Oligonucleotides with strongly fluorescent groups π-conjugated to a nucleobase: Syntheses, melting temperatures, and conformation

Article abstract of DOI:10.1016/S0960-894X(03)00555-9

Phosphoramidite 1 was prepared, incorporated into oligonucleotides, and these were studied via thermal denaturation and circular dichroism.

Full text of DOI:10.1016/S0960-894X(03)00555-9

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2785–2788
Oligonucleotides with Strongly Fluorescent Groups ꢀ-Conjugated
to a Nucleobase: Syntheses, Melting Temperatures, and
Conformation
Guan-Sheng Jiao and Kevin Burgess*
Department of Chemistry, PO Box 30012, Texas A & M University, College Station, TX 77842-3012, USA
Received 27 November 2002; accepted 25 April 2003
Abstract—Phosphoramidite 1 was prepared, incorporated into oligonucleotides, and these were studied via thermal denaturation
and circular dichroism.
# 2003 Elsevier Ltd. All rights reserved.
Fluorescently labelled oligonucleotides are central to
high throughput sequencing of genomic materials.1,2
Connection of dyes to DNAprimers almost invariably
features flexible, non-p-conjugated linkers. Many
groups have prepared nucleobases with rigid, p-con-
jugated linkers that attach them to fluorescent enti-
ties,3ꢀ5 metal complexes,6ꢀ11 EPR spin labels,12 and
electrochemically active tags.13 However, nucleobases
rigidly linked to the types of strongly fluorescent dyes
that are useful in DNAsequencing have not been
reported to date. This is surprising because rigid,
p-conjugated linkers could potentially be valuable in
that area. For instance, whereas attachment of fluor-
escent dyes to primers via flexible, saturated linkers does
not exclude the possibility that complications could
arise in the sequencing process as a result of intercala-
tion of the dyes into ds-DNA; this is less likely if the
linkers are rigid and p-conjugated (Fig. 1).
Phosphoramidite 1 was conceived as a first step to
exploring the characteristics of rigid, p-conjugated lin-
kers in dye-labelled primers. This Letter describes pre-
paration of this phosphoramidite, its incorporation into
oligonucleotides via solid-phase syntheses, melting tem-
peratures of the modified ds-DNAoligomers containing
this special nucleobase, and circular dichroism spectra
to probe conformational effects. The 6-linked fluor-
escein isomer of phosphoramidite 1 was also examined
in the same way.
Oligonucleotides p-conjugated to strongly fluorescent
dyes also have potential applications outside of DNA
sequencing. For instance, if energy transfer from the
nucleobase to the fluorescent dye is fast relative to non-
radiative processes, then emission from the fluorescent
group provides a means to monitor excitation of that
base, whether it occurs directly or via transmission
through the DNAstrand.
Scheme 1 outlines a synthesis of the phosphoramidite
1.14 Regioisomerically pure 5-iodofluorescein was
required for this synthesis. To obtain it, commercially
available 5-aminofluorescein was diazotized and treated
with potassium iodide. The product was then coupled
*Corresponding author. Tel.: +1-979-845-4345; fax: +1-979-845-
8839; e-mail: burgess@tamu.edu
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00555-9

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G.-S. Jiao, K. Burgess / Bioorg. Med. Chem. Lett. 13 (2003) 2785–2788
Figure 1. (a) Flexible linkers used in conventional attachment meth-
ods permit intercalation and exclude through-bond energy transfer,
whereas (b) intercalation is disfavored and through-bond energy
transfer is possible if rigid linkers are used.
Figure 2. Modified oligonucleotides and corresponding ds-DNA.
to assess purity, phosphodiesterase hydrolysis followed
by HPLC to assess nucleobase composition, and elec-
trospray mass spectrometry for confirmation of their
molecular masses.18
Stability changes caused by incorporating the Z residue
into ds-oligonucleotides were assayed by monitoring
thermal denaturation via UV. Representative data for
derivatives of oligonucleotide 4 and some control
sequences are shown in Figure 3. This shows there are
relatively small differences in stabilities, and that the
modified oligo 9 is less stable than the natural ds-oligo
10 but is more stable than the A-A mismatched oligo
11 (Tm=59.0, 63.0, 56.1 ꢁC, respectively). When the Z
residue was instead placed at the 50-terminus in 12,
then the melting temperature was almost the same as
when it was centrally situated (60.4 vs 59.0 ꢁC). The
shorter oligos 6–8 have similar but more profound
stability trends, as do oligos 9–11 (Tm=47.0, 51.1,
38.5 ꢁC, respectively).
Scheme. 1. Preparation of the phosphoramidite 1.
with 5-ethynyl thymidine 2115 via Sonogashira’s proce-
dure.16 Conversion of the protected-fluorescein-labelled
thymidine derivative into the corresponding phosphor-
amidite was performed using standard methods.15,17
In the solid-phase syntheses of the oligonucleotides 3–5,
controlled pore glass was used as the support in the
conventional phosphoramidite method.17 O-Acylation
of the fluorescein label protects it from iodination, and
this masking is conveniently removed in the course of
the standard resin cleavage procedure (treatment with
aqueous ammonia). The product oligonucleotides con-
tain the modified nucleobase derived from 1, here given
the symbol ‘Z’ (Fig. 2). These were analyzed by HPLC
Aparallel study was carried out to determine if the
position of the fluorescein linker is influential (two isomers
of substituted fluoresceins are common, the 5-isomer fea-
tured above, and the 6-isomer). 6-Aminofluorescein
thus was converted into regioisomerically pure 6-iodo-
fluorescein using the methods developed for the

G.-S. Jiao, K. Burgess / Bioorg. Med. Chem. Lett. 13 (2003) 2785–2788
2787
Figure 3. Thermal denaturation curves for the natural and modified
ds-DNAstrands 9–11.
Figure 4. CD spectra of oligonucleotide duplexes 9, 10, and 12.
5-isomer. The sequence shown in Scheme 1 was repe-
ated, but using 6-iodofluorescein to give an isomeric
phosphoramidite 10, then oligonucleotide 40 containing
the fragment Z0. In the series of ds-DNA 90, 10, and 11,
the Tm values obtained were 58.0, 63.0, and 56.1ꢁC,
respectively. Thus with identical sequences but containing
Z and Z0 (i.e., 9 and 90) the Tm values measured were very
similar (59.0 and 58.0 ꢁC), and the order of stabilities
(natural >9 or 90 >A–A mismatch) is the same.
Oligos 9, 10, and 12 give similar spectra, and all resemble
19
that expected for the B-DNAconformation.
In summary, T-derivatives with protected fluorescein
residues directly p-conjugated via rigid linkers are
accessible via a relatively direct synthesis. 30-Phosphor-
amidites of these can be formed, and di-O-acetate pro-
tection of the fluorescein moiety renders it compatible
with solid phase methods to produce modified DNA
oligomers (containing Z and Z0). Thermal denaturation
studies prove that the modified nucleobases do not
decrease the stability of the ds-DNAsignificantly, less,
in fact, than the corresponding A–A mismatch. The CD
studies indicate that incorporation of the Z fragment
does not perturb the ds-DNAB-form conformation.
Ds-DNAfrom the modified oligonucleotides with the
fluorescein p-conjugated in the 5- or 6-position have
similar stabilities. These studies lay the foundations for
experiments to probe the applications of rigid dye-
labelled primers in DNAsequencing, and in depth
studies of fluorescence energy transfer along DNA
strands.20
Acknowledgements
Financial support for this work was provided by The
National Institutes of Health (HG 01745) and by The
Robert A. Welch Foundation. The TAMU/LBMS-
Applications Laboratory, and useful discussions with
Lars H. Thoresen and Dr. Shane Tichy are acknowl-
edged.
References and Notes
Circular dichroism (CD) studies were performed to
probe if incorporation of fragment Z disrupts the DNA
conformation. Figure 4 shows the CD spectra obtained.
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