Synthesis and solution conformation studies of 3-substituted uridine and pseudouridine derivatives

Article abstract of DOI:10.1016/j.bmc.2007.11.039

A series of 3-substituted uridine and pseudouridine derivatives, based on the naturally occurring 3-(3-amino-3-carboxypropyl) modification, were synthesized. Their aqueous solution conformations were determined by using circular dichroism and NMR spectroscopy. Functional group composition and chain length were shown to have only a subtle influence on the distribution of syn/anti conformations of the modified nucleosides. The dominating factor appears to be the glycosidic linkage (C- vs. N-glycoside) in determining the nucleoside conformation.

Full text of DOI:10.1016/j.bmc.2007.11.039

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2676–2686
Synthesis and solution conformation studies of 3-substituted
uridine and pseudouridine derivatives
Yu-Cheng Chang, Jayatilake Herath, Tony H.-H. Wang and Christine S. Chow*
Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI 48202, USA
Received 24 September 2007; revised 9 November 2007; accepted 13 November 2007
Available online 19 November 2007
Abstract—A series of 3-substituted uridine and pseudouridine derivatives, based on the naturally occurring 3-(3-amino-3-carboxy-
propyl) modification, were synthesized. Their aqueous solution conformations were determined by using circular dichroism and
NMR spectroscopy. Functional group composition and chain length were shown to have only a subtle influence on the distribution
of syn/anti conformations of the modified nucleosides. The dominating factor appears to be the glycosidic linkage (C- vs. N-glyco-
side) in determining the nucleoside conformation.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
other RNAs. The role of acp3U is still unclear, since
the translation process is not affected by the presence
or absence of this modified nucleoside in tRNA.11 The
biological function of the related derivative m1acp3W is
also not known. This hypermodified nucleoside is found
in 18S rRNAs of eukaryotes,8,9,12,13 including humans
and yeast. It is also the only known modified nucleoside
in helix 31 of 18S rRNA in eukaryotes.14 A methylated
pseudouridine derivative (m1W) was found to be a
metabolite in Streptomyces platensis, then later discov-
ered in tRNA of archaebacteria.15,16 A 20-O-methyl-
pseudouridine derivative (Wm) is found in 18S and 28S
wheat embryo tRNAs and rRNAs.17
Modified nucleosides have the ability to regulate the
function, stability, or structures of RNA.1,2 Over 100
different post-transcriptional modifications occur in nat-
ural RNAs.3 Synthesis of the individual modified
nucleosides is useful in order to examine the unique
influences of the modifying groups on base and/or sugar
conformation.4 Modified nucleosides also have the abil-
ity to function as antiviral reagents by interfering with
polymerases or reverse transcriptase;5 however, a large
number of synthetic analogues have low activity or
excessive toxicity. Therefore, it is important to deter-
mine the level of conformational flexibility of the modi-
fied nucleosides and to correlate this information with
active site binding to a target protein or enzyme in order
to design better antiviral agents.6
In this study, a series of 3-substituted uridine and
pseudouridine analogues (acp3U, m1acp3W, 3-(4-ami-
no-4-carboxybutyl)uridine or acb3U, 3-(3-carboxypro-
pyl)uridine or cp3U, 3-(4-hydroxybutyl)uridine or
hb3U, and 1-methyl-3-(4-hydroxybutyl)pseudouridine
or m1hb3W, Fig. 1) were prepared by using a Mitsunobu
reaction in the key step, and their solution conforma-
tions were examined by using a variety of biophysical
techniques, namely circular dichroism (CD) and NMR
nuclear Overhauser effect (NOE) spectroscopy. The uri-
dine analogue cp3U was synthesized and compared with
acp3U to better understand the role of the homoserine
amino group in influencing sugar/base conformation.
For the hb3U and m1hb3W derivatives, the homoserine
carboxylate and amino groups were replaced with a
hydroxyl group. The acb3U derivative was prepared to
study the role of the amino acid side chain length (propyl
vs. butyl). In addition, two methylated pseudouridine
Methylation is the most common and simple nucleoside
modification.7 More complex modifications, such as
L-homoserine addition, are found in both uridine
(3-(3-amino-3-carboxypropyl)uridine, or acp3U) and
pseudouridine (1-methyl-3-(3-amino-3-carboxypropyl)
pseudouridine, or m1acp3W) at the N3 position in natu-
ral RNA sequences.8,9 Early studies revealed the pres-
ence of acp3U in Escherichia coli tRNAPhe
,
as well as
10
Keywords: Pseudouridine; Uridine; Homoserine; Methylation;
Conformation.
*
Corresponding author. Tel.: +1 313 577 2594; fax: +1 313 577
8822; e-mail: csc@chem.wayne.edu
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.039

Y.-C. Chang et al. / Bioorg. Med. Chem. 16 (2008) 2676–2686
2677
H
O
O
O
N
N
N
NH
NH
N
+
NH3
O
O
O
HO
HO
HO
O
O
O
-
CO2
HO OH
HO OH
HO OH
m1ψ
m1acp3ψ
β-pseudouridine (ψ)
H
O
N
O
O
N
NH
N
N
+
N
O
O
O
NH3
HO
HO
HO
O
O
O
-
CO2
OH
HO OCH3
HO OH
HO OH
acp3U
ψm
m1hb3ψ
O
O
O
N
N
N
O
N
N
O
N
O
HO
HO
HO
O
O
O
O-
-
CO2
+H3N
OH
HO OH
HO OH
O
HO OH
cp3U
acb3U
hb3U
Figure 1. The structures of pseudouridine (W), 1-methylpseudouridine (m1W), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (m1acp3W), 20-
O-methylpseudouridine (Wm), 1-methyl-3-(4-hydroxybutyl)pseudouridine (m1hb3W), 3-(3-amino-3-carboxypropyl)uridine (acp3U), 3-(4-amino-4-
carboxybutyl)uridine (acb3U), 3-(4-hydroxybutyl)uridine (hb3U), and 3-(3-carboxybutyl)uridine (cp3U) are shown.
derivatives, 1-methylpseudouridine (m1W) and 20-O-
methylpseudouridine (Wm), were prepared for compari-
son with W and m1acp3W.
and coupled with protected 1-methylpseudouridine 7
using the Mitsunobu reaction to give compound 10 in
>90% yield. The deprotection of compound 10 was
accomplished by saponification of the ester with
0.67 N NaOH(aq) and acid hydrolysis with TFA/H2O
(9/1) to give compound 11, m1acp3W (Scheme 1).
2. Results and discussion
2.1. Synthesis of pseudouridine derivatives
The m1hb3W derivative (deamino and decarbonyl ana-
logue of m1acp3W) was synthesized in four steps using
the same procedure as for compound 11. Prior to cou-
pling, commercially available 1,4-butanediol was
monosilylated with tert-butyldimethylsilyl chloride to
give compound 12 in 68% yield (Supplementary,
Scheme S1). The alcohol derivative 12 was then re-
acted with protected 1-methylpseudouridine 7 to give
compound 13 in 77% yield (Scheme 1). The silyl pro-
tecting groups were removed with tetrabutylammo-
nium fluoride (TBAF) followed by hydrolysis under
acidic conditions to afford the desired product 15.
The 20-O-methylpseudouridine (Wm) derivative was
prepared from pseudouridine following a literature
procedure.25
Pseudouridine was synthesized from compound 1,18
which was stereoselectively reduced with ZnCl2 and L-
selectride followed by cycloetherification using the Mits-
unobu reaction as adapted from Hanessian and
Machaalani19 to give the protected nucleoside 3 in
>80% yield (Scheme 1). The pyrimidine protective
groups were removed by sodium iodide treatment under
acidic and refluxing conditions to give compounds 4 and
5, followed by sugar deprotection with trifluoroacetic
acid (9/1 TFA/H2O) solution20 to give b-pseudouridine
6 (Scheme 1).
1-Methylpseudouridine was prepared from compound 5
(Scheme 1), which was treated with the bulky base N,O-
bis(trimethylsilyl)acetamide (BSA) and iodomethane
under refluxing conditions or at room temperature to
selectively methylate the N1 position.21,22 The protective
groups of compound 7 were removed under acidic con-
ditions to give the desired 1-methylpseudouridine (m1W)
8 in 90% yield. Next, the Mitsunobu reaction23 was uti-
lized to couple a suitable amino acid or alcohol moiety
to compound 7 and generate the 3-substituted deriva-
tives. The main advantage of this approach is that the
reaction is carried out under mild conditions with high
efficiency and without any side reactions on the homo-
serine or alcohol functionalities. A boc-protected homo-
serine methyl ester 9 was prepared from commercially
available homoserine using a literature procedure,24
2.2. Synthesis of uridine derivatives
Synthesis of the modified uridine analogues began with
commercially available uridine. The 20- and 30-hydroxyl
groups of uridine were protected with isopropylidene, to
generate intermediate 16, followed by 50 silylation with
tert-butyldiphenylsilyl chloride and imidazole to give
uridine derivative 17 (Supplementary, Scheme S2).26
Synthesis of 3-(3-amino-3-carboxypropyl)uridine (acp3U)
followed the protocol for synthesis of m1acp3W.20,24 The
protected uridine 17 and homoserine derivative 9 were
coupled under Mitsunobu conditions to give 18 in
>90% yield. The two-step deprotection employed
0.67 N NaOH(aq) followed by treatment with TFA/

2678
Y.-C. Chang et al. / Bioorg. Med. Chem. 16 (2008) 2676–2686
OMe
OMe
N
N
N
N
OH
O
OH OCH3
N
TBDPSO
TBDPSO
ii
O
i
OMe
TBDPSO
OMe
OH
O
O
O O
N
OCH3
O
O
2
3
1a and 1b
H
H
O
H
N
O
N
O
N
NH
NH
NH
O
O
v
HO
iii
O
TBDPSO
TBDPSO
+
O
O
O
HO OH
β-pseudouridine (Ψ)
O
O
HO OH
4
5
6
iv
5%
95%
vi
O
N
O
N
N
O
NH
TBDPSO
x
12
O
O
TBDPSO
vii
O
ΟΤΒS
O
O
O
O
O
O
N
NH
13
7
O
xi
HO
viii
O
9
O
HO OH
m1Ψ
O
N
N
N
8
O
N
HO
O
NHBoc
TBDPSO
O
O
OH
O
CO2Me
O
O
14
10
ix
xii
O
O
N
N
N
N
NH2
O
O
HO
HO
O
O
CO2H
OH
HO OH
HO OH
m1acp3Ψ
m1hb3Ψ
15
11
Scheme 1. Reagents and conditions: (i) ZnCl2, L-selectride, CH2Cl2, ꢀ72 ꢁC, 19 h, 92% yield; (ii) DIAD, PPh3, THF, 0 ꢁC to rt, 19 h, 91% yield; (iii)
CH3COOH, NaI, reflux, 35 min; (iv) concd H2SO4, acetone, rt, 1 h, 94%; (v) TFA/H2O (9/1), 1 h, rt, 100%; (vi) a—BSA (2.5 equiv), CH2Cl2, rt, 1 h;
b—CH3I (1.5 equiv), reflux, 120 h, 80%; (vii) TFA/H2O (9/1), 1 h, rt, 90%; (viii) 9, DIAD, PPh3, THF, rt, 1 h, 94%; (ix) a—0.67 N NaOH(aq),
dioxane, rt, 25 min; b—TFA/H2O (9/1), 1 h, rt, 92% in two steps; (x) 12, DIAD, PPh3, THF, rt, 1 h, 77%; (xi) tetrabutylammonium fluoride, THF, rt,
2.5 h, 89%; (xii) TFA, H2O/acetone (9/1, v/v), rt, 2 h, 99%.
H2O to give the desired acp3U 19 (Supplementary,
Scheme S3).
oxidized to the corresponding carboxylate 25 in high
yield using pyridinium dichromate (PDC).28 The protec-
tive groups were removed under acidic conditions (TFA/
H2O) to give the desired product 26 in good yield (71%
from compounds 17 and 22).
To investigate the relationship between the functional
group modification and nucleoside conformation, a ser-
ies of uridine derivatives were synthesized. The homo-
logue 3-(4-amino-4-carboxybutyl)uridine (acb3U) was
synthesized to study the role of amino acid chain length.
A protected amino alcohol was used in the coupling
reaction. Compound 2027 was treated with benzoyl chlo-
ride to protect the 1 position, followed by desilylation to
give the alcohol derivative 22 (Scheme 2). Compound 22
was coupled with uridine derivative 17 followed by re-
moval of the benzoyl group under basic conditions
(>90% yield) (Scheme 3). The resulting alcohol 24 was
The 3-(4-hydroxybutyl)uridine (hb3U) was synthesized
to determine the effects of removing the amino and
carbonyl groups of acp3U on nucleoside conforma-
tion. Synthesis of hb3U employed the same protocol
as m1hb3W synthesis. Protected uridine 17 was used
in place of m1W (7, Scheme 1), and coupled with 12
to give protected hb3U (27) in 92% yield (Supplemen-
tary, Scheme S4). The silyl protecting groups were re-
moved by treatment with TBAF followed by

Y.-C. Chang et al. / Bioorg. Med. Chem. 16 (2008) 2676–2686
2679
NHBoc
OBz
22
NHBoc
OBz
NHBoc
OH
ii
i
HO
TBDPSO
TBDPSO
20
21
Scheme 2. Reagents and conditions: (i) benzoyl chloride, pyridine, rt, 16 h, 98%; (ii) tetrabutylammonium fluoride, THF, rt, 1.5 h, 83%.
O
O
N
N
N
N
O
O
i
v
30
TBDPSO
TBDPSO
TBDPSO
17
O
O
22
OH
O
O
O
O
BocHN
OBz
23
31
ii
vi
O
N
O
N
N
N
O
O
TBDPSO
TBDPSO
O
O
OH
O
O
O
O
BocHN
O
OH
24
O
32
iii
vii
O
N
O
N
N
N
O
O
HO
O
CO2H
HO
O
O
BocHN
OH
HO OH
cp3U
O
25
iv
O
33
N
N
O
O
CO2H
HO OH
H2N
acb3U
26
Scheme 3. Reagents and conditions: (i) 22, DIAD, PPh3, THF, rt, 1 h, 96%; (ii) NaOMe, MeOH, rt, 16 h, 84%; (iii) PDC, DMF, rt, 20 h, 94%; (iv)
TFA/H2O (9/1), 1 h, rt, 94%; (v) a—30, DIAD, PPh3, THF, rt, 1 h; b—c TFA, CH3Cl, rt, 30 min, 51% yield in two steps; (vi) PDC, DMF, rt, 20 h,
80%; (vii) TFA/H2O (9/1), 2 h, rt, 90%.
hydrolysis under acidic conditions to afford the de-
sired product 29.
2.3. Circular dichroism studies of modified nucleosides
The solution conformations of the modified pseudouri-
dine and uridine nucleosides were studied by using circu-
lar dichroism (CD) spectroscopy. The CD spectrum of
pseudouridine (W) has a peak minimum at 276 nm
(Fig. 2), consistent with previous studies.30 This key fea-
ture is suggestive of a syn conformation for pseudouri-
dine.31 In the case of m1W, a peak minimum occurs at
281 nm, also consistent with the syn orientation. The
5 nm shift in the peak minimum compared to W is likely
due to the presence of the methyl group, and is similar to
that observed with m3W (282 nm).32 Overall, the pres-
ence of the methyl group at the N1 position does not ap-
pear to influence the preference of W for the syn
conformation. In contrast, the modified nucleoside
m3U prefers the anti conformation,32 consistent with
the fact that molecular orbital interactions play a role
in the nucleoside conformation.4 It has been implicated
that the orbital of the H5–H6 double bond in the pyrim-
idine ring of uridine has interactions with the lone pair
of the sugar O40 which results in an anti conformational
preference.33,34 The orbital overlap would clearly be dif-
To further utilize the Mitsunobu reaction, the acid deriv-
ative cp3U was synthesized from a dimethoxytrityl pro-
tected alcohol and protected uridine. 1,4-Butanediol
was treated with dimethoxyltrityl chloride to give mono-
protected compound 30 (Supplementary, Scheme S5).
This compound was coupled with uridine derivative 17
under Mitsunobu conditions followed by removal of
the dimethoxytrityl group to give alcohol 31 in one-pot
(Scheme 3). The dimethoxytrityl protecting group was
used on 1,4-butanediol, because it is acid labile and can
be removed without affecting the fluoride-labile 50-tert-
butyldiphenylsilyl group on uridine. The alcohol deriva-
tive 31 was oxidized to the corresponding acid with PDC
to give carboxylic acid 32. The protective groups were
removed in one step under acidic conditions to give the
desired product 33 (Scheme 3). Although the synthesis
of 3-(3-carboxypropyl)uridine (cp3U) was previously
reported by Seela and coworkers,29 we chose to use the
Mitsunobu reaction to extend the substrate scope and
further demonstrate the generality of the method.

2680
Y.-C. Chang et al. / Bioorg. Med. Chem. 16 (2008) 2676–2686
modified W nucleosides are predominately syn. This
2
1
A
study shows that the conformation of pseudouridine is
not significantly influenced by the presence of functional
groups on the pyrimidine ring (N1 or N3) or sugar moi-
ety, and that the dominating contribution comes from
the glycosidic linkage between the base and ribose (i.e.,
C- vs. N-glycoside).
0
-1
-2
-3
-4
-5
-6
Similarly, N3 modifications have minimal effects on the
CD spectra of the uridine derivatives (Fig. 3). The spec-
trum for the modified nucleoside acp3U has a peak max-
imum at 269 nm, and the other uridine modified
nucleosides, acb3U, cp3U, and hb3U, show CD peak
maxima at 268 nm. For comparison, the uridine spec-
trum also has a peak maximum at 268 nm. These data
suggest that the uridine nucleosides are all predomi-
nately in the anti conformation in solution. The shapes
of the CD spectra of the uridine analogues are similar,
suggesting that the functional groups do not make a sig-
nificant contribution to the conformational preference.
The derivatives of pseudouridine and uridine consis-
tently show opposite absorption bands due to the C–C
and C–N linkages between the sugar and base, which
lead to different molecular electronic dipole and molec-
ular magnetic dipole transition moments. The opposing
CD spectra for the pseudouridine and uridine deriva-
tives are in agreement with previous results.31,35 In addi-
tion, the CD spectra of the naturally modified
nucleosides, m1W, m1acp3W, and acp3U, do not show
significant changes with pH (data are not shown), pro-
viding further support that the amino and carboxyl
functionalities have little influence on the nucleoside
conformations.
____ Ψ
_ _ _ m1Ψ
_ _ _ m1acp3Ψ
260
270
280
290
300
310
320
wavelength, nm
2
1
B
0
-1
-2
-3
-4
-5
-6
____ Ψ
_ _ _ m1Ψ
_ _ _ m1hb3Ψ
260
270
280
290
300
310
320
wavelength, nm
C
2
1
2.4. NMR studies of uridine and pseudouridine derivatives
NMR NOE experiments were done to study the effects
of modification on the base orientation and sugar puck-
er. The syn and anti conformations of the bases can be
determined by four protons (H6, H10, H20, and
H30).36,37 NOE experiments on the pseudouridine deriv-
atives were done in D2O at room temperature. The uri-
dine derivatives were studied in D2O at 40 ꢁC because of
overlap between the H10 and H5 peaks at the lower tem-
perature. The syn conformation is expected to give a
strong NOE at H10 when H6 is irradiated. In contrast,
the same irradiation typically gives a strong NOE to
H20 and H30 if the nucleoside is in the anti conformation
(Fig. 4).
0
-1
-2
-3
-4
-5
-6
____ Ψ
_ _ _ m1Ψ
_ _ _ Ψm
260
270
280
290
300
310
320
wavelength, nm
Figure 2. CD spectra of m1acp3W (A), m1hb3W (B), and Wm (C)
(dashed black lines) in H2O are compared with W (solid lines) and m1W
(dashed gray lines) at room temperature. The molar ellipticities were
normalized from concentrations of 10, 60, and 20 mM, respectively,
based on an extinction coefficient of 1.09 · 104 cmꢀ1 Mꢀ1 at 260 nm.
Each curve represents the average of five scans.
The derivatives m1W, m1acp3W, and m1hb3W show
stronger H10 NOE effects when H6 is irradiated com-
pared to W (Table 1), revealing a preference for the
syn conformation. The strongest H10 NOE effect is ob-
served for m1W, suggesting that the syn conformation
is more highly favorable for this nucleoside. These data
are consistent with the results from CD spectroscopy.
The observation of weaker NOEs between H10 and H6
of Wm reveals that the syn conformation is not as
strongly preferred for the 20-O-methylated derivative.
In contrast, strong NOE effects at H20 and H30 are ob-
served for acp3U, acb3U, and hb3U upon irradiation at
H6 (Table 1). This suggests that the anti conformation is
ferent for W and corresponding derivatives, due to the
C-glycosidic linkage and altered spacial arrangement
of the pyrimidine ring. The CD spectra of the modified
nucleosides m1acp3W, m1hb3W and Wm (Fig. 2) show
peak minima at 280, 279, and 276 nm, respectively.
Our results reveal that the conformations of the four

Y.-C. Chang et al. / Bioorg. Med. Chem. 16 (2008) 2676–2686
2681
A
C
B
25
25
20
15
10
5
20
15
10
5
0
0
-5
-5
____
_ _ _
____
U
U
acp U
3
3
-10
-10
_ _ _
acb U
-15
210
-15
210
230
250
270
290
310
230
250
270
290
310
wavelength, nm
wavelength, nm
D
25
20
15
10
5
25
20
15
10
5
0
0
-5
-5
____
_ _ _
-10
-15
-20
-10
-15
-20
____
_ _ _
U
cp U
U
3
hb3U
210
230
250
270
290
310
210
230
250
270
290
310
wavelength, nm
wavelength, nm
Figure 3. CD spectra of acp3U (A), acb3U (B), cp3U (C), and hb3U (D) (dashed lines) in H2O are compared with U (solid lines) at room temperature.
The molar ellipticities were normalized from concentrations of 10, 14, 37, and 17 mM, respectively, based on an extinction coefficient of
1.33 · 104 cmꢀ1 Mꢀ1 at 260 nm. Each curve represents the average of five scans.
O
O
H
H
N
H
N
H
N
N
H(3') H(6)
O
H(3')
O
H(6)
HOH2C
H(4')
HOH2C
H(4')
H(2')
H(2')
O
O
H(1')
H(1')
O(3')
O(3')
O(2')
O(2')
syn
anti
H(3')
Ψ
Ψ
H(2')
HOH2C
H(4')
HOH2C
H(2')
O(2')
H(3')
O
O
H(1')
O(3')
H(1')
O(2')
H(4')
O(3')
C3'-endo (N)
C2'-endo (S)
0
Figure 4. The possible conformations (anti and syn; C3 -endo(N) and C2 -endo(S)) of pseudouridine, which can be determined by 1D NOE
0
experiments, are represented.
preferred for these nucleosides, which again is in agree-
ment with the CD data. The cp3U nucleoside shows
stronger H10 and weaker H20 and H30 NOE effects than
the other modified uridines. Thus, the anti conformation
is less preferred for cp3U compared to the other modi-
fied uridines. Also, the H10 to H6 NOEs are greater
for m1W, W, and m1hb3W compared to m1acp3W. This
trend suggests that the syn conformation is slightly more
favored when N3 substituents are less bulky or absent.
A similar trend is observed with the uridine derivatives
in which the more bulky substituents more strongly pre-
fer the anti conformation.

2682
Y.-C. Chang et al. / Bioorg. Med. Chem. 16 (2008) 2676–2686
Table 1. Irradiation and NOE data for pseudouridine at 25 ꢁC and uridine derivatives at 40 ꢁC
Compound
Proton irradiated
H6
NOE (%)
Compound
Proton irradiated
H6
NOE (%)
W
H10 (5.8)
H20 + H30 (1.9)
H6 (5.9)
U
anti
H10 (4.3)
H20 + H30 (10.2)
H6 (4.5)
syn
H10
H20
H30
H10
H20
H30
H6 (2.2)
H6 (0.8)
H6 (6.9)
H6 (2.5)
m1W
syn
H6
H10 (9.1)
H20 + H30 (2.4)
H6 (7.6)
acp3U
anti
H6
H10 (4.0)
H20 + H30 (9.7)
H6 (4.0)
H10
H20
H30
H10
H20
H30
H6 (2.6)
H6 (2.1)
H6 (6.7)
H6 (2.1)
m1acp3W
syn
H6
H10 (6.5)
H20 + H30 (3.6)
H6 (5.1)
acb3U
anti
H6
H10 (5.0)
H20 + H30 (9.2)
H6 (4.9)
H10
H20
H30
H10
H20
H30
H6 (2.2)
H6 (0.7)
H6 (7.3)
H6 (2.1)
m1hb3W
syn
H6
H10 (7.9)
H20 + H30 (4.3)
H6 (5.5)
hb3U
anti
H6
H10 (4.4)
H20 + H30 (8.1)
H6 (4.0)
H10
H20
H30
H10
H20
H30
H6 (2.5)
H6 (0.5)
H6 (5.5)
H6 (1.9)
Wm
syn
H6
H10 (3.5)
H20 + H30 (1.2)
H6 (1.4)
ND
cp3U
anti
H6
H10 (5.2)
H20 + H30 (7.5)
H6 (4.8)
H10
H20
H30
H10
H20
H30
H6 (6.5)
H6 (2.1)
ND
ND, not detectable.
0
0
0
Table 3. Percentages of C3 -endo (N) versus C2 -endo (S) conformers
and equilibrium constants (N/S) for pseudouridine and uridine
derivatives at 25 ꢁC
The ribose moieties of RNA normally adopt C2 -endo
0
(S) or C3 -endo (N) conformations (Fig. 4). The confor-
mations or sugar puckers can be defined by the
JH1 ꢀH2 and JH3 ꢀH3 coupling constants.37 The per-
centage of these two conformers can be derived from
0
0
Compound
% C3 -endo (N)
% C2 -endo (S)
Keq (N/S)
0
0
0
0
Wa
48
46
48
52
43
53
60
55
58
58
52
54
52
48
57
47
40
45
42
42
0.9
0.9
0.9
1.1
0.8
1.1
1.5
1.2
1.4
1.4
0
m1W
0
0
0
0
0
0
the0 equations: [C2 -endo] = J 1 ,2 /(J1 ,2 + J3 ,4 ) and
0
[C3 -endo] = 1-[C2 -endo]. The coupling constants for
the modified nucleosides are shown in Table 2. The uri-
m1acp3Wa
m1hb3W
Wm
0
dine derivatives show a slight preference for C3 -endo
Ua
(N) conformers (55–60%) compared to pseudouridine
derivatives (43–52%) (Table 0 3). The C-glycoside
nucleosides normally favor C2 -endo (S) compared to
N-glycoside nucleosides due to the decreased anomeric
effect.4,38 The conformation of sugars in the W and U
analogues is in agreement with previous conforma-
tional studies of other C–C and C–N nucleo-
sides.33,36,39 The exception is m1hb3W, which has a
Keq (N/S) value of 1.1.
acp3Ua
acb3U
hb3U
cp3U
aRefs. 35 and 36.
3. Conclusions
A simple and efficient experimental procedure for syn-
thesizing 3-substituted uridine and pseudouridine deriv-
atives has been presented. The Mitsunobu coupling
reaction between a simple alcohol and nucleoside gives
excellent yields (over 90% yield) and high efficiency com-
pared to the somewhat harsher conditions using bromi-
nated or tosylated substrates (typically ꢁ70%
yields).10b,35 A number of recent studies have shown that
modification of nucleobases can be achieved in an effi-
cient and practical way using the Mitsunobu reaction.39
The mild reaction conditions of this procedure are
highly tolerated by a broad range of nucleoside func-
tional or modified groups. Furthermore, the highly
Table 2. 1H–1H coupling constants for pseudouridine and uridine
derivatives at 25 ꢁC
0
J1 ,2
0
0
J2 ,3
0
0
J3 ,4
0
0
J4 ,5
0
0
J4 ,5
0 0
Compound
m1W
5.6
5.5
5.0
6.4
5.2
5.0
5.5
5.2
4.8
5.0
5.5
4.8
3.2
3.0
3.5
3.2
4.4
3.5
4.5
4.4
m1acp3W
m1hb3W
Wm
acp3U
acb3U
hb3U
cp3U
4.0
4.5
4.0
4.0
5.5
5.5
6.0
5.5
6.0
5.5
5.5
5.5
3.0
3.0
3.0
3.0
4.5
4.5
4.5
4.5

Y.-C. Chang et al. / Bioorg. Med. Chem. 16 (2008) 2676–2686
2683
water-soluble phosphine reagent can be used to facilitate
the purification process.40 The CD and NMR NOE
studies allow the syn and anti conformational assign-
ments for the modified nucleosides to be made and com-
pared. In m1W, the N1 methyl group provides steric
constraints to suppress the anti conformation and the
syn conformation is highly favored. This was observed
in circular dichroism spectra and NMR NOE studies.
The functional group and length of the side chain at
N3 have only a slight influence on the syn/anti confor-
mations of the modified uridine and pseudouridine
nucleosides examined in this study. The conformation
of the ribose sugars was also investigated. The C-C
drous THF (200 mL) at 0 ꢁC under argon. The reaction
was slowly warmed to rt and stirred for 19 h. The sol-
vent was removed under reduced pressure. The residue
was purified by column chromatography using EtOAc/
hexane (15–30%) to give 3 (1.79 g, 91%) as a colorless
1
oil: Rf 0.58 (EtOAc/hexane 1:1); H NMR (400 MHz,
CDCl3) d 8.30 (s, 1H), 7.66 (m, 4 H), 7.39 (m, 6H),
4.98 (d, J = 4.0 Hz, 1H), 4.71 (dd,J = 6.4, 4.8 Hz, 1H),
4.65 (dd,J = 6.4, 4.8 Hz, 1H), 4.13 (m, 1H), 3.98 (s,
3H), 3.94 (s, 3H), 3.89 (m, 1H), 3.82 (dd, J = 11.2,
4.8 Hz, 1H), 1.59 (s, 3H), 1.35 (s, 3H), 1.06 (s, 9H);
13C NMR (400 MHz, CDCl3) d 168.8, 165.4, 157.2,
135.9, 135.8, 133.41, 133.37, 130.00, 129.97, 128.0,
114.5, 113.4, 85.4, 84.9, 81.8, 80.6, 77.5, 64.2, 55.1,
54.3, 27.9, 27.1, 25.9, 22.0, 19.5; ESI-MS (ES+) m/z calcd
for C30H38N2O6Si 550.25, found 551.19 (M+H+), 573.15
(M+Na+); HRMS calcd for C30H38N2O6Si (M+ꢀC4H9)
493.1795, found 493.1790.
nucleosides show a slightly higher population of the
0
0
C2 -endo (S) conformation, whereas the C3 -endo (N)
conformation is slightly more favored for C–N nucleo-
sides. This observation is in agreement with the confor-
mation studies of other C–C and C–N nucleosides. The
development of efficient syntheses of modified nucleo-
sides will allow for generation of phosphoramidites or
nucleotide triphosphates, followed by incorporation
onto oligoribonucleotides for further biophysical studies
of modified RNA sequences.
4.1.3. 50-O-(tert-Butyldiphenylsilyl)pseudouridine (4), 50-
O-(tert-Butyldiphenylsilyl)-20,30-O-(isopropylidene)pseu-
douridine (5), 5-(b-D-ribofuranosyl)uracil (6, W),
1-methyl-50-O-(tert-butyldiphenylsilyl)-20,30-O-(isopropyli-
dene)pseudouridine (7), and 1-methylpseudouridine (8,
m1W). Compounds 4–8 were prepared according to the
literature procedures.18,21,22
4. Experimental
4.1. Synthesis of modified nucleosides
4.1.4. 1-Methyl-3-[(S)-3-N-(tert-butoxycarbonyl)-amino-
3-methyl-carboxypropyl]-50-O-(tert-butyldiphenylsilyl)-20,
30-O-(isopropylidene)pseudouridine (10). Diisopropyl
azodicarboxylate (0.12 mL, 0.80 mmol, 1.3 equiv) was
added to a stirred solution of 7 (0.33 g, 0.61 mmol, 1.0
4.1.1. 5-[(1R,2S,3R,4S)-50-O-(tert-Butyldiphenylsilyl)- 20,
30-O-isopropylidene-1,4-pentandiol]-2,4-dimethoxypyrim-
idine (2).
A 1.0 M solution of ZnCl2 (7.15 mL,
7.15 mmol) in diethyl ether was added dropwise to a
solution of 1a and 1b (2.70 g, 4.77 mmol) in anhydrous
dichloromethane (110 mL) at ꢀ72 ꢁC under argon.
After stirring at ꢀ72 ꢁC for 30 min, a 1.0 M solution
of L-selectride (18.12 mL, 18.12 mmol) in THF was
added slowly over 30 min. The reaction was warmed
to rt and stirred for 19 h. The reaction mixture was
quenched by addition of EtOH (5 mL), water (5 mL),
30% H2O2 (5 mL), and 5 N NaOH (5 mL). After work-
up, the crude product was purified by column chroma-
tography using EtOAc/hexane (30–50%) to give 2
(2.50 g, 92%) as a colorless oil: Rf0.25 (EtOAc/hexane
equiv), boc-protected homoserine methyl ester 9
(0.14 g, 0.61 mmol, 1.0 equiv), and triphenylphosphine
(0.19 g, 0.74 mmol, 1.2 equiv) in anhydrous THF
(20 mL) at rt under argon. The reaction mixture was
stirred for 1 h. The solvent was removed under reduced
pressure. The residue was purified by column chroma-
tography using EtOAc/hexane (40–60%) to give 10
(0.43 g, 94%) as a white foam: Rf 0.23 (EtOAc/hexane
1
1:1); mp 53–56 ꢁC; H NMR (500 MHz, CDCl3) d 7.66
(m, 4H), 7.44 (m, 2H), 7.37 (m, 4 H), 7.28 (d,
J = 1.0 Hz, 1H), 5.44 (d, J = 9.0 Hz, 1H), 4.91 (d,
J = 3.5 Hz, 1H), 4.74 (dd, J = 6.5, 4.5 Hz, 1H), 4.65
(dd, J = 6.5, 3.5 Hz, 1H), 4.37 (m, 1H), 4.13 (dd,
J = 7.5, 4.0 Hz, 1H), 4.04 (m, 2H), 3.98 (dd, J = 11.5,
3.5 Hz, 1H), 3.84 (dd, J = 12.0, 4.5 Hz, 1H), 3.65 (s,
3H), 3.07 (s, 3H), 2.12 (m, 2H), 1.59 (s, 3H), 1.45 (s,
9H), 1.37 (s, 3H), 1.06 (s, 9H); 13C NMR (500 MHz,
CDCl3) d 172.9, 162.0, 155.8, 151.6, 140.3, 135.7,
135.6, 133.7, 133.2, 130.2, 130.2, 128.1, 128.0, 114.4,
112.4, 85.6, 85.0, 81.2, 80.9, 80.1, 64.1, 52.5, 51.7, 37.9,
37.0, 29.7, 28.6, 27.8, 27.1, 25.8, 19.6; ESI-MS (ES+)
m/z calcd for C39H53N3O10Si 751.4, found 774.4
(M+Na+); HRMS calcd for C39H53N3O10SiNa+
(M+Na)+ 774.3392, found 774.3424.
1
1:1); H NMR (500 MHz, CDCl3) d 8.38 (s, 1H), 7.68
(m, 4 H), 7.41 (m, 6H), 5.29 (d, J = 4.0 Hz, 1H), 4.36
(m, 1H), 4.26 (m, 2H), 3.96 (m, 6H), 3.91 (dd,
J = 10.0, 3.0 Hz, 1H), 3.83 (dd, J = 10.0, 5.0 Hz, 1H),
3.24 (d, J = 6.0 Hz, 1H), 3.12 (d, J = 3.5 Hz, 1H), 1.48
(s, 3H), 1.31 (s, 3H), 1.08 (s, 9H); 13C NMR
(500 MHz, CDCl3) d 168.0, 164.8, 157.2, 135.8, 135.7,
133.14, 133.07, 130.2, 130.1, 128.1, 128.0, 115.6, 108.8,
78.2, 77.6, 76.5, 69.9, 65.6, 64.9, 54.9, 54.3, 27.1, 27.0,
25.1, 19.5; ESI-MS (ES+) m/z calcd for C30H40N2O7Si
568.26, found 575.19 (M+Li+), 607.20 (M+K+); HRMS
calcd for C26H31N2O7Si (M+ꢀC4H9) 511.1901, found
511.1896.
4.1.5. 1-Methyl-3-(3-amino-3-carboxypropyl)pseudouri-
dine (11, m1acp3W). To a solution of compound 10
(0.48 g, 0.64 mmol) in dioxane (3 mL) was added
0.67 M (aq) NaOH (2.37 and 1.59 mL). The reaction
was stirred for 0.5 h at rt. The mixture was diluted with
water (15 mL) and acidified with 10% HCl (aq). The
4.1.2. 5-[50-O-(tert-Butyldiphenylsilyl)-20,30-O-isopropyli-
dene-b-D-ribofuranosyl]-2,4-dimethoxypyrimidine
(3).
Diisopropyl azodicarboxylate (1.38 mL, 7.14 mmol)
was added to a stirred solution of 2 (2.03 g, 3.57 mmol)
and triphenylphosphine (1.87 g, 7.14 mmol) in anhy-

2684
Y.-C. Chang et al. / Bioorg. Med. Chem. 16 (2008) 2676–2686
aqueous solution was extracted with ethyl acetate (3·
50 mL). The combined organic extracts were dried over
Na2SO4 and concentrated under reduced pressure to
give a colorless oil. The remaining protecting groups
were removed using TFA/H2O solution to give com-
pound 11 (0.21 g, 92%) as a white solid: mp 205–
208 ꢁC; IR (KBr) m 3184, 1700, 1661, 1624, 1460,
(m, 2H), 1.55 (m, 2H); 13C NMR (500 MHz, CD3OD)
d 163.2, 151.8, 142.9, 111.0, 84.2, 80.5, 74.4, 71.2, 61.8,
61.4, 40.9, 36.1, 29.8, 24.0; ESI-MS (ES+) m/z calcd
for C14H22N2O7 330.1, found 353.3 (M+Na+); HRMS
calcd for C14H22N2O7 (M+) 330.1427, found 330.1436.
4.1.9. 20,30-O-(Isopropylidene)uridine (16) and 50-O-(tert-
butyldiphenylsilyl)-20,30-O-(isopropylidene)uridine (17).
Compounds 16 and 17 were generated according to lit-
erature procedures.26
1
1105 cmꢀ1; H NMR (500 MHz, D2O) d 7.61 (s, 1H),
4.57 (d, J = 5.5 Hz, 1H), 4.13 (t, J = 5.0 Hz, 1H), 4.00
(t, J = 6.5 Hz, 1H), 3.95 (m, 2H), 3.87 (m, 1H), 3.73
(dd, J = 13.0, 3.5 Hz, 1H), 3.61 (m, 2H), 3.27 (s, 3H),
2.10 (m, 2H); 13C NMR (500 MHz, D2O) d 173.0,
164.1, 152.7, 144.4, 110.2, 83.2, 79.5, 73.7, 70.7, 61.4,
52.0, 37.6, 37.3, 27.8; ESI-MS (ES+) m/z calcd for
C14H21N3O8 359.13, found 360.13 (M+H+), 382.09
(M+Na+); Anal. Calcd for C14H21N3O8: C, 46.80; H,
5.89; N, 11.69; O, 35.62. Found: C, 44.66; H, 5.68; N,
10.08; O, 35.08.
4.1.10. 3-(3-Amino-3-carboxypropyl)uridine (19, acp3U).
The same procedure for compound 11 was employed for
compound 18.
4.1.11. 5-O-(tert-Butyldiphenylsilyl)-1-O-benzoyl-(S)-2-
N-(tert-butoxycarbonyl)-pentane (21). To a solution of
compound 20 (0.51 g, 1.12 mmol) in pyridine (6 mL)
was added benzoyl chloride (0.16 mL, 1.34 mmol)
and stirred for 16 h at rt. The solvent was evaporated
under reduced pressure in a hot water bath. The res-
idue was taken up by ethyl acetate (50 mL) and water
(10 mL). The organic layer was washed with brine and
dried over Na2SO4. The solvent was evaporated to
yield crude product that was purified by column chro-
matography using ethyl acetate/hexane (10–25%) to
4.1.6. 1-O-(tert-Butyldimethylsilyl)-butane-4-ol (12) and
1-O-(dimethoxytrityl)-butane-4-ol (30). Compounds 12
and 30 were prepared using excess amount of 1,4-
butanediol relative to the protective groups (Supplemen-
tary data).
Compounds 13, 18, 23, and 27. The procedure for gener-
ating compounds 13, 18, 23, and 27 was the same as for
10 using the appropriate alcohol derivative and nucleo-
side 7 or 17 (Supplementary data).
give 21 (0.61 g, 98%) as a colorless oil: Rf 0.34
1
(EtOAc/hexane 1:4); H NMR (500 MHz, CDCl3)
d
8.05 (m, 2H), 7.66 (m, 4H), 7.56 (m, 1H), 7.40 (m,
8H), 4.64 (d, J = 9.0 Hz, 1H), 4.32 (m, 2H), 4.00 (m,
1H), 3.70 (t, J = 6.0 Hz, 1H), 1.68 (m, 4 H), 1.42 (s,
9H), 1.04 (s, 9H); 13C NMR (500 MHz, CDCl3) d
166.7, 155.7, 135.8, 134.0, 133.3, 130.2, 129.9, 129.9,
128.6, 127.9, 79.7, 67.1, 63.6, 49.9, 29.11, 28.6, 27.1,
19.43; ESI-MS (ES+) m/z calcd for C33H43NO5Si
561.3, found 584.2 (M+Na+), 600.2 (M+K+); HRMS
calcd for C29H34NO4Si (M+ꢀC4H9O) 488.2257, found
488.2267.
4.1.7. 1-Methyl-3-[4-hydroxybutyl]-20,30-O-(isopropyli-
dene)pseudouridine (14). To a solution of compound 13
(0.20 g, 0.28 mmol) in dry THF (10 mL) was added tet-
rabutylammonium fluoride (1.0 M solution in THF,
0.61 mL, 0.61 mmol) at rt under argon. The reaction
mixture was stirred for 2.5 h. The solvent was removed
under reduced pressure and the crude product was puri-
fied by column chromatography using methanol/methy-
lene chloride (0–20%) to give 14 (91 mg, 89%) as a
colorless oil: Rf 0.38 (MeOH/CH2Cl2 1:9); 1H NMR
(400 MHz, CD3OD) d 7.70 (s, 1H), 4.72 (m, 3H), 4.04
(m, 1H), 3.94 (m, 2H), 3.75 (m, 1H), 3.65 (dd,
J = 12.0, 4.8 Hz, 1H), 3.56 (t, J = 6.4 Hz, 2H), 3.38 (s,
3H), 1.66 (m, 2H), 1.55 (m, 5H), 1.32 (s, 3H); 13C
NMR (400 MHz, CD3OD) d 162.8, 151.8, 143.1,
114.0, 110.7, 85.2, 84.4, 82.0, 81.8, 62.2, 61.4, 40.9,
36.1, 29.8, 26.7, 24.6, 24.0; ESI-MS (ES+) m/z calcd
for C17H26N2O7 370.2, found 393.4 (M+Na+); HRMS
calcd for C17H26N2O7 (M+) 370.1740, found 370.1754.
4.1.12. 3-[5-Hydroxy-(S)-4-N-(tert-butoxycarbonyl)-ami-
no-pentyl]-50-O-(tert-butyldiphenylsilyl)-20,30-O-(isopro-
pylidene)uridine (24). To a solution of compound 23
(570 mg, 0.688 mmol) in anhydrous methanol (5 mL)
was added sodium methoxide (1.0 M solution in
MeOH, 1.38 mL, 1.38 mmol) and stirred for 16 h at
rt. The solvent was removed under reduced pressure.
The residue was purified by column chromatography
using MeOH/CH2Cl2 (1–10%) to give 24 (420 mg,
84%) as a white foam:Rf 0.58 (MeOH/CH2Cl2 1:9);
mp 66–69 ꢁC; 1H NMR (500 MHz, CD3OD) d 7.67
(m, 5H), 7.39 (m, 6H), 5.82 (d, J = 2.0 Hz, 1 H), 5.53
(d, J = 8.0 Hz, 1H), 4.91 (dd, J = 6.5, 2.0 Hz, 1H),
4.78 (dd, J = 6.0, 3.5 Hz, 1H), 4.27 (m, 1H), 3.95 (dd,
J = 11.5, 3.5 Hz, 1H), 3.81 (m, 3H), 3.45 (m, 3H),
3.30 (m, 1H), 1.57 (m, 6H), 1.42 (s, 9H), 1.32 (s,
3H), 1.04 (s, 9H); 13C NMR (500 MHz, CD3OD) d
163.7, 157.1, 150.8, 140.8, 135.6, 135.5, 133.2, 132.8,
130.0, 130.0, 127.8, 127.8, 113.8, 100.9, 94.4, 88.0,
85.1, 81.0, 78.8, 64.4, 64.2, 52.4, 52.3, 40.8, 28.5,
27.7, 26.4, 26.3, 24.5, 24.1, 21.2, 18.9; ESI-MS (ES+)
m/z calcd for C38H53N3O9Si 723.4, found 762.2
(M+K+); HRMS calcd for C37H50N3O9Si 708.3316,
found 708.3294.
4.1.8. 1-Methyl-3-(4-hydroxybutyl)pseudouridine, (15,
m1hb3W). To a solution of compound 14 (86 mg,
0.23 mmol) in H2O/acetone (9:1, 7 mL) was added
TFA (3 mL) and stirred for 2 h. The solvent was evapo-
rated under reduced pressure in a hot water bath and
coevaporated with toluene. The residue was purified
by column chromatography using MeOH/methylene
chloride (10–30%) to give 15 (76 mg, 99%) as a light yel-
low oil: Rf 0.44 (MeOH/CH2Cl2 1:4); 1H NMR
(500 MHz, CD3OD) d 7.72 (s, 1H), 5.62 (d, J = 5.0 Hz,
1H), 4.14 (m, 1 H), 4.07 (t, J = 5.0 Hz, 1H), 3.93 (m,
3H), 3.81 (dd, J = 12.5, 3.0 Hz, 1H), 3.67 (dd, J = 12.0,
3.5 Hz, 1H), 3.56 (t, J = 6.0 Hz, 2H), 3.38 (s, 3H), 1.66

Y.-C. Chang et al. / Bioorg. Med. Chem. 16 (2008) 2676–2686
2685
4.1.13. 3-[(S)-4-N-(tert-Butoxycarbonyl)-amino-4-carbo-
xybutyl]-50-O-(tert-butyldiphenylsilyl)-20,30-O-(isopropyl-
idene)uridine (25). To a solution of compound 24 (392
mg, 0.54 mmol) in anhydrous N,N-dimethylformamide
(10 mL) was added pyridinium dichromate (1.22 g,
3.25 mmol) and stirred for 20 h at rt. Water (50 mL)
was added to the reaction mixture and then extracted
with ethyl acetate (3· 50 mL). The combined organic
layers were dried over Na2SO4. The solvent was evapo-
rated under reduced pressure. The residue was purified
by column chromatography using MeOH/CH2Cl2 (5–
15%) to give 25 (370 mg, 94%) as a colorless oil: Rf
0.22 (MeOH/CH2Cl2 1:9); 1H NMR (400 MHz, CDCl3)
d 7.61 (m, 5H), 7.42 (m, 6H), 5.92 (m, 1H), 5.53 (d,
J = 8.4 Hz, 1H), 5.12 (br s, 1H), 4.75 (s, 2H), 4.36 (br
s, 1H), 4.30 (m, 1H), 3.96 (m, 3H), 3.82 (dd, J = 12.0,
4.0 Hz, 1H), 1.70 (m, 4 H), 1.58 (s, 3H), 1.44 (s, 9H),
1.35 (s, 3 H), 1.06 (s, 9H); 13C NMR (400 MHz, CDCl3)
d 163.2, 150.9, 138.7, 135.8, 135.6, 132.9, 132.5, 130.4.
130.4, 128.2, 128.2, 114.4, 102.0, 93.2, 86.9, 85.7, 80.5,
64.2, 40.7, 29.9, 28.6, 27.5, 27.2, 25.6, 23.8, 19.5; ESI-
MS (ES+) m/z calcd for C38H51N3O10Si 737.3, found
776.2 (M+K+).
4.1.17. 3-(3-Carboxypropyl)-50-O-(tert-butyldiphenylsi-
lyl)-20,30-O-(isopropylidene)uridine (32) and 3-(3-carb-
oxypropyl)uridine (33, cp3U). The procedures are the
same as for compounds 25 and 26.
4.2. CD and NMR studies of modified nucleosides
4.2.1. Sample preparation. Each modified nucleoside was
dissolved in ddH2O for circular dichroism studies. Their
concentrations were determined using Beer-Lambert’s
law: A = e Æ C Æ k, in which k is the pathlength of the cuv-
ette. The extinction coefficient used for pseudouridine
nucleosides W, m1W, Wm, m1acp3W, and m1hb3W is
1.09 · 104 cmꢀ1 Mꢀ1 at 260 nm. The extinction coefficient
used for uridine nucleosides U, acp3U, acb3U, cp3U, and
hb3U is 1.33 · 104 cmꢀ1Mꢀ1 at 260 nm. The molar ellip-
ticity was normalized using equation De = h/(32.98 · C),
in which h is the CD absorbance of each nucleoside. The
modified nucleosides were dissolved in deuterium oxide
(0.25 M concentrations) for NMR experiments.
4.3. Circular dichroism and NMR spectroscopy
CD spectra were recorded on a Chirascan circular
dichroism spectrometer equipped with a water bath to
control the temperature at 25 ꢁC. The molar ellipticities
were normalized from a concentration of 10 mM for
each nucleoside. 1D NMR NOE spectra were recorded
on a 500 MHz NMR spectrometer (Varian-500S). The
NMR experiments of pseudouridine derivatives were
performed at room temperature and of uridine deriva-
tives were done at 40 ꢁC in order to avoid overlap of
the H10 and H5 peaks. One-dimensional NOE data at
40 ꢁC and room temperature show no significant
changes; for example, the derivative acp3U gave NOEs
to H10 (4.0%), H20 (7.1%), and H30 (2.9%) when H6
was irradiated at room temperature compared to H10
(4.0%), H20 (7.0%), and H30 (2.7%) at 40 ꢁC.
4.1.14. 3-(4-Amino-4-carboxybutyl)uridine, TFA salt (26,
acb3U). The protective groups of compound 25 were re-
moved using TFA/H2O solution (Supplementary data).
4.1.15. 3-(4-Hydroxybutyl)-20,30-O-(isopropylidene)uri-
dine (28) and 3-(4-hydroxybutyl)uridine (29, hb3U). The
procedures were the same as for compounds 14 and 15.
4.1.16. 3-(Butan-4-ol)-50-O-(tert-butyldiphenylsilyl)-20,30-
O-(isopropylidene)uridine (31). Diisopropyl azodicar-
boxylate (0.26 mL, 1.33 mmol) was added to a stirred
solution of 17 (0.53 g, 1.02 mmol), 30 (0.40 g, 1.02
mmol), and triphenylphosphine (0.240 g, 1.22 mmol)
in anhydrous THF (15 mL) at rt under argon. The
reaction was stirred for 1 h. The solvent was removed
under reduced pressure. The residue was dissolved in
EtOAc (100 mL) and washed with saturated solution
of NaHCO3 and brine. The organic layer was dried
over anhydrous Na2SO4. The solvent was removed un-
der reduced pressure and the residue was dissolved in
chloroform (10 mL). TFA (3 mL) was added to the
solution dropwise and stirred for 30 min. The orange-
red solution was evaporated to dryness and the residue
was purified by column chromatography using ethyl
acetate/hexane (50–70%) to give 31 (0.31 g, 51%) as a
colorless oil:Rf 0.24 (EtOAc/hexane 3:7); 1H NMR
Acknowledgments
This work was supported by the National Institutes of
Health (GM54632). We thank J.-P. Desaulniers, A.
Feig, and B. Ksebati for technical assistance and helpful
discussions.
Supplementary data
(400 MHz, CDCl3)
J = 8.0 Hz, 1 H), 7.40 (m, 6H), 5.92 (d, J = 2.4 Hz,
1H), 5.50 (d, J = 8.0 Hz, 1H), 4.74 (m, 2H), 4.29 (dd,
d
7.60 (m,
4
H), 7.55 (d,
Schemes for compounds 15, 17, 19, 29, and 30, experi-
mentals for compounds 12, 13, 18, 19, 22, 23, 26–30,
32, 33, and NMR spectra for compounds 15, 26, 29,
and 33 are available. Supplementary data associated
with this article can be found, in the online version, at
doi:10.1016/j.bmc.2007.11.039.
J = 6.4, 2.4 Hz,
1 H), 3.93 (m, 3H), 3.80 (dd,
J = 11.2, 4.0 Hz, 1H), 3.66 (t, J = 6.4 Hz, 2H), 1.62
(m, 7 H), 1.33 (s, 3H), 1.05 (s, 9H), 13C NMR
(400 MHz, CDCl3) d 162.9, 151.0, 138.5, 135.8, 135.6,
132.9, 132.5, 130.4, 130.3, 128.2, 128.2, 114.4, 102.1,
93.2, 86.9, 85.7, 80.6, 64.2, 62.7, 40.9, 29.9, 27.5,
27.2, 25.6, 24.2, 19.5; ESI-MS (ES+) m/z calcd for
C32H42N2O7Si 594.3, found 617.2 (M+Na+), 633.1
(M+K+); HRMS calcd for C31H39N2O7Si (M+ꢀCH3)
579.2527, found 579.2537.
References and notes
1. Lane, B. G., Modification and Editing of RNA. In
Grosjean, H., Eds.; ASM Press: Washington, DC, 1998;
pp 1–20.

2686
Y.-C. Chang et al. / Bioorg. Med. Chem. 16 (2008) 2676–2686
2. Agris, P. F. Prog. Nucleic Acid Res. Mol. Biol 1996, 53,
79–129.
3. Rozenski, J.; Crain, P. F.; McCloskey, J. A. Nucleic Acids
Res. 1999, 27, 196–197.
21. Bhattacharya, B. K.; Devivar, R. V.; Revankar, G. R.
Nucleosides Nucleotides 1995, 14, 1269–1287.
22. Chui, H. M.-P.; Desaulniers, J.-P.; Scaringe, S. A.; Chow,
C. S. J. Org. Chem. 2002, 67, 8847–8854.
4. Davis, D. R. Modification and Editing of RNA. In
Grosjean, H., Benne, R., Eds.; ASM Press: Washington,
DC, 1988; pp 85–102.
23. (a) Mitsunobu, O. Synthesis 1981, 1–28; (b) Hughes, D. L.
Org. React. 1992, 42, 335–656.
24. Howarth, N. M.; Wakelin, L. P. G. J. Org. Chem. 1997,
62, 5441–5450.
5. See for example: Eldrup, A. B.; Allerson, C. R.; Bennett,
C. F.; Bera, S.; Bhat, B.; Bhat, N.; Bosserman, M. R.;
Brooks, J.; Burlein, C.; Carroll, S. S.; Cook, P. D.; Getty,
K. L.; MacCoss, M.; McMasters, D. R.; Olsen, D. B.;
Prakash, T. P.; Prhavc, M.; Song, Q.; Tomassini, J. E.;
Xia, J. J. Med. Chem. 2004, 47, 2283–2295.
6. See for example: Marquez, V. E.; Ben-Kasus, T.; Barchi, J.
J., Jr.; Green, K. M.; Nicklaus, M. C.; Agbaria, R. J. Am.
Chem. Soc. 2004, 126, 543–549.
7. So¨ll, D.; Kline, L. The Enzymes. In Boyer, P. D., Ed.;
Academic Press: New York, NY, 1982; pp 557–566.
8. Saponara, A. G.; Enger, M. D. Biochim. Biophys. Acta
1974, 349, 61–77.
9. Maden, B. E. H.; Forbes, J.; de Jonge, P.; Klootwijk, J.
FEBS Lett. 1975, 59, 60–63.
10. (a) Nishimura, S.; Taya, Y.; Kuchino, Y.; Ohashi, Z.
Biochem. Biophys. Res. Commun. 1974, 57, 702–708; (b)
Ohashi, Z.; Maeda, M.; McCloskey, J. A.; Nishimura, S.
Biochemistry 1974, 13, 2620–2625.
25. Ross, B. S.; Vasquez, G.; Manalili, S.; Lesnik, E.; Griffey,
R. Nucleosides Nucleotides 1997, 16, 1547–1549.
26. (a) Hampton, A. J. Am. Chem. Soc. 1961, 83, 3640–3645;
(b) Hanessian, S.; Lavalee, P. Can. J. Chem. 1975, 53,
2975–2977.
27. Kadota, I.; Kawada, M.; Muramatsu, Y.; Yamamoto, Y.
Tetrahedron: Asymmetry 1997, 8, 3887–3893.
28. Collier, P. N.; Campbell, A. D.; Patel, I.; Raynham, T. M.;
Taylor, R. J. K. J. Org. Chem. 2002, 67, 1802–1815.
29. Seela, F.; Thi, Q. H. T.; Hasselmann, D. Chem. Br. 1979,
112, 700–707.
30. Schweizer, M. P.; Thedford, R.; Slama, J. Biochim.
Biophys. Acta 1971, 232, 217–226.
31. Rabczenko, A.; Jankowski, K.; Zakrzewska, K. Biochim.
Biophys. Acta 1974, 353, 1–15.
32. Desaulniers, J.-P.; Chui, H. M.-P.; Chow, C. S. Bioorg.
Med. Chem. 2005, 13, 6777–6781.
33. Thibaudeau, C.; Plavec, J.; Watanabe, K. A.; Chat-
topadhyaya, J. J. Chem. Soc. Chem. Commun. 1994,
537–540.
11. Friedman, S. J. Biol. Chem. 1979, 254, 7111–7115.
12. Brand, R. C.; Klootwijk, J.; Planta, R. J.; Maden, B. E. H.
Biochem. J. 1978, 169, 71–77.
´
34. Neumann, J. M.; Bernassau, J. M.; Gueron, M.; Tran-
13. Fuke, M.; Busch, H. Nucleic Acids Res. 1979, 7, 1131–1135.
14. (a) Maden, B. E. Prog. Nucleic Acid Res. Mol. Biol. 1990,
39, 241–303; (b) Maden, B. E. H.; Wakeman, J. A.
Biochem. J. 1988, 249, 459–464.
15. Argoudelis, A. D.; Mizsak, S. A. J. Antibiot. 1976, 29,
818–823.
16. Pang, H.; Ihara, M.; Kuchino, Y.; Nishimura, S.; Gupta,
R.; Woese, C. R.; McCloskey, J. A. J. Biol. Chem. 1982,
257, 3589–3592.
17. Hudson, L.; Gray, M.; Lane, B. G. Biochemistry 1965, 4,
2009–2016.
Dinh, S. Eur. J. Biochem. 1980, 108, 457–463.
35. Smith, W. S.; Nawrot, B.; Malkiewicz, A.; Agris, P. F.
Nucleosides Nucleotides 1992, 11, 1683–1694.
36. Rosemeyer, H.; Toth, G.; Golankiewicz, B.; Kazimierc-
zuk, Z.; Bourgeois, W.; Kretschmer, U.; Muth, H.-P.;
Seela, F. J. Org. Chem. 1990, 55, 5784–5790.
37. Altona, C.; Sundaralingam, M. J. Am. Chem. Soc. 1973,
95, 2333–2344.
38. Lipnick, R. L.; Fissekis, J. D.; O’Brien, J. P. Biochemistry
1981, 20, 7319–7327.
´
39. (a) de Champdore, M.; De Napoli, L.; Di Fabio, G.;
18. Grohar, P. J.; Chow, C. S. Tetrahedron Lett. 1999, 40,
2049–2052.
19. Hanessian, S.; Machaalani, R. Tetrahedron Lett. 2003, 44,
8321–8323.
Messere, A.; Montesarchio, D.; Piccialli, G. J. Chem. Soc.
Chem. Commun. 2001, 2598–2599; (b) Lu, W.; Sengupta,
S.; Petersen, J. L.; Akhmedov, N. G.; Shi, X. J. Org.
Chem. 2007, 72, 5012–5015.
´
20. Chen, J. J.; Drach, J. C.; Townsend, L. B. J. Org. Chem.
2003, 68, 4170–4178.
40. Veliz, E. A.; Beal, P. A. Tetrahedron Lett. 2006, 47, 3153–
3156.