Synthesis of selenium-derivatized cytidine and oligonucleotides for X-ray crystallography using MAD

Article abstract of DOI:10.1021/ol0365077

Synthesis of the novel 2′-Se-cytidine phosphoramidite was achieved via transformation of the uridine analogue to the cytidine derivative in high yield. This 2′-Se-cytidine phosphoramidite was used to synthesize selenium-derivatized DNA and RNA oligonucleo

Full text of DOI:10.1021/ol0365077

ORGANIC
LETTERS
2004
Vol. 6, No. 7
1099-1102
Synthesis of Selenium-Derivatized
Cytidine and Oligonucleotides for X-ray
Crystallography Using MAD
Yuri Buzin, Nicolas Carrasco, and Zhen Huang*
Department of Chemistry, Brooklyn College, and Program of Biochemistry and
Chemistry, The Graduate School, The City UniVersity of New York,
2900 Bedford AVenue, Brooklyn, New York 11210
zhuang@brooklyn.cuny.edu
Received December 25, 2003
ABSTRACT
Synthesis of the novel 2′-Se-cytidine phosphoramidite was achieved via transformation of the uridine analogue to the cytidine derivative in
high yield. This 2′-Se-cytidine phosphoramidite was used to synthesize selenium-derivatized DNA and RNA oligonucleotides for X-ray
crystallography using MAD. The nucleotide coupling yield using this novel phosphoramidite was over 99% when 5-benzylmercaptotetrazole
(5-BMT) was used as the coupling reagent.
Derivatization of DNA and RNA for phase determination
in X-ray crystallography is a long-standing problem even
though several conventional approaches are commonly used,
such as heavy atom soaking, cocrystallization, and halogen
derivatization.1 Derivatization efforts via soaking and co-
crystallization often fail, probably due to nucleotide cleavage
by the heavy metal ions or nonspecific binding to polyanionic
nucleic acids. Halogen derivatization is often limited to
oligonucleotides.2 Therefore, selenium derivatization of
nucleic acids is of great importance because of the potential
application in determination of 3-D structures of DNAs,
ribozymes, other functional RNAs and nucleic acid-protein
complexes by X-ray crystallography.3
replacement of oxygen in nucleotide with selenium using
multiwavelengh anomalous dispersion (MAD).4-8 Previous
X-ray crystallographic studies on the oligonucleotides con-
taining the 2′-methylselenouridine have indicated that the
2′-methylseleno-substituted furanoses display C3′-endo puck-
ers, consistent with the A-form geometry of RNA and
A-form DNA. Furthermore, the duplexes of the modified
oligonucleotides were as stable as the native ones. These
results demonstrated that the 2′-selenium functionality is
suitable for RNA and A-form DNA derivatization for X-ray
(3) (a) Kondo, J.; Umeda, S.; Sunami, T.; Takenaka, A. Nucleic Acids
Res. Suppl. 2003, 3, 223. (b) Song, J. J.; Liu, J.; Tolia, N. H.; Schneiderman,
J.; Smith, S. K.; Martienssen, R. A.; Hannon, G. J.; Joshua-Tor, L. Struct.
Biol. 2003, 10, 1026. (c) Lei, M.; Podell, E. R.; Baumann, P.; Cech, T. R.
Nature 2003, 426, 198.
Recently, Huang, Egli, and co-workers have established
the principle of nucleic acid X-ray crystallography by the
(4) Carrasco, N.; Ginsburg, D.; Du, Q.; Huang, Z. Nucleosides, Nucleo-
tides, Nucleic Acids 2001, 20, 1723-1734.
(5) Du, Q.; Carrasco, N.; Teplova, M.; Wilds, C. J.; Egli, M.; Huang, Z.
J. Am. Chem. Soc. 2002, 124, 24-25.
(1) (a) Holbrook, S. R.; Kim, S.-H. Biopolymers 1997, 44, 3. (b)
Utsunomiya, R.; Kumar, P. K.; Fujimoto, Z.; Mizuno, H. Nucleic Acids
Res. Suppl. 2003, 3, 225-6.
(2) (a) Ogata, C. M.; Hendrickson, W. A.; Gao, X.; Patel, D. J. Abstr.
Am. Cryst. Assoc. Mtg. 1989, Ser. 2, 17, 53. (b) Xiong, Y.; Sundaralingam,
M. Nucl. Acids Res. 2000, 28, 2171.
(6) Teplova, M.; Wilds, C. J.; Du, Q.; Carrasco, N.; Huang, Z.; Egli, M.
Biochimie 2002, 84, 849-858.
(7) Wilds, C. J.; Pattanayek, R.; Pan, C.; Wawrzak, Z.; Egli, M. J. Am.
Chem. Soc. 2002, 124, 14910-14916.
(8) Carrasco, N.; Huang, Z. J. Am. Chem. Soc. 2004, 126, 448-449.
10.1021/ol0365077 CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/10/2004

crystallography.5,6 To further apply this strategy in nucleic
acid derivatization, we designed and synthesized a novel
building block, 2′-Se-cytidine phosphoramidite 5, for de-
rivatization of DNAs and RNAs using the solid-phase
synthesis. We report here syntheses of the cytidine derivative
with oxygen replacement with selenium at the 2′-position,
the corresponding phosphoramidite, and the Se-DNAs and
Se-RNAs for X-ray crystallography using MAD.
Analogous to the 2′-Se-uridine synthesis,5 the synthesis
of the 2′-Se-cytidine derivatives was first attempted. Due to
the low yield in the introduction of the methylseleno group,
an alternative route (Scheme 1) was explored by converting
Figure 1. Reversed-phase HPLC analysis of crude DMTr-on 2′-
Se-C RNA8mer (5′-DMTr-GGCSeGUGCC-3′) after deprotection of
the bases, backbone, and 2′-TOM groups. Its retention time is 22.1
min.
Scheme 1. Synthesis of the 2′-Se-Cytidine Phosphoramidite
and Its Incorporation into DNA and RNA Oligonucleotidesa
the 3′-hydroxyl group using 1-(trimethylsilyl)imidazole
(TMS-Im) in dry acetonitrile, the position 4 of the uridine
derivative was activated via formation of the triazolide (2)
in dry acetonitrile. Intermediate 2 was directly converted to
the cytidine derivative (3) without purification by aqueous
ammonia treatment. The transient 3′-TMS protection was
concomitantly removed during this treatment. The yield over
these reactions in one-pot was 86%. After silylation of the
3′-OH group of 3 with TMS-Im in dry tetrahydrofuran
(THF), the amino group of the cytidine derivative was
acetylated using acetic anhydride in the presence of dry TEA
and a catalytic amount of N,N-dimethylaminopyridine
(DMAP). Under the same acetylation conditions, the acyl-
ation using benzoyl chloride was attempted without success.
The transient silyl protection on the 3′-OH was removed to
give 4 by tetrabutylammonium fluoride treatment in THF.
Cytidine derivative 4 was converted to 2′-Se-cytidine phos-
phoramidite 5 in 92% yield by reacting it with 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite in the presence of
N,N-diisopropylethylamine, in dry CH2Cl2.5
a Reagents and conditions: (a) TMS-Im, then POCl3-triazole-
TEA in CH3CN; (b) NH4OH; (c) TMS-Im, then Ac2O, TEA, and
DMAP in THF; (d) 2-cyanoethyl N,N-diisopropyl-chlorophosphor-
amidite and N,N-diisopropylethylamine in CH2Cl2; (e) synthesis
of oligonucleotides on solid phase.
uridine to cytidine.9 Transformation of uridine to cytidine
consists of two major steps: activation of the position 4 of
uridine and ammonia treatment. Initially, we encountered loss
of the selenium functionality in the activation step. The
deselenization probably was caused by the highly reactive
species in the reagent-generating reaction, where phosphorus
oxytriazolide was made in situ using phosphorus oxychloride
(POCl3) and triazole. This difficulty was later overcome by
extending the reagent-making time and adding a large excess
of dry triethylamine (TEA). After the in situ protection of
Figure 2. HPLC analysis of purified DMTr-off 2′-Se-C DNA12mer
(5′-TATCGTTAATCSeT-3′). The sample was analyzed on a 300SB-
C8 column (4.5 × 150 mm), eluted (1 mL/min) with buffer A (5
mM ammonium acetate, pH 6.5) for 2 min, and then eluted with a
linear gradient from buffer A to 100% buffer B (60% acetonitrile
and 40% of buffer A) in 13 min. Its retention time is 7.2 min.
(9) McGee, D. P. C.; Vanghn-Settle, A.; Vargeese, C. Zhai, Y., J. Org.
Chem. 1996, 61, 781-785.
1100
Org. Lett., Vol. 6, No. 7, 2004

end, where it experienced multiple cycles of the I2 oxidation
(data not shown). In addition, like the 2′-Se-U phosphor-
amidite, the solid-phase coupling yields of RNAs and DNAs
using this novel 2′-Se-C phosphoramidite were higher than
99%. The reversed-phase HPLC analysis of the crude
RNA8mer synthesized using 2′-Se-C phosphoramidite 5 is
shown in Figure 1.
Chemically synthesized RNA and DNA oligonucleotides
containing 2′-Se-C derivatization were purified twice by
HPLC (DMTr-on and DMTr-off). The reversed-phase HPLC
purifications were performed by a Zorbax C8 column (21.2
× 250 mm). Samples were eluted (10 mL/min, Figure 1)
with a linear gradient from buffer A [50 mM triethylammo-
nium acetate (TEAAc), pH 7.1] to 90% buffer B (50%
aqueous acetonitrile, 50 mM TEAAc, pH 7.1) in 25 min. A
typical HPLC profile of purified Se-RNAs and Se-DNAs is
shown in Figure 2. Purified 2′-Se-C-derivatized oligonucleo-
tides were confirmed by electrospray mass spectrometry. MS
spectral examples of 2′-Se-C-RNAs and 2′-Se-C-DNAs are
shown in Figure 3, and the molecular peaks with several
different charges are observed. The MS analytical data are
shown in Table 1.
Table 1. MS Analytical Data of RNA and DNA
Oligonucleotides
entry
a
Se-oligonucleotides
2′-Se-C RNA8mer
(5′-GGCSeGUGCC-3′)
77H98N31O54P7Se: FW 2617.3
measured (calcd) m/z
M
M
2-: 1307.8 (1307.7)
3-: 871.5 (871.4)
C
b
c
d
2′-Se-C RNA14mer
(GCSeUGACGAUACACC)
M
M
3-: 1500.6 (1500.5)
4-: 1125.3 (1125.2)
C
134H169N54O93P13Se: FW 4504.6
2′-Se-C DNA8mer
(5′-ACSeTGACAG-3′)
M
M
2-: 1254.7 (1254.7)
3-: 836.1 (836.1)
Figure 3. Electrospray MS analysis of 2′-Se-C oligonucleotides:
(A) DMTr-off 2′-Se-C RNA8mer (C77H98N31O54P7Se), isotopic
mass 2617.3, M-2 1307.8 (1307.7), M-3 871.5 (871.4); (B) DMTr-
off 2′-Se-C DNA12mer (C119H153N38O73P11Se), isotopic mass
3702.6, M-2 1850.6 (1850.3), M-3 1233.4 (1233.2), M-4 924.7
(924.6), M-5 739.6 (739.5), M-6 616.2 (616.1).
C
79H100N33O44P7Se: FW 2511.4
DMTr-on 2′-Se-C DNA12mer
(DMTr-TATCGTTAATCSeT)
formula: C140H171N38O75P11Se
formula isotopic weight: 4004.7
M
M
M
M
M
M
M
M
M
M
M
2-: 2001.8 (2001.4)
3-: 1334.2 (1333.9)
3-: 1334.2 (1333.9)
4-: 1000.3 (1000.2)
5-: 800.1 (799.9)
6-: 666.6 (666.5)
2-: 1850.6 (1850.3)
3-: 1233.4 (1233.2)
4-: 924.7 (924.6)
5-: 739.6 (739.5)
6-: 616.2 (616.1)
e
DMTr-off 2′-Se-C DNA12mer
(5′-TATCGTTAATCSeT-3′)
formula: C119H153N38O73P11Se;
formula isotopic weight: 3702.6
To demonstrate the compatibility of 5 with the solid-phase
synthesis, several DNA and RNA oligonucleotides were
designed and synthesized. Selenium-derivatized RNAs were
synthesized using 2′-O-triisopropylsilyloxymethyl (TOM)-
protected nucleoside phosphoramidites,10 while Se-deriva-
tized DNAs were synthesized by following standard solid-
phase synthesis and using 5-benzylmercaptotetrazole (5-
BMT) as the coupling reagent.11 The coupling yield using
5-BMT activator was higher than that using tetrazole (data
not shown). Like the previous report on the synthesis of 2′-
Se-U DNAs,5 the protected selenide functionality in 2′-Se-C
oligonucleotides was also stable under mild I2 treatment (20
mM, 20 s) for the phosphite oxidation. This selenium
functionality was stable even when it was close to the 3′-
To investigate the duplex stability of RNA containing the
2′-Se derivatization, the UV melting study was performed
using the RNA8mer and its analogues (Table 2). Solutions
of the duplex RNAs (1.5 µM) were prepared by dissolving
the RNAs in a buffer containing NaCl (200 mM), sodium
phosphate (5 mM, pH 7.4), and EDTA (1 mM). The solutions
were then heated to 95 °C for 1 min, cooled slowly to room
temperature, and stored at 5 °C overnight before measure-
ment.12 Denaturation curves were acquired at 260 nm at a
heating rate of 0.5 °C/min (from 10 to 70 °C) using a 8453
UV-vis Spectrometer from Agilent Technologies, which was
(10) Pitsch, S.; Weiss, P. A.; Wu, X.; Ackermann, D.; Honegger, T. HelV.
Chim. Acta 1999, 82, 1753.
(11) Welz, R.; Muller, S. Tetrahedron Lett. 2002, 43, 795.
Org. Lett., Vol. 6, No. 7, 2004
1101

RNAs and DNAs by the solid-phase synthesis. As the large-
scale synthesis of Se-derivatized oligonucleotides is possible,
the chemical synthesis of Se-DNAs and Se-RNAs is of great
importance in nucleic acid X-ray crystallography using
MAD. As one selenium atom has anomalous phasing power
over approximately 30 nucleotides, long DNAs and RNAs
(e.g., over 100 nt) containing multiple selenium labels can
be prepared through a ligation of one synthetic fragment
containing multiple selenium atoms with a polymerized DNA
or transcribed RNA fragment.13,14 In addition, the derivati-
zation strategy of nucleic acids with selenium can be used
to derivatize nucleic acid-protein complexes via derivati-
zation of nucleic acids instead of the protein counterparts
for X-ray crystallography.
Table 2. UV Melting Temperatures of the RNA8mers
melting
entry
a
RNA8mer
temperature (°C)
native RNA8mer
36.6
(5′-GGCGUGCC-3′)
2′-MeO-RNA8mer
(5′-GGCOGUGCC-3′)
2′-MeSe-RNA8mer
(5′-GGCSeGUGCC-3′)
2′-MeSe-RNA8mer
(5′-GGCGUSeGCC-3′)
b
c
d
32.9
31.7
32.4
equipped with a Peltier temperature controller. The result
shows that the melting temperatures of the 2′-Se-C and 2′-
Se-U RNA8mers were almost the same as that of the 2′-
MeO RNA8mer (Table 2), which was slightly lower than
that of the native. This melting study indicates that the 2′-
Se modification had just minor effect on RNA duplex
stability. This is consistent with the stability study of the
A-form DNA duplex derivatized with the 2′-Se functionality
investigated previously, which indicated that the selenium
derivatization had no significant effect on stability of the
A-form DNA duplex.6
Acknowledgment. We thank Dr. Clifford E. Soll at
Hunter College for assisting in MS data collection. This work
was supported by PSC-CUNY Awards (Nos. 4203-00-33 and
65356-00-34), CUNY Project Programs (Nos. 80210-0302
and 80209-0408), and National Institute of Health grant (No.
GM069703).
Supporting Information Available: Experimental pro-
1
cedures and H, 13C, and 31P NMR, and HRMS analytical
data. This material is available free of charge via the Internet
at http://pubs.acs.org.
In conclusion, we have developed a route to synthesize
the novel 2′-Se-cytidine phosphoramidite via conversion of
the uridine to the 2′-Se-cytidine derivative. This 2′-Se-C
phosphoramidite has been successfully used to derivatize
OL0365077
(13) Sherlin, L. D.; Bullock, T. L.; Nissan T. A.; Perona, J. J.; Lariviere
F. J.; Uhlenbeck, O. C.; Scaringe, S. A. RNA 2001, 7, 1671.
(14) Kurata, S.; Ohtsuki, T.; Suzuki, T.; Watanabe, K. Nucleic Acids
Res. 2003, 31, e145.
(12) Puglisi, J. D.; Tinoco, I., Jr. Methods Enzymol. 1989, 180, 304-
325.
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