Palladium-catalyzed base-selective H-D exchange reaction of nucleosides in deuterium oxide

Article abstract of DOI:10.1055/s-2005-868489

We have developed an efficient and extensive deuterium incorporation method using a heterogeneous Pd/C-D₂O-H₂ system into the base moiety of nucleosides. The results presented here provide a deuterium gas-free, totally catalytic, and post-synthetic deuterium labeling method in D ₂O media. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2005-868489

LETTER
1385
Palladium-Catalyzed Base-Selective H–D Exchange Reaction of Nucleosides in
Deuterium Oxide
P
alladium-Catalyz
i
e
d
Ba
s
r
ove H–DExc
n
R
eacti
a
on of
N
ucle
o
osides inDeuterium
S
Oxide ajiki,* Hiroyoshi Esaki, Fumiyo Aoki, Tomohiro Maegawa, Kosaku Hirota
Laboratory of Medicinal Chemistry, Gifu Pharmaceutical University, Mitahora-higashi, Gifu 502-8585, Japan
Fax +81(58)2375979; E-mail: sajiki@gifu-pu.ac.jp
Received 5 April 2005
cedure for the H–D exchange reaction at the base moiety
of nucleosides applying the Pd/C–D2O–H2 system with
heating conditions.
Abstract: We have developed an efficient and extensive deuterium
incorporation method using a heterogeneous Pd/C–D2O–H2 system
into the base moiety of nucleosides. The results presented here pro-
vide a deuterium gas-free, totally catalytic, and post-synthetic deu-
terium labeling method in D2O media.
To explore the scope of our method for the H–D exchange
of nucleic acids, the reaction of a number of substrates
was investigated (Tables 1– 4).
Key words: nucleosides, palladium, reaction, H–D exchange,
base-selective
Table 1 H–D Exchange Reaction of Adenine Derivatives
NH2
NH2
Nucleoside analogs are noteworthy as biologically active
targets for the development of potential antiviral and anti-
tumor agents1 and as synthetic oligonucleotide probes.2
Deuterium-labeled compounds have a number of impor-
tant uses in many different branches of science, including
analysis of drug metabolism, investigation of reaction
mechanisms, kinetics, and so on.3,4 Deuterium-labeled nu-
cleosides have proven valuable in metabolic studies5 and
structural analyses of DNA.6 For the preparation of base-
selectively deuterium-labeled nucleic acids, the previous
methods are chiefly categorized under the following two
types: the multi-step synthetic method starting from orig-
inally deuterium-labeled small synthons,7 and the post-
synthetic H–D exchange displacement of the hydrogen
bound to the carbon of an unlabeled compound by deute-
rium using a catalytic method.8 It is apparent that the latter
process is highly effective and accessible for the prepara-
tion of deuterium-labeled nucleosides. However, such
conventional post-synthetic procedures for the incor-
poration of deuterium into the base-moieties of nucleo-
sides are often limited to activated positions of the
molecules,8a–8c,e,g leading to low levels of deuterium incor-
poration,8e,f and require a vast amount of the catalyst,8d,f,g
addition of acidic or basic additives,8a–8c,e,f and/or expen-
sive deuterium atmosphere.8d,f,g,9
N
N
10% Pd/C, H2
N
N
(8)
D
D2O, Temp, 24 h
N
R
N
R
N
N
D
(2)
Entry R
Temp
(°C)
D content (%)a
Yield
(%)
99
2-D
8-D
1
2
H
110
110
96b
95
96b
92
99
HO
O
OH OH
3
4
160
160
95
96
96
96
98
81
HO
HO
O
OH OH
O
OH
a Determined by 1H NMR spectroscopy using 3-trimethylsilyl-1-pro-
panesulfonic acid sodium salt (DSS) as an internal standard.
b Indicates the average D content.
O
O
>98
0
NH
We have recently reported an efficient and chemoselec-
tive exchange of deuterium derived from D2O with hydro-
gen atoms on a benzylic carbon using Pd/C as a
heterogeneous catalyst in the presence of a catalytic
amount of hydrogen gas at room temperature.10 We also
found that application of heat could promote the catalyst
activity of the Pd/C–D2O–H2 system and lead to a H–D
exchange reaction even on non-activated carbon.11,12
Herein, we describe a distinctly general and selective pro-
100
97
NH
N
O
TBDMSO
N
O
O
Me
OTBDMS
OTBDMS
1 (93% isolated yield)
2 (100% isolated yield)
Figure 1 Deuterium efficiency of 1 and 2 by the H–D exchange
reaction at 160 °C for 24 hours.
Typically, the reaction is carried out in 1.0 mL of D2O us-
ing 0.25 mmol of the substrate and 10% Pd/C (10 wt%) at
110–160 °C under a hydrogen atmosphere. The reactions
are usually very clean and no chromatographic separation
SYNLETT 2005, No. 9, pp 1385–1388
Advanced online publication: 27.04.2005
DOI: 10.1055/s-2005-868489; Art ID: U06205ST
© Georg Thieme Verlag Stuttgart · New York
0
1.
0
6.
2
0
0
5

1386
H. Sajiki et al.
LETTER
Table 2 H–D Exchange Reaction of Guanosine, Inosine, and Hypoxanthine
10% Pd/C, H2, D2O
Substrate
Substrate-dn
110 °C (reflux), 24 h
Entry
1
Substrate
D content (%)a
Yield (%)
99
100
92
O
O
N
N
N
N
NH
NH
95
O
N
N
NH2
NH2
HO
HO
O
OH OH
OH OH
2
3
O
N
O
N
N
N
N
NH
NH
96
97
N
HO
HO
O
O
OH OH
O
OH OH
O
N
N
NH
N
NH
98
97
N
H
N
H
N
a Determined by 1H NMR spectroscopy using DSS as an internal standard.
Table 3 H–D Exchange Reaction of Uracil and Cytosine Derivatives
X
X
(5)
(6)
D
D
10% Pd/C, H2
NH
NH
D2O, Temp, Time
N
R
O
N
R
O
Entry
X
R
Temp (°C)
Time (h)
D content (%)a
5-D
Yield (%)
6-D
–
1b
2
O
O
O
H
140
160
140
48
24
48
–
–
H
98
–
97
–
100
–
3b
HO
O
OH OH
4
O
160
24
94
35
100
HO
O
OH OH
5b
6
NH
NH
NH
H
110
160
140
24
48
48
–
–
–
H
96
93
96
35
98
100
7
HO
O
OH OH
a Determined by 1H NMR spectroscopy using DSS as an internal standard.
b Partial hydrogenation of the 5,6-double bond was observed.
Synlett 2005, No. 9, 1385–1388 © Thieme Stuttgart · New York

LETTER
Palladium-Catalyzed Base-Selective H–D Exchange Reaction of Nucleosides in Deuterium Oxide
1387
Table 4 H–D Exchange Reaction of Thymine Derivatives
O
O
(5)
H3C
D3C
10% Pd/C, H2
NH
NH
D2O, Temp, 24 h
(6)
N
R
O
D
N
R
O
Entry
R
H
Temp (°C)
D content (%)
Yield (%)
5-CD3
6-D
96
–
1
110
140
97
–
89
–
2b
HO
O
OH
a Determined by 1H NMR spectroscopy using DSS as an internal standard.
b Hydrolytic cleavage of deoxyribose was observed.
is required to obtain spectrally pure deuterated products in
excellent yields.13,14 When uracil, uridine, or cytosine was
used as the substrate, partial hydrogenation of the 5,6-
double bond was observed at a relatively lower tempera-
ture (110–140 °C, Table 3, entries 1, 3, and 5). It is note-
worthy that this drawback can be overcome by raising the
temperature to 160 °C (Table 3, entries 2, 4, and 6). The
5-methyl group of thymine was deuterated entirely, to-
gether with the 6-position at 110 °C without partial hydro-
genation (Table 4, entry 1). No competitive deuterium
incorporation into the sugar moieties was observed in all
cases.15 It should be noted that the exchange reaction us-
ing pyrimidine nucleosides, such as uridine and cytidine,
led to lower deuterium incorporation at the 6-position
(Table 3, entries 4 and 7) although the use of uracil and
cytosine, which lack the sugar moiety, gave excellent deu-
terium efficiency (Table 3, entries 2 and 6). For these rea-
sons, it may be concluded that the steric hindrance arising
from the 5¢-hydroxy group lowered deuterium incorpora-
tion; also no deuterium incorporation into the 6-position
of the more hindered 2¢,3¢,5¢-tris-O-TBDMS-uridine (1)
under the reaction conditions confirmed this while the
deuteration of 1-methyluracil (2) possessing a small
methyl substituent at the 1-position gave excellent deuter-
ium efficiencies at both 5- and 6-positions (Figure 1).12a
Acknowledgment
The authors thank the Research Foundation of Gifu Pharmaceutical
University.
References
(1) For examples, see: (a) Townsend, L. B. Chemistry of
Nucleosides and Nucleotides; Plenum Press: New York,
1988. (b) Nucleosides and Nucleotides as Antitumor and
Antiviral Agents; Chu, C. K.; Baker, D. C., Eds.; Plenum
Press: New York, 1993. (c) Haraguchi, K.; Itoh, Y.; Tanaka,
H. J. Synth. Org. Chem. Jpn. 2003, 61, 974. (d) Matsuda,
A.; Sasaki, T. Cancer Sci. 2004, 95, 105.
(2) (a) Ruth, T. L. Oligonucleotides and their Analogues; IRL
Press: London, 1991. (b) Giese, B.; Imwinkelried, P.;
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(4) Very recently an attractive method for monitoring reaction
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Omar, M. M. Chem. Commun. 2002, 66.
(5) (a) Gani, D.; Young, D. W. J. Chem. Soc., Chem. Commun.
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A limitation of this methodology is that thymidine, a
deoxy-pyrimidine nucleoside, decomposed with complete
hydrolysis at the glycosyl bond (Table 4, entry 2) even
though nearly quantitative deuteration efficiency was
achieved in 2¢-deoxyadenosine without hydrolysis
(Table 1, entry 4).
In summary, the present D2 gas-free and selective H–D
exchange reaction retains sufficient usefulness in nucleic
acid chemistry. It discloses a convenient route to the post-
synthetic introduction of deuterium atoms into the base
moiety of nucleosides with high deuterium efficiency un-
der neutral reaction conditions. Studies to further eluci-
date the scope of this incorporation method are currently
underway.
Synlett 2005, No. 9, 1385–1388 © Thieme Stuttgart · New York

1388
H. Sajiki et al.
LETTER
Chem. Commun. 1975, 125. (f) Kiritani, R.; Asano, T.;
Fujita, S.; Dohmaru, T.; Kawanishi, T. J. Labelled Compd.
Radiopharm. 1986, 23, 207. (g) Roček, J.; Sváta, V.;
Lešetický, L. Collect. Czech. Chem. Commun. 1985, 50,
1244.
[a]D20 –55 (c 0.38, H2O) [adenosine Lit.16 [a]D11 –62 (c 0.71,
H2O)]. 1H NMR (400 MHz, DMSO-d6): d = 8.37 (s, 0.053
H), 8.12 (s, 0.042 H), 7.34–7.30 (br s, 2 H), 5.90 (d, J = 6.4
Hz, 1 H), 5.45–5.41 (m, 2 H), 5.20 (d, J = 4.9 Hz, 1 H), 4.63
(dd, J = 4.9, 6.4 Hz, 1 H), 4.16 (dd, J = 3.4, 4.4 Hz, 1 H),
3.99 (dd, J = 3.4, 3.4 Hz, 1 H), 3.72–3.67 (m, 1 H), 3.60–
3.54 (m, 1 H). 13C NMR (100 MHz, DMSO-d6): d = 156.2,
152.3 (small peak), 149.0, 139.9 (small peak), 119.3, 87.9,
85.9, 73.4, 70.6, 61.6. 2H NMR (400 MHz, DMSO): d = 8.02
(br). MS (ES+): m/z (%) = 269 (3) [M + 2].
(9) D2 gas [¥ 31300/10 L of D2 gas (Aldrich 36840-7) in lecture
bottle] is purchased as a lecture bottle or a cylinder charged
by high-pressure.
(10) Sajiki, H.; Hattori, K.; Aoki, F.; Yasunaga, K.; Hirota, K.
Synlett 2002, 1149.
19
(11) (a) Sajiki, H.; Aoki, F.; Esaki, H.; Maegawa, T.; Hirota, K.
Org. Lett. 2004, 6, 1485. (b) Maegawa, T.; Akashi, A.;
Esaki, H.; Aoki, F.; Sajiki, H.; Hirota, K. Synlett 2005, 845.
(12) Matsubara et al. also reported interesting Pd/C-catalyzed H–
D exchange reactions under hydrothermal conditions, see:
(a) Matsubara, S.; Yokota, Y.; Oshima, K. Chem. Lett. 2004,
33, 294. (b) Yamamoto, M.; Yokota, Y.; Oshima, K.;
Matsubara, S. Chem. Commun. 2004, 1714. (c) Yamamoto,
M.; Oshima, K.; Matsubara, S. Org. Lett. 2004, 6, 5015.
(13) Typical Procedure for Deuteration of Adenosine (Table
1, entry 3): Adenosine (66.8 mg, 0.25 mmol) and 10% Pd/C
(6.7 mg, 10 wt% of the substrate, Aldrich) in D2O (1 mL)
was stirred at 160 °C in a sealed tube under a H2 atmosphere
for 24 h. After cooling, the reaction mixture was filtered
using a membrane filter (Millipore Millex®-LG). The
filtered catalyst was washed with boiling water (50 mL) and
the combined filtrates were concentrated in vacuo to give
adenosine-d2 as a white powder (66.3 mg, 98%). The
deuterium content (%) was determined by 1H NMR using 3-
trimethylsilyl-1-propanesulfonic acid sodium salt (DSS) as
an internal standard and confirmed by mass spectroscopy.
(14) Specific rotations of nucleosides: Table 1, entry 2 [a]D
–60 (c 0.38, H2O) {adenosine Lit.16 [a]D11 –62 (c 0.71,
H2O)}; Table 1, entry 3 [a]D20 –55 (c 0.38, H2O) {adenosine
Lit.16 [a]D11 –62 (c 0.71, H2O)}; Table 1, entry 4 [a]D20 –19
(c 0.36, CH3OH) {deoxyadenosine [a]D20 –20 (c 0.36,
CH3OH)}; Table 2, entry 1 [a]D20 –59 (c 0.25, 0.02 N NaOH)
{guanosine [a]D20 –61 (c 0.30, 0.02 N NaOH)}; Table 2,
entry 2 [a]D21 –46 (c 0.34, H2O) {inosine Lit.16 [a]D18 –49 (c
0.9, H2O)}; Table 3, entry 4 [a]D21 +5 (c 0.27, H2O) {uridine
Lit.16 [a]D20 +4 (c 2)}; Table 3, entry 7 [a]D21 +25 (c 0.26,
H2O) {cytidine Lit.16 [a]D25 +31 (c 0.7, H2O)}; 1 [a]D21 +18
(c 0.74, CH3Cl) {2¢,3¢,5¢-tris-O-TBDMS-uridine [a]D22 +22
(c 0.83, CH3Cl)}.
(15) Although Matsubara et al. recently reported quite interesting
H–D exchange reaction of primary alcohols at the a-position
using RuCl2(PPh3)2 as a catalyst, we observed no
competitive deuterium incorporation into the sugar moieties,
see: Takahashi, M.; Oshima, K.; Matsubara, S. Chem. Lett.
2005, 34, 192.
(16) The Merck Index 13th Ed.; Merck & Co., Inc.: Whitehouse
Station, 2001.
Synlett 2005, No. 9, 1385–1388 © Thieme Stuttgart · New York