Synthesis of a 2′-Se-uridine phosphoramidite and its incorporation into oligonucleotides for structural study

Article abstract of DOI:10.1021/ol052270y

(Chemical Equation Presented) We report here the synthesis of the 5′-[benzhydryloxybis(trimethylsilyloxy)]silyl-2′-methylseleno- 2′-deoxyuridine phosphoramidite and its incorporation into oligonucleotides by solid-phase synthesis. The coupling yield of th

Full text of DOI:10.1021/ol052270y

ORGANIC
LETTERS
2005
Vol. 7, No. 25
5645-5648
Synthesis of a 2′-Se-uridine
Phosphoramidite and Its Incorporation
into Oligonucleotides for Structural
Study
Jozef Salon, Guexiong Chen, Yoani Portilla, Markus W. Germann, and
Zhen Huang*
Department of Chemistry, Georgia State UniVersity, Atlanta, Georgia 30303, and
Brooklyn College, Brooklyn, New York 11210
huang@gsu.edu
Received September 19, 2005
ABSTRACT
We report here the synthesis of the 5
′
-[benzhydryloxybis(trimethylsilyloxy)]silyl-2
′
-methylseleno-2
′
-deoxyuridine phosphoramidite and its
incorporation into oligonucleotides by solid-phase synthesis. The coupling yield of this phosphoramidite into oligonucleotides is higher than
99%. We also demonstrate that this 2 -methylselenophosphoramidite is compatible with the 5 -silyl-2 -ACE chemistry, for longer Se-RNA solid-
-Se-DNA has revealed a USe A base pair and a duplex structure formation
′
′
′
phase synthesis. Our preliminary NMR study on the synthesized 2
′
−
when its complementary strand was present.
Nucleic acids are important biomacromolecules in living
systems and are involved in genetic information storage,
expression, and regulation. Chemical modifications and
structural studies have helped to reveal many insights on their
biological functions and mechanisms.1-5 To get insights of
the structure and function of nucleic acids, we have recently
developed selenium derivatization of DNA and RNA for
phase determination,6-10 a long-standing problem,11 in X-ray
crystallography. We have incorporated selenium atoms into
nucleic acids chemically and enzymatically by replacing the
nucleotide oxygen, such as the 5′, 2′, and nonbridging
phosphate oxygen atoms6,9,12 for X-ray crystal structure
determination. Recently, several crystal structures of DNA
and RNA molecules derivatized with the Se-labeling strategy
have been reported by several laboratories using multiwave-
length anomalous dispersion (MAD) phasing.8,13,14
(7) Du, Q.; Carrasco, N.; Teplova, M.; Wilds, C. J.; Egli, M.; Huang, Z.
J. Am. Chem. Soc. 2002, 124, 24-25.
(8) Teplova, M.; Wilds, C. J.; Wawrzak, Z.; Tereshko, V.; Du, Q.;
Carrasco, N.; Huang, Z.; Egli, M. Biochimie 2002, 84, 849-858.
(9) Carrasco, N.; Huang, Z. J. Am. Chem. Soc. 2004, 126, 448-449.
(10) Carrasco, N.; Buzin, Y.; Tyson, E.; Halpert, E.; Huang, Z. Nucleic
Acids Res. 2004, 32, 1638-1646.
(1) Adams, P. L.; Stahley, M. R.; Kosek, A. B.; Wang, J.; Strobel, S. A.
Nature 2004, 430, 45-50.
(2) Ban, N.; Nissen, P.; Hansen, J.; Moore, P. B.; Steitz, T. A. Science
2000, 289, 905-920.
(3) Benoff, B.; Yang, H.; Lawson, C.; Parkinson, G.; Liu, J.; Blatter,
E.; Ebright, Y. W.; Berman, H. M.; Ebright, R. H. Science 2002, 297, 1562-
1566.
(11) Holbrook, S. R.; Kim, S.-H. Biopolymers 1997, 44, 3-21.
(12) Buzin, Y.; Carrasco, N.; Huang, Z. Org. Lett. 2004, 6, 1099-
1102.
(4) Suryadi, J.; Tran, E. J.; Maxwell, E. S.; Brown, B. A., II. Biochemistry
2005, 44, 9657-9672.
(13) Wilds, C. J.; Pattanayek, R.; Pan, C.; Wawrzak, Z.; Egli, M. J. Am.
Chem. Soc. 2002, 124, 14910-14916.
(14) Serganov, A.; Keiper, S.; Malinina, L.; Tereshko, V.; Skripkin, E.;
Ho¨bartner, C.; Polonskaia, A.; Phan, A. T.; Wombacher, R.; Micura, R.;
Dauter, Z.; Ja¨schke A.; Patel, D. J. Nat. Struct. Mol. Biol. 2005, 12, 218-
224.
(5) Haeberli, P.; Berger, I.; Pallan, P. S.; Egli, M. Nucleic Acids Res.
2005, 33, 3965-3975.
(6) Carrasco, N.; Ginsburg, D.; Du, Q.; Huang, Z. Nucleosides Nucle-
otides Nucleic Acids 2001, 20, 1723-1734.
10.1021/ol052270y CCC: $30.25
© 2005 American Chemical Society
Published on Web 11/16/2005

Our early experimental results on the structural determi-
nation of the Se-oligonucleotides by X-ray crystallography
indicated that the 2′-Se modification favors the sugar pucker
with the 2′-exo conformation, which is identical to that of
A-form DNA or RNA.8 Reliable and efficient synthesis of
longer RNAs with this 2′-Se modification is desirable for
X-ray crystal structural studies of catalytic RNAs. Preparation
of longer RNAs with this Se derivatization is currently
achieved via chemical synthesis of short RNA fragements
and enzymatic ligation of these synthesized fragments.15 This
approach requires several purification steps and ligation steps,
which is labor-intensive and also suffers low yield. Obvi-
ously, the current strategy is not practical for routine synthesis
of large quantity (several miligrames) of longer RNAs, which
limits the wide applications of the Se-derivatization of longer
functional RNAs.
As the 5′-silyl-2′-ACE chemistry is currently the most
effective strategy for synthesizing longer RNAs,16,17 we have
recently explored this ACE chemistry using the previously
developed 5′-O-DMTr-2′-Se-Me uridine and cytidine phos-
phoramidites.7,10 Due to differences in the 5′-deprotection
and phosphite oxidation steps, however, these two Se-
phosphoramidites could not be conveniently used in the 5′-
silyl-2′-ACE chemistry. To overcome these shortcomings and
to meet the needs of longer Se-RNA synthesis, we have
synthesized 5′-benzhydryl-2′-methylselenophosphoramidite
(4, Scheme 1). This development allows milligram-scale
C, and G) for the ACE chemistry. To conveniently demon-
strate that 5′-BzH-2′-Se-uridine phosphoramidite 4 is com-
patible with 5′-silyl-2′-ACE chemistry, syntheses of 2′-Se-
RNAs and 2′-Se-DNAs were designed as a model system to
examine the compatibility of 4 with the reagents and
conditions used in the 5′-silyl-2′-ACE chemistry. The
synthesized Se-DNAs were also studied by NMR to obtain
the insight of duplex formation.
We report here the synthesis of the 5′-O-BzH-2′-Se-Me-
uridine phosphoramidite (4), its incorporation into oligo-
nucleotides via solid-phase synthesis, and Se-DNA duplex
study by NMR. Uridine derivative 1, synthesized previ-
ously,10 was treated with 80% acetic acid to remove the 5′-
DMTr group (96% yield), followed by protection of the 5′-
hydroxy group with benzhydryloxybis(trimethylsilyloxy)silyl
(BzH) group using BzH-Cl in dry DMF (86% yield).17
Partially protected uridine derivative 3 was converted to
the corresponding phosphoramidite 4 in dry CH2Cl2 solvent
using bis(N,N-diisopropylamino)methoxyphosphine (DI-
PAMP) in the presence of 5-(benzylthio)-1H-tetrazole (88%
yield).10, 17
The incorporation cycle of phosphoramidite 4 in oligo-
nucleotide solid-phase synthesis10 was modified in order to
successfully synthesize the 2′-Se-derivatized DNAs and
RNAs, which also served as testing models for the compat-
ibility with 5′-silyl-2′-ACE chemistry. These following modi-
fications represent the reagents and treatments in the 5′-silyl-
2′-ACE chemistry. First, 5-benzylmercapto-1H-tetrazole was
used as the reagent for the coupling reaction of phosphora-
midite 4,18 instead of 1H-tetrazole. Second, after the coupling
of Se-modified phosphoramidite 4, we examined the phos-
phite oxidation with tert-butyl hydroperoxide (BHPO) in
toluene for 45 s.17 This BHPO treatment did not cause oxida-
tion of the 2′-selenide functionality, which is consistent with
the iodine oxidation.10 MS and HPLC analyses indicated that
the selenide oxidation was not detectable (Figures 1 and 2).
Third, the 5′-BzH deprotection was performed with triethyl-
ammonium fluoride in DMF for 60 s, instead of the 5′-DMTr
deprotection with 3% trichloroacetic acid. Fourth, after the
oligonucleotide synthesis was completed, the immobilized
oligonucleotides were treated with disodium 2-carbamoyl-
2-cyanoethylene-1,1-dithiolate trihydrate17 in DMF for 15
min to remove the methoxy group on the 3′-phosphate group
of the Se-modified nucleotide, followed by water and
acetonitrile washing. Finally, the oligonucleotide RNAs and
DNAs containing the 2′-Se derivatization were cleaved off
the support and fully deprotected with concentrated ammonia
at 55 °C overnight for DNAs and with methylamine for
RNAs.10 The stability of the Se-oligonucleotides under these
conditions and treatments used in the 5′-silyl-2′-ACE chem-
istry indicates the compatibility of this Se-uridine phos-
phoramidite (4) with the ACE chemistry for RNA synthesis.
The crude 2′-Se-derivatized DNA and RNA oligonucle-
otides with DMTr-on were analyzed by HPLC. Representa-
tive reversed-phase HPLC elution profiles are shown in
Scheme 1. Synthesis of 5′-BzH-2′-Se-Me-uridine
Phosphoramidite (4) and Oligonucleotides Containing the Se
Derivatization (5)
synthesis of longer RNAs derivatized with selenium, without
ligation. Similarly, this synthesis can be extended to syn-
theses of the other three Se-derivatized phosphoramidites (A,
(15) a. Hobartner, C.; Micura, R. J. Am. Chem. Soc. 2004, 126, 1141-
1149; b. Hobartner, C.; Rieder, R.; Kreutz, C.; Puffer, B.; Lang, K.;
Polonskaia, A.; Serganov, A.; Micura, R. J. Am. Chem. Soc. 2005, 127,
12035-12045.
(16) Scaringe, S. A.; Wincott, F. E.; Caruthers, M. H, J. Am. Chem.
Soc. 1998, 120, 11820-1182.
(17) Scaringe, S. A. Methods 2001, 23, 206-217.
(18) Welz, R.; Muller, S. Tetrahedron Lett. 2002, 43, 795-797.
5646
Org. Lett., Vol. 7, No. 25, 2005

Figure 1. Reversed-phase HPLC analysis of Se-oligonucleotides.
(A) Crude DMTr-on 2′-Se-DNA (5′-DMTr-GAGCUSeCCAT-3′)
after cleavage of the oligonucleotide from the solid support and
the deprotection of the bases and backbone. The sample was
analyzed on a Zorbax SB-C18 column (4.6 × 250 mm), eluted (1
mL/min) with a linear gradient from buffer A (20 mM triethylam-
monium acetate, pH 7.1) to 100% buffer B (50% acetonitrile, 20
mM triethylammonium acetate, pH 7.1) in 30 min. Its retention
time is 20.4 min. (B) crude DMTr-on 2′-Se-RNA (5′-DMTr-
AUGGUSeGCUC-3′) after cleavage of the oligonucleotide from the
solid support and the deprotection of the bases, 2′-TOM groups,
and backbone. The sample was analyzed on the same column with
the same buffers A and B. The sample was eluted (1 mL/min) with
a linear gradient from buffer A to 100% buffer B in 45 min. Its
retention time is 35.9 min.
Figure 2. MALDI-TOF MS analysis of the Se-oligonucleotides:
(A) DMTr-off 2′-Se-DNA9mer (5′-GAGCUSeCCAT-3′; molecular
formula: C87H111N33O52P8Se), FW 2777.8; [M + H]+: 2779 (calcd
2778.8). (B) DMTr-off 2′-Se-RNA9mer (5′-AUGGUSe-GCUC-3′;
molecular formula: C86H108N32O62P8Se), FW 2908.7; [M - H]-
2907 (calcd 2907.7).
Typical MS spectra of DMTr-off DNAs and RNAs are
shown in Figure 2, and all MS data are collected in Table 1.
To understand the effect of Se modification on oligo-
nucleotide structure, we examined the consequence of a
single derivatization. The host DNA duplex forms a B-type
duplex with 9 base pairs (5′-GAGCTCCAT-3′ and 5′-
ATGGAGCTC-3′, Figure 3A).19 Figure 3 shows the imino
proton NMR spectra of the unmodified host duplex, the Se-
modified duplex and the constituent strands. The Se-modified
single strand (5′-GAGCUSeCCAT-3′) is largely unstructured
in solution (Figure 3B), while the complement strand (Figure
3C) exhibits some base pair formation by itself, indicative
of homoduplex formation. In the presence of the Se-modified
counterpart, however, the heteroduplex is formed (Figure
3D). The minor peak observed at 14.05 ppm in Figure 3D
indicates the presence of small amount of homoduplex.
Figure 1. Less than 7% short oligonucleotides were formed
on average, which suggested a high coupling yield per
synthetic cycle (higher than 99% on average). As expected,
the coupling yields of this Se-phosphoramidite in both Se-
DNA and Se-RNA syntheses are the same due to the almost
identical coupling of the Se-phosphoramidite incorporation
into both Se-DNA and Se-RNA. The synthesized oligonucle-
otides were purified twice by HPLC (DMTr-on and DMTr-
off). The reversed-phase HPLC purification was performed
by a Zorbax C18 column (21 × 250 mm), eluted (10 mL/
min) with a linear gradient from buffer A (20 mM triethyl-
ammonium acetate, pH 7.1) to 100% buffer B (50% aceto-
nitrile, 20 mM triethylammonium acetate, pH 7.1) in 30 min.
These oligonucleotides were also confirmed by MS analysis.
(19) Aramini, J. M.; Kalisch, B. W.; Pon, R. T.; van de Sande, J. H.;
Germann, M. W. Biochemistry 1996, 35, 9355-9365.
Org. Lett., Vol. 7, No. 25, 2005
5647

Table 1. MALDI-TOF MS Analytical Data of
Se-oligonucleotides
entry
a
Se-oligonucleotides
Se-DNA1
measured (calcd) m/z
[M + H]+: 2779 (2778.8)
(5′-GAGCUSeCCAT-3′)
C87H111N33O52P8Se:
FW 2777.8
Se-DNA2
nontreated with BHPO
(5′-ATGGUSeGCTC-3′)
88H112N32O54P8Se:
b
c
[M + H]+: 2810 (2809.9)
[M + H]+: 2810 (2809.9)
C
FW 2808.9
Se-DNA2
treated with BHPO
(5′-ATGGUSeGCTC-3′)
C88H112N32O54P8Se:
FW 2808.9
Se-DNA3
nontreated with BHPO
(5′-CCTUSeGACAAAG-3′)
d
e
f
[M + H]+: 3405 (3400 avg)
[M + Na]+: 3427 (3422 avg)
Figure 3. NMR study of Se-DNA, the complementary strand, and
Se-derivatized DNA duplex (100 µM each sequence) in 50 mM
NaCl, 0.1 mM EDTA, 10 mM NaH2PO4-Na2HPO4 (pH 6.5) at
283 K: (A) NMR spectrum of the unmodified heteroduplex as a
control; (B) NMR spectrum of 5′-GAGCUSeCCAT-3′ single strand;
(C) NMR spectrum of 5′-ATGGAGCTC-3′ single strand; (D) NMR
spectrum of the modified heteroduplex.
C
107H135N43O62P10Se:
FW 3404.2
Se-DNA3
[M + H]+: 3405 (3400 avg)
[M + Na]+: 3427 (3422 avg)
treated with BHPO
(5′-CCTUSeGACAAAG-3′)
C107H135N43O62P10Se:
FW 3404.2
Se-RNA9mer
treated with BHPO
(5′-AUGGUSeGCUC-3′)
[M + H]-: 2907 (2907.7)
[M - H]-: 3549 (3549.1)
this phosphoramidite (4) and the synthesized 2′-Se-oligo-
nucleotides are stable and compatible with the reagents used
in the 5′-silyl-2′-ACE chemistry for RNA synthesis. As
longer Se-RNA synthesis with satisfactory quantity and
quality is desired in studying functional RNAs, this phos-
phoramidite (4) meets the need for site-specific RNA
derivatization with selenium in X-ray crystal structure study
of longer RNAs.20
C86H108N32O62P8Se:
FW 2908.7
g
Se-RNA11mer
treated with BHPO
(5′-CCUUSeGACAAAG-3′)
C106H133N43O72P10Se:
FW 3550.1
Our NMR data demonstrate the formation of a DNA
duplex structure in aqueous solution between the 2′-Se-U
modified sequence and its unmodified complementary strand.
The USe-A base pair formation is consistent with our
previous crystal structure study.8 Further NMR experiments
will provide conformation parameters of 2′-Se derivatized
nucleotides in solution and will also help in determining
optimal positions of Se derivatization for nucleic acid X-ray
crystal structure study. Moreover, our results have suggested
that the 2′-Se labels can be useful in NMR spectrum
assignment and structure determination, especially for longer
RNAs (over 60 nt). The selenium derivatization of DNA and
RNA will significantly facilitate structure and mechanism
studies of nucleic acids.
Although the Se-modified duplex is thermodynamically less
stable than the control duplex, we clearly observe the
presence of nine base pairs. The imino protons of the 5 G-C
base pairs resonate at 12.6-12.9 ppm, while the 3 A-T and
1 A-USe base pairs are in the region of 13-14.3 ppm. As
anticipated, the spectrum of the control duplex (5 G-C and
4 A-T, Figure 3A) is similar to the modified duplex. In
particular, the chemical shifts of 1, 2, 8 A-T base pairs
remain the same. While the chemical shift of the no. 5 A-T
imino proton in native duplex is at 13.9 ppm, the modified
A-USe base pair is at 13.7 ppm. This chemical shift
demonstrates that the central Se-modified U is capable of
base pairing with A in the context of a B type host duplex.
This comparison of the Se-modidified and control duplexes
reveals that the Se-modification does not cause significant
perturbation, which is consistent with the UV-melting
temperature study.12 Further NMR study will reveal the
structural nature of the modified duplex.
Acknowledgment. This work was supported by the GSU
Research Program and the National Institutes of Health
(GM069703). We thank Dr. Abdalla Hassan and Julianne
Caton-Williams for carefully reading this manuscript.
Supporting Information Available: Experimental pro-
cedures, 1H and 13C NMR, HPLC, and HRMS analytical data.
This material is available free of charge via the Internet at
http://pubs.acs.org.
In conclusion, we have synthesized the 2′-Se-uridine
phosphoramidite (4). More importantly, we have successfully
incorporated it into oligonucleotides with a high coupling
yield (over 99%). As expected, the 2′-Se functionality is
stable under conventional oligonucleotide synthesis, indicated
by HPLC and MS analyses. We have also demonstrated that
OL052270Y
(20) Holbrook, S. R. Curr. Opin. Struct. Biol. 2005, 15, 302-308.
Org. Lett., Vol. 7, No. 25, 2005
5648