Synthesis and crystallographic analysis of 5-Se-thymidine DNAs

Article abstract of DOI:10.1021/ol9004867

We investigated the possibility of the interaction of 5-CH₃ of thymidine and its 5′-phosphate backbone (C-H···O ^--P0₃ interaction) in DNA via the insertion of the atomic probe (a selenium atom) into the exo-5-position of t

Full text of DOI:10.1021/ol9004867

ORGANIC
LETTERS
2009
Vol. 11, No. 12
2503-2506
Synthesis and Crystallographic Analysis
of 5-Se-Thymidine DNAs
Abdalla E. A. Hassan, Jia Sheng, Jiansheng Jiang, Wen Zhang, and Zhen Huang*
Department of Chemistry, Georgia State UniVersity, Atlanta, Georgia 30303
Huang@gsu.edu
Received March 8, 2009
ABSTRACT
We investigated the possibility of the interaction of 5-CH3 of thymidine and its 5′-phosphate backbone (C-H···O--PO3 interaction) in DNA via
the insertion of the atomic probe (a selenium atom) into the exo-5-position of thymidine (5-Se-T). 5-Se-T was synthesized for the first time, via
Mn(OAc)3 assisted electrophilic addition of CH3SeSeCH3 to 3′,5′-di-O-benzoyl-2′-deoxyuridine. The 5-Se-T phosphoramidite was subsequently
synthesized and incorporated into DNA in over 99% coupling yield. Biophysical and structural investigations of the 5-Se-T DNAs revealed that
the Se-modified and nonmodified DNAs are virtually identical. In addition, the crystallographic analysis of a 5-Se-T DNA strongly suggests a
hydrogen-bond formation between the 5-CH3 and 5′-phosphate groups (CH3···PO4- interaction).
Hydrogen bonds are formed between hydrogen-bond accep-
tors and donors (X-H). In a classical hydrogen bond, X is
an atom with strong electron-negativity (oxgen, nitrogen,
etc.). However, recently hydrogen bonds where X is an atom
with weak electron-negativity (e.g., carbon) are gaining more
acceptance and importance, for instance carbon in C-H···OdC
hydrogen bond.1,2 The interactions between C-H and hy-
drogen-bond acceptors (electron donors), such as C-H···OdC
hydrogen bond in proteins,1a C-H···OdC in uracil crystal,1b
and C-H···Cl in a guest-host system,3 and other noncon-
ventional interactions (such as H···π interaction in RNA)4a
have played critical roles in molecular recognition, catalysis,
and DNA duplex stability within chemical and biological
systems.1-5 Since a negatively charged phosphate group is
an excellent electron donor and the phosphorylation and
dephosphorylation are common cellular regulation mecha-
nisms,5 we investigated whether a C-H (or CH3) group is
capable of forming a hydrogen bond with a phosphate group.
In DNA duplexes,6 the 5-methyl group of thymidine is
normally 4-5 Å away from the closest oxygen (pro-Sp
oxygen) of the 5′-phosphate group (O--PO3). In order to
extend the CH3 group closer to the phosphate and to give
the CH3 more rotational flexibility at the same time, we
inserted an atomic linker between the methyl group and the
C5 carbon of thymidine. If the CH3 and phosphate (O--PO3)
groups are able to form a genuine hydrogen bond, they will
(1) (a) Singh, S. K.; Babu, M. M.; Balaram, P. Proteins 2003, 51, 167.
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(3) Li, Y.; Flood, A. H. Angew. Chem., Int. Ed. 2008, 47, 2649.
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Nature 1994, 370, 575. (b) Yudushki, I. A.; Schleifenbaum, A.; Kinkha-
bwala, A.; Neel, B. G.; Schultz, C.; Bastiaens, P. I. Science 2007, 315,
(4) (a) Sarkhel, S.; Rich, A.; Egli, M. J. Am. Chem. Soc. 2003, 125,
8998. (b) Tewari, A. K.; Dubey, R. Bioorg. Med. Chem. 2008, 16, 126. (c)
Wang, S.; Kool, E. T. Biochemistry 1995, 34, 4125. (d) Gesteland, R. F. C.;
Cech, T. R.; Atkins, J. F. Eds. The RNA World: The Nature of Modern
RNA Suggests a Prebiotic RNA, ed. 3; Cold Spring Harbor Laboratory Press
Cold Spring Harbor, NY, 2006; (e) Umezawa, Y.; Nishio, M. Nucleic Acids
Res. 2002, 30, 2183.
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(6) (a) Wing, R.; Drew, H.; Takano, T.; Broka, C.; Tanaka, S.; Itakura,
K.; Dickerson, R. E. Nature 1980, 287, 755. (b) Jain, S.; Zon, G.;
Sundaralingam, M. Biochemistry 1989, 28, 2360. (c) Jiang, J.; Sheng, J.;
Carrasco, N.; Huang, Z. Nucleic Acids Res. 2007, 35, 477.
10.1021/ol9004867 CCC: $40.75
Published on Web 05/26/2009
 2009 American Chemical Society

be observed in a closer proximity and with strong electron
density between them in a crystal structure. If there is no
such stabilizing interaction between them, the methyl group
would prefer to turn away from the phosphate due to the
steric-hindrance and point into the major groove. Since the
size and geometry are essential requirements for this
investigation, we thus inserted a selenium atom as an atomic
probe7 between the 5-methyl group and C5 carbon. Herein
we report the first synthesis of 5-Se-thymidine phosphora-
midite (1, Scheme 1), its chemical incorporation into DNAs,
shows λmax absorption at 309 nm, red-shifted by 44 nm when
compared with thymidine (Supporting Information). This
large red-shift is caused by the electron-donating effect of
5-SeCH3 on the nucleobase π-system. Following the trity-
lation of 4, 5 was converted to 1 by the standard phosphora-
midite synthesis.7c
5-Se-thymidine (5-Se-T) phosphoramidite 1 was found
compatible with the conditions of the solid-phase synthesis,
including the coupling reaction, acetylation capping, I2
oxidation, and trichloroacetic acid and concentrated ammonia
treatments. The stability of the 5-Se-T moiety allows us to
successfully synthesize the Se-oligonucleotides using the
normal phosphoramidites. A typical HPLC profile of the
synthesized crude Se-DNAs is shown in Figure 1, which is
Scheme 1. Synthesis of 5-Se-T Phosphoramidite 1 and
Oligonucleotides (6) Containing 5-Se-T
Figure 1.
HPLC analysis of crude 5′-DMTr-TT-5-SeT-T-3′ (15.3
min).
virtually identical to that of the corresponding native DNA
(Supporting Information). We determined that the coupling
yield of the 5-Se-T phosphoramidite was over 99%. The
synthesized Se-DNAs were purified and analyzed by HPLC
and MS (Table 1 and Supporting Information).
and the biophysical and structural studies of the DNAs
containing the Se-extended 5-CH3. Excitingly, we have
discovered a novel methyl/phosphate hydrogen bond
(CH···O--PO3) for the first time.
We started novel synthesis of the 5-Se-thymidine phos-
phoramidite (1) from 3′,5′-di-O-benzyol-2′-deoxyuridine
(2).8a Though the arylselenylation at the 5-position of
pyrimidines was reported over a decade ago,8b the incorpora-
tion of alkylselenyl substitutions (such as CH3-Se) at the
5-position has not been reported in the literature, probably
due to the instability of the alkylselenyl intermediate. After
several attempts of screening Lewis acids as electrophile
activators, Mn(OAc)3 was found effective to promote the
methyselenation at the 5-position of 2 with dimethyldis-
elenide CH3SeSeCH3. Treatment of 2 with CH3SeSeCH3 (in
the presence of Mn(OAc)3 in AcOH at 90 °C) gave 5-Se-
thymidine derivative 3 in good yield. NMR analysis of 3
showed the disappearance of the H-5 peak and the appear-
ance of the H-6 singlet peak, and displayed the characteristic
1H and 13C chemical shifts of the 5-SeCH3 moiety at 2.06
and 7.24 ppm, respectively. Deprotection of 3 with NaOCH3
in MeOH gave 4 in a quantitative yield. UV spectrum of 5
Table 1. MALDI-TOF MS Data of the 5-Se-T DNAs
entry
Se-oligonucleotides
measured (calcd.) m/z
[M]+: 1234.5 (1234.1)
a
b
c
5′-TT-5-SeT-T-3′
5′-G-5-SeT-GTACAC-3′
[M - H+]-: 2487.4 (2487.4)
5′-GCG-5-SeT-ATACGC-3′ [M - H+]-: 3106.7 (3105.5)
d
e
f
5′-ATGG-5-SeT-GCTC-3′
5′-CTCCCA-5-SeT-CC-3′
[M - H+]-: 2808 (2808.4)
[M + H+]+: 2674.5 (2674.4)
5′-CTTCT-5-SeT-GTCCG-3′ [M + H+]+: 3352.4 (3352.6)
g
GdU2′-SeG-5-SeT-ACAC
[M + K+ - 2H+]-: 2605.1 (2605.2)
Our UV-melting data (Table 2) show that the melting
temperature differences of the native and Se-modified DNA
duplexes are small (less than (1.0 °C per modification),
whereas the melting temperature of the S-modified (5-Me-
S-T) DNA duplex is the lowest stable (approximately 3 °C
lower than that of the native8c). Lower stability of the 5-S-T
DNA duplex is probably due to the shorter S-C bond length
compared with the Se-C one, which may better facilitate
the 5-methyl/phosphate backbone interaction. In most cases
that we examined (Table 2), the native duplexes are slightly
more stable than the corresponding Se duplexes. Our results
(7) (a) Carrasco, N.; Ginsburg, D.; Du, Q.; Huang, Z. Nucleosides,
Nucleotides Nucleic Acids 2001, 20, 1723. (b) Du, Q.; Carrasco, N.; Teplova,
M.; Wilds, C. J.; Egli, M.; Huang, Z. J. Am. Chem. Soc. 2002, 124, 24. (c)
Salon, J.; Sheng, J.; Jiang, J.; Chen, G.; Caton-Williams, J.; Huang, Z. J. Am.
Chem. Soc. 2007, 129, 4862.
2504
Org. Lett., Vol. 11, No. 12, 2009

We also closely examined the geometry of the 5-CH3
moiety extended by the selenium insertion (Figures 2 and
3). Excitingly, the 5-methyl group clearly points to the 5′-
Table 2. UV Melting Temperatures of the 5-Se-T DNAs
melting
temp.
entry
DNA/DNA pairs
(°C, native)
a
b
c
d
e
5′-G-5-SeT-GTACAC-3′ (self-complementary)
5′-GCG-5-SeT-ATACGC-3′ (self-complementary)
5′-ATGG-5-SeT-GCTC-3′ 3′-TACC---A-CGAG-5′
5′-CTCCCA-5-SeT-CC-3′ 3′-GAGGGT---A-GG-5′
27.9 (27.5)
26.5 (28.0)
39.3 (40.3)
36.3 (36.5)
5′-CTTCT-5-SeT-GTCCG-3′ 3′-GAAGA---A-CAGGC-5′ 43.9 (44.9)
suggest that the insertion of a Se atom at the exo-5-position
of thymidine does not cause significant structure perturbation.
It is counterintuitive that the larger selenium atom causes
less destabilization than smaller sulfur atom, although both
disrupt the 5-CH3/π interaction with the preceding 5′-
nucleobase. It appears that the larger Se-linker allows the
methyl/phosphate stabilizing interaction.
Similar to other Se-derivatized nucleic acids (SeNA),7,9
these novel 5-Se-T DNAs are also stable. The crystal growth
of the self-complementary Se-DNA (5′-G-dU2′-Se-G-5-SeT-
ACAC-3′)2 was successfully facilitated by the utilization of
2′-Me-Se-dU.6c Superimposition of the determined Se-DNA
crystal structure (1.80 Å) over the corresponding native in
the same tetragonal space group6b reveals that these two
structures are virtually identical (Figure 2A), which is
Figure 3. Global and local structures of the 5-Se-T-DNA [(5′-GdU2′-
Se-G-5-SeT-ACAC-3′)2]. (A) The duplex structure of the modified
DNA (3BM0, in cyan). (B) Single-strand structure. (C) Methyl
group and pro-Sp-oxygen in the local structure.
phosphate backbone (Figure 3), and strong experimental
electron density exists between them (Figure 2B), although
the major groove is wide open and available to accommodate
the 5-Me-Se moiety. As a typical hydrogen-bond length
(X-H···Y) is between 2.8 and 3.2 Å, the distance (2.93 Å)
between the carbon of the 5-methyl group and the pro-Sp
oxygen of the 5′-phosphate (Figure 2C) indicates formation
of a methyl/phosphate hydrogen bond (CH3···O--PO3). In
addition, our observation on the CH3···O--PO3 interaction
is consistent with the interactions between C-H and other
electron donors, such as C-H···OdC hydrogen bond in
proteins [bond length (3.14 Å) and angle (149°)]1a and
C-H···OdC interaction in uracil crystal [hydrogen-bond
length (3.21 Å) and angle (161°)].1b The angle of the
C-H···O in the 5-Me-Se-T DNA structure is 144°, which
further supports the C-H···O hydrogen-bond formation. The
CH3 rotation about the single bond between the Se and C
allows the three hydrogen atoms to have equal opportunity
to interact with the pro-Sp oxygen (Figure 2B), which
strengthens the C-H···O--PO3 interaction.
Figure 2. Global and local structures of the 5-Se-T-DNA [(5′-GdU2′-
Se-G-5-SeT-ACAC-3′)2]. (A) Duplex structure of the modified DNA
(3BM0, in cyan) is superimposed over the native (1DNS, in pink).
(B) The experimental electron density map of the 5-SeT4:A5 base
pair and CH3···O--PO3 hydrogen-bond formation. (C) The super-
imposition of the Se-modified (in cyan) and native (in pink) local
5-SeT4:A5 structures.
Furthermore, in our structural comparison with the native
structure, we found that the Se insertion disrupts the weak
5-CH3/π interaction4e in the native structure,6b which results
in slight reduction of the duplex stability. On the other hand,
the intramolecular hydrogen bond slightly stabilizes the
intermolecular interaction (the duplex stability) due to the
possible preorganization (slight entropy decrease of the Se-
modified single strand). Therefore, our UV-melting observa-
consistent with our UV-melting study. Moreover, we ob-
served that the 5-Se-T and A form a base pair as well as the
native T-A pair (Figure 2, B and C). The lengths of these
two hydrogen bonds of the 5-SeT-A are 2.79 and 2.76 Å.
(8) (a) Fox, J. J.; Praag, D. V.; Wempen, I.; Doerr, I. L.; Cheong, L.;
Knoll, J. E.; Eidinoff, M. L.; Bendich, A.; Brown, G. B. J. Am. Chem. Soc.
1959, 81, 178. (b) Lee, C. H.; Kim, Y. H. Tetrahedron Lett. 1991, 32,
2401. (c) Ahmadian, M.; Zhang, P. M.; Bergstrom, D. E. Nucleic Acids
(9) (a) Carrasco, N.; Huang, Z. J. Am. Chem. Soc. 2004, 126, 448. (b)
Moroder, H.; Kreutz, C.; Lang, K.; Serganov, A.; Micura, R J. Am. Chem.
Soc. 2006, 128, 9909. (c) Caton-Williams, J.; Huang, Z. Angew. Chem.,
Int. Ed. 2008, 47, 1723. (d) Salon, J.; Jiang, J.; Sheng, J.; Gerlits, O. O.;
Huang, Z. Nucleic Acids Res. 2008, 36, 7009.
Res. 1998, 26, 3127
.
Org. Lett., Vol. 11, No. 12, 2009
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tion on the slight destabilization or stabilization of the Se-
DNA duplexes ((1.0 °C per modification) is consistent with
these two conflicting interactions, with the sequence and
modification dependences, in the presence of the Se-
modification and the C-H···O--PO3 hydrogen bond. Fur-
thermore, lower stability of the S-DNA duplex8c is consistent
with this C-H···O--PO3 interaction in the Se-DNA duplex.
As showned in Figure 3, negatively charged oxide orientates
the 5-methyl group toward DNA backbone, instead of the
major groove, for better interaction. Thus, our results indicate
the first observation of the C-H···O--PO3 hydrogen bond.
In conclusion, we have discovered a novel C-H···O
hydrogen bond (or CH3···O--PO3 interaction) Via the
biophysical and structural studies and the synthesis of the
5-Se-thymidine and the 5-Se-derivatized DNAs. Our experi-
mental results have revealed that the Se-modification does
not cause significant perturbation of the native DNA
structure. This unique CH3···O--PO3 interaction can be used
to advantage in designing inhibitors for the backbone
digestion. Our research results have suggested a new research
direction to design and develop nuclease-resistant DNAs and
RNAs functioning as antisense DNAs, siRNAs, or microR-
NAs. Moreover, our exciting discovery of the C-H···phosphate
hydrogen bond may provide new insight into atomic mech-
anisms of phosphorylation and dephosphorylation, which are
common strategies in cellular signal transduction regula-
tions.5 Furthermore, this selenium-derivatization has great
potential in the determination of nucleic acid crystal struc-
tures Via multiwavelength anomalous dispersion (MAD) or
single-wavelength anomalous (SAD) phasing.
Acknowledgment. We thank Dr. Anand Saxena at NSLS
beamline X12C. This work was supported by Georgia Cancer
Coalition (GCC) and the NSF (MCB-0824837 and CHE-
0750235).
Supporting Information Available: Detailed experimen-
tal procedures, 1H, 13C NMR, and MALDI-MS spectra, UV-
melting, HPLC, and crystal diffraction data. This material
is available free of charge Via the Internet at http://pubs.acs.org.
OL9004867
2506
Org. Lett., Vol. 11, No. 12, 2009