Biomedical Engineering Reference
In-Depth Information
Synthesis of 2
0
-Se-derivatized uridine phosphoramidite, DNAs, and RNAs. (
a
)
Scheme 3.2
(
Ms
Methanesulfonyl) MsCl, THF, Et
3
N; 95%. (
b
) Toluene, tetrahexylammonium hydrogen
sulfate,
D
Na
2
CO
3
(sat.).
(
c
)(Bu)
4
NF;
95%.
(
d
)
NaHSe,
then
MeI,
or
NaSeMe;
96%.
(
e
)
PCl(OCH
2
CH
2
CN)N(i-Pr)
2
; 92%. (
f
) Solid-phase synthesis
nucleophile (NaHSe or NaSeCH
3
). The nucleoside was also successfully introduced
into oligonucleotide by standard phosphoramidite chemistry and automated solid-
phase DNA synthesis (Scheme
3.2
).
Through the collaboration between Huang and Egli research labs, we suc-
cessfully determined the first structure of nucleic acid based on selenium MAD
phasing by selenium derivatization [
25
,
26
]. The crystal of the Se-modified decamer
(5
0
-GCGTA
U
2
0
Se
ACGC-3
0
)
2
(PDB ID: 1MA8) (shown in Fig.
3.2
) diffracted to
1.3 A resolution. The modified nucleoside maintains a C3
0
-endo sugar pucker in
an A-form duplex. In-depth study of crystal structure revealed that the 2
0
-MeSe
(2
0
-Se) groups located in the minor groove of DNA. Even though selenium is more
hydrophobic than oxygen, there is no significant change on minor groove hydration
as well as the overall structure when compared to native. This was the first proof of
principle that selenium-derivatized nucleic acid in combination with MAD or SAD
can largely facilitate structure determination of nucleic acid.
Later study [
27
] further proved the principle by directly comparing 2
0
-SeMe-U
DNA with the bromo-derivatization (5-Br-uridine), which is the currently conven-
tional phasing strategy for nucleic acids. From the crystallographic point of view,
the Se-DNA (5
0
-G
U
2
0
Se
GTACAC-3
0
)
2
(PDB ID: 1Z7I) showed many advantages
over the Br-DNA (5
0
-
GTGU
5-Br
ACAC-3
0
)
2
(PDB ID: 2H05), including less local
structure perturbation, higher resolution (1.28
˚
Avs.1.8 A), and faster crystal
growth.
In order to allow large-scale synthesis of 2
0
-Se-U, an improved synthetic method
was developed by utilizing a one-step cyclization reaction to convert uridine to
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