogues, ed. by
S. Agrawal, Humana Press, New Jersey, USA, 1993, Vol. 33.
Mishra, Roli
