Hydrogen-bonding controlled rigidity of an isoindoline-derived nitroxide spin label for nucleic acids

Article abstract of DOI:10.1039/c2cc36389e

Nucleosides spin-labelled with isoindoline-derived benzimidazole ( ^ImU) and benzoxazole (^OxU) moieties were synthesized and incorporated into DNA oligonucleotides. Both labels display limited mobility in duplex DNA but ^ImU

Full text of DOI:10.1039/c2cc36389e

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Hydrogen-bonding controlled rigidity of an
isoindoline-derived nitroxide spin label for
nucleic acids†‡
Cite this: Chem. Commun., 2013,
49, 999
Received 2nd September 2012,
Accepted 21st November 2012
Dnyaneshwar B. Gophane and Snorri Th. Sigurdsson*
DOI: 10.1039/c2cc36389e
www.rsc.org/chemcomm
Nucleosides spin-labelled with isoindoline-derived benzimidazole
ImU) and benzoxazole (OxU) moieties were synthesized and incor-
(
porated into DNA oligonucleotides. Both labels display limited
mobility in duplex DNA but ImU was less mobile, which was
attributed to an intramolecular hydrogen bond between the N–H
of the imidazole and O4 of the uracil nucleobase.
Long-range distance measurements using pulsed electron
paramagnetic resonance (EPR) spectroscopy, such as pulsed
electron–electron double resonance (PELDOR, also called DEER),
are now routinely used in structural studies of biopolymers.1
The distances are measured between two radicals that are
usually incorporated using site-directed spin labelling (SDSL),
in which aminoxyl radicals, usually called nitroxides, are linked
to specific sites.2 The majority of the currently available
spin-labelling techniques rely on covalent attachment of the
nitroxide using a tether that contains single bonds. Rotation
about those single bonds usually results in displacement of the
nitroxide relative to the anchoring site. This flexibility
decreases the accuracy of distance measurements and renders
EPR studies of dynamics more challenging. Therefore, consid-
erable effort has been put into the preparation of spin labels
with reduced mobility.3
Fig. 1 Spin-labelled nucleosides. (A) Rigid spin labels Ç3e and Çm.3i (B) A
nitroxide-derived nucleoside for conformationally unambiguous-spin labelling.8
(C) Isoindoline derived spin labels ImU and OxU. (D) Base pairing of ImU with A.
The intramolecular H-bond between the imidazole N–H and O4 of the
uracil nucleobase restricts the rotation around the bond connecting the nitroxide
to the base.
Optimally, the spin label probe should be immobilized on
the biopolymer. Such rigid labels have been prepared for
nucleic acids, for example Ç3e and Çm3i (Fig. 1A) for DNA and
RNA, respectively. In these cases, the nitroxide moiety has been
Godt and coworkers have coined the term ‘‘conformationally
fused to the nucleobase, which in turn is immobilized in a unambiguous spin labelling’’ for probes that have internal
nucleic acid duplex through hydrogen bonding and base-stacking. rotation about single bonds that do not change the spatial
These labels have the added advantage of enabling studies of positioning of the nitroxide relative to the labelled molecule
orientation4 and dynamics.5 Syntheses of these rigid spin labels and demonstrated their usefulness for distance measurements
are non-trivial and, therefore, there is still a need for readily in model compounds.3g Nucleoside 13g (Fig. 1B) is one example
prepared labels with limited mobility.
that illustrates the basis of the design: the N–O bond lies on the
same axis as the rotatable bonds and, therefore, rotation around
those bonds does not change the position of the nitroxide.
However, this nucleoside is not suitable for nucleic acid label-
ling through chemical synthesis, because the phthalimide
functional group is not stable under basic conditions,6 required
for oligonucleotide deprotection.
University of Iceland, Department of Chemistry, Science Institute, Dunhaga 3,
107 Reykjavik, Iceland. E-mail: snorrisi@hi.is
† In memory of Professor Har Gobind Khorana (1922–2011), acknowledging his
legacy to the scientific community.
‡ Electronic supplementary information (ESI) available: Synthetic protocols and
other experimental details. See DOI: 10.1039/c2cc36389e
c
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Scheme 1 Synthesis of diamino isoindoline 4.
In this communication we describe two nucleosides that con-
tain nitroxide spin labels with limited mobility, an isoindoline-
derived benzimidazole (ImU) and a benzoxazole (OxU) (Fig. 1C).
In addition to the ease of synthesis, in particular of ImU, they
enable base-pairing with A, whereas Ç pairs with G. The
nitroxides are linked to the nucleobase by a single bond that
nearly lies on the axis of the N–O bond and should, therefore,
be good probes for distance measurements. We show by conti-
nuous wave (CW) EPR that these labels have limited mobility in
DNA duplexes. Furthermore, we demonstrate that ImU is less
mobile than OxU, presumably due to an intramolecular hydrogen
bond between the N–H of the imidazole moiety and the O4 of the
nucleobase (Fig. 1D), which restricts rotation around the single
bond linking the label to the base.
The synthesis of ImU started with the preparation of
4,5-diamino-1,1,3,3-tetramethylisoindoline4 (4, Scheme 1),
which had previously been prepared in four steps from 2.6
We discovered that 4 could be synthesized in only two steps
by direct amination of 5-nitro-1,1,3,3-tetramethylisoindoline
(2)7 with 1,1,1 trimethylhydrazinium iodide, followed by
hydrogenation.
Reaction of diamine 4 with 30,50-di-O-acetyl-5-formyl-20-deoxy-
uridine (5)8 in the presence of K3Fe(CN)6 yielded the benz-
9
Scheme 2 (A) Synthesis of benzimidazole derivative 6. (B) Synthesis of benzoxazole
derivative 8. (C) Synthesis of nucleosides ImU and OxU and their corresponding
phosphoramidites. Yields: 6 (47%), 8 (40%), 9A (56%), 9B (50%), ImU (77%),
OxU (80%), 10A (51%), 10B (44%).
imidazole derivative 6 (Scheme 2A). Oxidation of 6 proved to be
somewhat challenging; different oxidizing agents under a vari-
ety of conditions led to decomposition of the starting material.
We argued that oxidation of the uracil 5,6-double bond could
be the cause of the side reaction(s) and might be avoided by
inclusion of a nucleophile that could add reversibly to the
6-position.10 Indeed, sodium azide facilitated mCPBA oxidation
of 6 to give 9A in a moderate yield. Deprotection, tritylation
and phosphitylation subsequently gave the ImU spin-labelled
phosphoramidite 10A. The phosporamidite of OxU was pre-
pared in a similar manner (Scheme 2B and C), except that the
isoindoline-derived amino phenol 73e was oxidatively coupled
to 5 in the presence of iodobenzene diacetate to yield 8
(Scheme 2B).11
The spin labelled nucleosides ImU and OxU were incorpo-
rated into the 14-mer DNA oligonucleotide 50-d(GAC CTC GXUA
TCG TG) by solid-phase synthesis and purified by denaturing
polyacrylamide gel electrophoresis (DPAGE). DPAGE analysis
showed that these oligomers migrated slower than the unmodi-
fied 14-mer (T instead of XU) (Fig. S1, ESI‡), consistent with
incorporation of the spin labels, which was confirmed by mass
spectrometry (Fig. S2, ESI‡). Circular dichroism (CD) spectra of
the ImU and OxU containing 14-mer duplexes were consistent
with right-handed B-DNA (Fig. S3, ESI‡). ImU and OxU slightly
destabilized the DNA duplexes, as judged by a decrease in the
melting temperature (TM) of 4 and 6 1C, respectively.
Fig. 2 CW EPR spectra of single stranded (upper) and duplex DNA (lower)
containing ImU (left) and the rigid spin label Ç (right) at 20 1C (10 mM phosphate,
100 mM NaCl, 0.1 mM Na2EDTA, pH 7.0).
The EPR spectra of ImU-labelled single stranded and duplex
DNA are shown in Fig. 2. Broadening of the spectrum upon
c
1000 Chem. Commun., 2013, 49, 999--1001
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detect its base-pairing by EPR spectroscopy, due to the limited
motion of the spin label relative to the base.
In summary, we have synthesized novel nitroxide-labelled
benzimidazole (ImU) and benzoxazole (OxU) derivatives of
20-deoxyuridine as spin probes for nucleic acids. Both ImU
and OxU had limited mobility in duplex DNA, in particular
ImU, indicating that rotation around the single bond linking the
spin label to the uracil is restricted. This is, to our knowledge,
the first example of using intramolecular hydrogen-bonding to
restrict spin label mobility. ImU should not only be a good label
for accurate distance measurements in oligonucleotides, but
also yield information about the relative orientation of the
labels.4 Distance measurements by pulsed EPR using these
spin probes are in progress and the results will be reported in
due course.
Fig. 3 CW EPR spectra of ImU- and OxU-containing single-stranded (left) and
duplex DNA (right) at 0 1C (10 mM phosphate, 100 mM NaCl, 0.1 mM Na2EDTA,
pH 7.0).
This work was supported by The Icelandic Research Fund
(080041023). We thank members of the Sigurdsson research
group for helpful discussions.
Notes and references
duplex formation is striking and indicates that there is limited
motion of the label independent of the nucleic acid itself.
In fact, at 20 1C the mobility of the ImU is similar to that of
the rigid spin label Ç in the same DNA sequence (Fig. 2) and at
ꢀ10 1C the spectra of the two become superimposable (Fig. S4,
ESI‡). We postulate that the low mobility of ImU is in part due to
an intramolecular hydrogen bond between the imidazolic
hydrogen and the O4 carbonyl of uracil (Fig. 1D).
To investigate if hydrogen bonding played a role in the
limited mobility of ImU, we compared the spectra of ImU- and
OxU-labelled single strands and duplexes (Fig. 3). The only
difference between these two labels is that the imidazole N–H
of ImU has been replaced by O in OxU. Therefore, OxU should be
more mobile if hydrogen bonding between the imidazole N–H
and the O4 of the uracil reduces the motion about the single
bond that connects the nitroxide to the nucleobase. Com-
parison of the EPR spectra of ImU- and OxU-labelled DNAs
shows that there is not much difference between the two labels
at 25 1C, while the spectra of the ImU-labeled DNA are notice-
ably broader than the spectra of the OxU oligomers at 0 1C
(Fig. 3). These data clearly indicate that hydrogen bonding
between the imidazole N–H and O4 of the uracil contributes
towards reducing the motion of the spin label at lower
temperatures.
1 (a) T. Prisner, M. Rohrer and F. MacMillan, Annu. Rev. Phys. Chem.,
2001, 52, 279; (b) O. Schiemann and T. F. Prisner, Q. Rev. Biophys.,
2007, 40, 1; (c) G. W. Reginsson and O. Schiemann, Biochem. Soc.
Trans., 2011, 39, 128.
2 (a) G. Z. Sowa and P. Z. Qin, in Prog. Nucleic Acid Res. Mol. Biol,
ed. P. M. Conn, 2008, vol. 82, p. 147; (b) S. A. Shelke and
S. T. Sigurdsson, Eur. J. Org. Chem., 2012, 2291; (c) S. A. Shelke
and S. T. Sigurdsson, Struct. Bonding, 2012, DOI: 10.1007/
430_2011_62, in press.
3 (a) A. Rassat and P. Rey, Bull. Soc. Chim. Fr., 1967, 3, 815;
(b) A. Spaltenstein, B. H. Robinson and P. B. Hopkins, J. Am. Chem.
Soc., 1988, 110, 1299; (c) M. Tominaga, S. R. Barbosa, E. F. Poletti,
J. Zukerman-Schpector, R. Marchetto, S. Schreier, A. Paiva and
C. R. Nakaie, Chem. Pharm. Bull., 2001, 49, 1027; (d) P. Z. Qin,
K. Hideg, J. Feigon and W. L. Hubbell, Biochemistry, 2003, 42, 6772;
(e) N. Barhate, P. Cekan, A. P. Massey and S. T. Sigurdsson, Angew.
Chem., Int. Ed., 2007, 46, 2655; ( f ) P. Cekan, A. L. Smith, N. Barhate,
B. H. Robinson and S. T. Sigurdsson, Nucleic Acids Res., 2008,
36, 5946; (g) M. Sajid, G. Jeschke, M. Wiebcke and A. Godt,
Chem.–Eur. J., 2009, 15, 12960; (h) S. Stoller, G. Sicoli, T. Y.
Baranova, M. Bennati and U. Diederichsen, Angew. Chem., Int. Ed.,
¨
2011, 50, 9743; (i) C. Hobartner, G. Sicoli, F. Wachowius,
D. B. Gophane and S. T. Sigurdsson, J. Org. Chem., 2012, 77,
7749.
4 O. Schiemann, P. Cekan, D. Margraf, T. F. Prisner and
S. T. Sigurdsson, Angew. Chem., Int. Ed., 2009, 48, 3292.
5 A. Marko, V. Denysenkov, D. Margraf, P. Cekan, O. Schiemann,
S. T. Sigurdsson and T. F. Prisner, J. Am. Chem. Soc., 2011, 133, 13375.
6 V. Colotta, D. Catarzi, F. Varano, O. Lenzi, G. Filacchioni,
C. Costagli, A. Galli, C. Ghelardini, N. Galeotti, P. Gratteri,
J. Sgrignani, F. Deflorian and S. Moro, J. Med. Chem., 2006, 49, 6015.
7 P. F. Pagoria, A. R. Mitchell and R. D. Schmidt, J. Org. Chem., 1996,
61, 2934.
8 X. S. Fan, X. Y. Zhang, L. H. Zhou, K. A. Keith, E. R. Kern and
P. F. Torrence, J. Med. Chem., 2006, 49, 3377.
9 J. Cheng, J. T. Xie and X. J. Luo, Bioorg. Med. Chem. Lett., 2005,
15, 267.
Spin labels with limited mobility can be used to study local
structural perturbations in nucleic acids.12 To determine if EPR
could be used to detect base-pairing of ImU, four DNA 14-mer
duplexes were prepared, in which ImU was paired with either A,
T, G or C. Overlay of the four EPR spectra revealed that they 10 H. Hayatsu, J. Biochem., 1996, 119, 391.
11 R. S. Varma, R. K. Saini and O. Prakash, Tetrahedron Lett., 1997,
could indeed all be distinguished from each other (Fig. S5A,
ESI‡), although ImUꢁG and ImUꢁC were similar. Since TꢁT mis-
matches are able to form metallo base-pairs with mercuric
ions,13 it was not surprising that the EPR spectrum of the
ImUꢁT-pair became nearly identical to that of ImUꢁA upon
addition of Hg2+-ions (Fig. S5B, ESI‡). Thus, ImU can clearly
38, 2621.
12 (a) P. Cekan and S. T. Sigurdsson, J. Am. Chem. Soc., 2009, 131,
18054; (b) U. Jakobsen, S. A. Shelke, S. Vogel and S. T. Sigurdsson,
J. Am. Chem. Soc., 2010, 132, 10424.
13 Y. Miyake, H. Togashi, M. Tashiro, H. Yamaguchi, S. Oda, M. Kudo,
Y. Tanaka, Y. Kondo, R. Sawa, T. Fujimoto, T. Machinami and
A. Ono, J. Am. Chem. Soc., 2006, 128, 2172.
c
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Chem. Commun., 2013, 49, 999--1001 1001