Oxygen replacement with selenium at the thymidine 4-position for the Se base pairing and crystal structure studies

Article abstract of DOI:10.1021/ja0680919

The T-A and C-G base pairing and stacking allow the formation of the stable DNA duplex structure for genetic information storage, transcription, and replication. To replace the oxygen of the nucleotide nucleobases with selenium for the studies of the base

Full text of DOI:10.1021/ja0680919

Published on Web 03/28/2007
Oxygen Replacement with Selenium at the Thymidine 4-Position for the Se
Base Pairing and Crystal Structure Studies
Jozef Salon, Jia Sheng, Jiansheng Jiang, Guexiong Chen, Julianne Caton-Williams, and
Zhen Huang*
Department of Chemistry, Georgia State UniVersity, Atlanta, Georgia 30303
Received November 15, 2006; E-mail: huang@gsu.edu
Scheme 1. Synthesis of 4-Se-Thymidine Phosphoramidite (4) and
Oligonucleotides Containing 4-Se-T (5)a
Nucleic acids participate in many important biological functions
in living systems, including genetic information storage, gene
expression, and catalysis.1-4 The T-A and C-G base pairing and
stacking allow the formation of the stable DNA duplex for genetic
information storage, transcription, and replication. The replacement
of oxygen on the nucleobases with sulfur5,6 has provided more
insights on the DNA duplex stability, recognition, and replication
at the atomic level.7,8 The 2-thio thymidine causes the slight
stabilization of the T-A paring and DNA duplex, while the 4-thio
thymidine causes the slight destabilization of them.7,8 Recent studies
on these sulfur modifications have revealed the enhanced base-
paring selectivity8a and the replication efficiency and fidelity,8b
especially with the 2-thio thymidine, probably due to discouraging
the undesired T-G wobble base pairing by the larger sulfur atom
at the 2-position of the thymine.
As selenium (atomic radius, 1.16 Å), another element member of
family VIA in the periodic table, is much larger than oxygen (0.73
Å), the replacement of O with Se on the nucleobases of nucleic
acids will most likely provide more advantages over the natives and
reveal more insights on the base-pairing selectivity, the duplex
stability and recognition, and the replication efficiency and fidelity.
Furthermore, the Se derivatization by replacing O can facilitate
X-ray crystal structure study of nucleic acids via multiwavelength
anomalous dispersion (MAD) phasing,9 and the nucleobase Se deriv-
atization can provide unique opportunities over the Se-derivatized
nucleic acids (SeNA) with Se on other positions for the structure
study.10,11 In addition, specific pyrimidines in natural tRNAs have
been derivatized by Se on the nucleobases, and the Se function has
been barely understood, though it was suggested that the Se substi-
tution might be involved in the tRNA anticodon.12 Despite the
synthesis of the Se-containing nucleobases and nucleosides several
decades ago,13 synthesis of the nucleic acids containing the Se
nucleobases remained a challenge. To explore the Se derivatization
on the nucleobases for the function and structure studies, we report
here the first synthesis of the 4-Se-thymidine phosphoramidite
(Scheme 1), its incorporation into oligonucleotides, the SeT-A base-
pair formation, and the thermostability and crystal structure studies
of the DNAs containing 4-Se-thymidine modification.
To replace the 4-oxygen with selenium, we activated the partially
protected thymidine derivative (1) at the 4-position via the formation
of triazolide 2 in two reactions (87% yield).14 We also developed
di(2-cyanoethyl) diselenide reagent for the Se introduction.15 After
reducing the diselenide reagent to the corresponding selenol with
NaBH4, the protected Se functionality was introduced to the
4-position of 2 by displacing the 4-triazolyl activating group,
achieving the simultaneous Se incorporation and protection for the
Se-T intermediate (3). Due to the undesired removal of the
2-cyanoethyl group on Se while removing the 3′-TMS group by
the fluoride treatment, we developed a mild condition to remove
the 3′-TMS group by using 10% triethylamine in methano (81%
a Reagents and conditions: (a) TMS-Im and CH3CN; (b) triazole-
POCl3-Et3N; (c) (NCCH2CH2Se)2/NaBH4, EtOH; (d) 10% Et3N in MeOH;
(e) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite and N,N-diisopro-
pylethylamine in CH2Cl2; (f) the solid-phase synthesis.
yield). Alternatively, we removed the 3′-TMS group first by the
fluoride treatment, followed by the Se incorporation (85% yield in
two reactions). Finally, Se-thymidine derivative 3 was converted
to the corresponding phosphoramidite 4 in 92% yield.10c
The 4-Se-T phosphoramidite (4) was examined on solid-phase
nucleotide synthesis. As expected, it is compatible with the coupling
reaction, acetylation, I2 oxidation, and trichloroacetic acid treatment
to remove the 5′-DMTr group, without causing deselenization.
Unfortunately, the concentrated ammonia treatment, which is
commonly used for deprotecting the nucleobases and the 2-cya-
noethyl groups from O and S functionalities,6 caused the desele-
nization. To prevent the deselenization, we found the deprotecting
condition by using the potassium carbonate in methanol, which
removes the 2-cyanoethyl group on Se in 2 h. The oligonucleotides
synthesized using the phosphoramidites containing the ultramild
nucleobase deprotection groups can also be fully deprotected
overnight with the same potassium carbonate solution.16 The
synthesized Se DNAs were purified and analyzed by HPLC and
MS (Table 1 and Supporting Information). A typical crude Se DNA
HPLC profile, which is almost identical to that of the corresponding
native, is shown in Figure 1. Less than 2% short oligonucleotides
were formed in this Se decamer synthesis, indicating the high yield
of the Se-thymidine coupling (99%).
The stability study of 5′-ATGGSeTGCTC-3′ indicated that
4-Se-T was stable at 60 °C over 3 h without significant
decomposition. In addition, the UV-melting temperature of the
duplexes of 5′-ATGGSeTGCTC-3′ and 5′-GAGCACCAT-3′ (38.6
°C) is close to that of the corresponding native duplex (39.2 °C),
suggesting no significant perturbation caused by the Se modification
on thymine. We have also successfully crystallized the 4-Se-T-
containing DNA (5′-G-dUSe-G-SeT-A-C-A-C-3′, self-complemen-
9
4862
J. AM. CHEM. SOC. 2007, 129, 4862-4863
10.1021/ja0680919 CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Table 1. MS Analysis of the DNAs Containing 4-Se-T
bond length: 2.8-3.2 Å) in the crystal structure is consistent with
the 4-Se DNA UV-melting study.
entry
Se oligonucleotides
measured (calcd) m/z
In summary, we have synthesized the 4-Se thymidine phosphor-
amidite (4) and incorporated it into oligonucleotides with quantita-
tive yield. The Se substitution on the nucleobase is relatively stable
under the elevated temperature. By the UV-melting and crystal
structure studies, we have further demonstrated that the O substitu-
tion with Se on the nucleobase does not cause the significant duplex
structure perturbation. The crystal structure study also reveals the
accommodation of the large Se atom on the thymine by the DNA
duplex and the formation of the Se-mediated hydrogen bond within
the T-A base pair. This work will stimulate the studies on the Se
substitution on other nucleobases and positions, opening a new ave-
nue for further exploring the base-paring selectivity governed by
the hydrogen bond and base shape, the duplex structure and flexi-
bility, the DNA replication efficiency and fidelity, and the poly-
merase recognition of the modified nucleobases. Moreover, the Se
derivatization on the nucleobases will largely facilitate X-ray crystal
structure studies of nucleic acids as well as their protein complexes.
1
5′-SeTT-3′
[M - H+]-: 609.0 (609.1)
C20H17N4O12PSe: FW 610.1
2
3
4
5
6
7
5′-DMTr-SeTSeTT-3′
[M - H+]-: 1279.0 (1279.1)
[M + H+]+: 1219.0 (1219.1)
[M + H+]+: 1523.0 (1523.2)
[M + H+]+: 2476 (2474)
C51H58N6O19P2Se2: FW 1280.1
5′-TSeTTT-3′
C40H53N8O25P3Se: FW 1218.1
5′-TTSeTTT-3′
C50H66N10O32P4Se: FW 1522.2
5′-GSeTGTACAC-3′
C78H99N30O45P7Se: FW 2473
5′-ATGGSeTGCTC-3′
[M + H+]+: 2792.5 (2793.4)
[M + H+]+: 3091.6 (3092.0)
C88H112N32O53P8Se: FW 2792.4
5′-GCGSeTATACGC-3′
C97H123N38O57P9Se: FW 3091.0
Acknowledgment. We thank Dr. Anand Saxena at NSLS
beamline X12C for his help in the data collection. This work was
supported by GSU and the NSF (MCB-0517092).
Supporting Information Available: Synthetic procedures, 1H and
13C NMR, HPLC, and MS analytical data. This material is available
free of charge via the Internet at http://pubs.acs.org.
Figure 1. HPLC analysis of crude 5′-DMTr-GCG(SeT)ATACGC-3′ after
cleavage from the solid support and the deprotection of the bases and
backbone (retention time: 21.0 min).
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Figure 2. The global and local structures of the 4-Se-T DNA [(5′-G-
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density.
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