Facile methods for the synthesis of 5-formylcytidine

Article abstract of DOI:10.1016/S0040-4039(00)02191-2

5′-O-Protected 5-methylcytidine 3 was oxidized with Na₂S₂O₈ to give a mixture of the corresponding 5-(hydroxymethyl)- and 5-formylcytidine derivatives, 4 and 5. The hydroxymethyl group of 4 was further oxidized to a formyl group by treatment with ceric(IV) ammonium nitrate (CAN). Alternatively, 2′,3′,5′-O-protected 5-(hydroxymethyl)cytidine 10 was directly oxidized with CAN to give the desired 5-formylcytidine derivative 11. After removal of the protecting groups in each intermediate, 5-formylcytidine (6) was obtained in good yield.

Full text of DOI:10.1016/S0040-4039(00)02191-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 1061–1063
Facile methods for the synthesis of 5-formylcytidine
Adel A.-H. Abdel Rahman, Takeshi Wada* and Kazuhiko Saigo*
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-8656, Japan
Received 21 September 2000; accepted 24 November 2000
Abstract—5%-O-Protected 5-methylcytidine 3 was oxidized with Na2S2O8 to give a mixture of the corresponding 5-(hydroxy-
methyl)- and 5-formylcytidine derivatives, 4 and 5. The hydroxymethyl group of 4 was further oxidized to a formyl group by
treatment with ceric(IV) ammonium nitrate (CAN). Alternatively, 2%,3%,5%-O-protected 5-(hydroxymethyl)cytidine 10 was directly
oxidized with CAN to give the desired 5-formylcytidine derivative 11. After removal of the protecting groups in each intermediate,
5-formylcytidine (6) was obtained in good yield. © 2001 Elsevier Science Ltd. All rights reserved.
The post-transcriptional processing of RNA produces
an exceptional number and structural diversity of
modified nucleosides.1,2 Recently, the functional roles
of modifications existing in biologically important
RNA molecules have been gradually revealed in paral-
lel with continuous discovery of new modified
nucleosides.3–5 Such a new nucleoside, 5-formylcytidine
(f5C), has been found at the first position of the anti-
codon of tRNAMet from bovine liver mitochondria.6
This situation led us to investigate the oxidation of
5%-O-protected 5-methylcytidine with an expectation
that the intermediary 5%-O-protected 5-(hydroxy-
methyl)cytidine would be easily oxidized to the corre-
sponding 5%-O-protected 5-formylcytidine, resulting in
the improvement of the total yield of the desired 5-
formylcytidine derivative. For this purpose we at first
synthesized 5-methylcytidine (5) as follows: 1-(tri-O-
benzoyl-b-D-ribofuranosyl)-5-methyl-2,4(1H,3H)-pyri-
midinedione (1) was synthesized in 99% yield by the
ribosylation of a silylated thymine9 using Sn(IV) as a
catalyst according to the method of Niedballa et al.10
Compound 1 was treated with 1,2,4-triazole, triethyl-
amine, and phosphorous oxychloride in acetonitrile to
give the triazolyl intermediate in 98% yield after chro-
matographic purification, which was converted to 2 in
95% yield by treatment with a mixture of dioxane and
ammonia at room temperature for 24 h and with
methanolic ammonia overnight. The physical data of 2,
thus obtained, were in agreement with those in the
literature.11 Treatment of 2 with t-butyldiphenylsilyl
chloride in the presence of imidazole in DMF gave the
5%-O-t-butyldiphenylsilylated 3 in 94% yield after chro-
matographic purification. Oxidation of 3 with sodium
peroxosulfate in a buffer solution at 70°C under argon
gave 5-(hydroxymethyl)cytidine derivative 4 and 5-
formylcytidine derivative 5 in 27 and 20% yields,
respectively. The 5-(hydroxymethyl)cytidine derivative
4 was easily converted to 5 by treatment with 1 M
ceric(IV) ammonium nitrate (CAN) applying the
method of Trahnovski et al.12 to give 5 in a total yield
of 45%. Deprotection of 5 by treatment with tetra-
butylammonium fluoride in THF at room temperature
gave the desired product, 5-formylcytidine (6), in 71%
yield (Scheme 1).
Moriya et al.6 have synthesized 5-formylcytidine from
5-(hydroxymethyl)cytosine; the oxidation of 5-(hydroxy-
methyl)cytosine with Ce(IV) gave 5-formylcytosine,
which was converted to the corresponding dimethyl
acetal and then ribosylated using a Sn(IV) catalyst.
Recently, Kasai and co-workers7 have reported a novel
method for the synthesis of 5-formyl-2%-deoxycytidine,
which is essentially applicable for the synthesis of 5-
formylcytidine, starting from 3%,5%-bis-O-t-butyl-
dimethyl-silyl-5-iodo-2%-deoxyuridine but through six
steps.
On the other hand, it has been reported8 that a 5-
methylcytidine derivative was oxidized with peroxosul-
fate in a buffer solution to give the desired 5-
formylcytidine derivative in low yield together with the
formation of 5-(hydroxymethyl)cytidine, 5-(hydroxy-
methyl)cytosine and 5-methycytosine as hardly separa-
ble side products. Moreover, in this case the selective
oxidation of the 5-(hydroxymethyl)cytidine derivative
to the corresponding 5-formyl derivative has been
difficult due to the presence of two primary hydroxyl
groups (5-hydroxymethyl and 5%-OH).
* Corresponding authors.
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)02191-2

1062
A. A.-H. A. Rahman et al. / Tetrahedron Letters 42 (2001) 1061–1063
O
N
NH2
NH2
N
CH3
CH3
CH3
HN
O
N
N
O
O
O
N
O
1. Dioxane/NH3
2. MeOH/NH3
Triazole, Et3N,
POCl3
TBDPSCl,
Imidazole
BzO
HO
TBDPSO
O
DMF
94%
MeCN
BzO OBz
HO OH
95%
98%
HO OH
1
2
3
NH2
NH2
N
NH2
CH2OH
CHO
CHO
N
N
N
O
N
O
O
N
O
Na2S2O8
TBDPSO
TBDPSO
HO
TBAF
O
O
+
pH 7/Argon
THF
71%
HO OH
HO OH
total 45%
HO OH
4
5
6
1 M CAN
Scheme 1.
In the next stage, in order to develop an alternative,
convenient and efficient method for the synthesis of
5-formylcytidine (6), we noted the facts that the oxida-
tion of 5-(hydroxymethyl)cytosine with 1 M CAN gave
5-formylcytosine in high yield6,12 and that 4 could be
easily oxidized by CAN to also give 5 in high yield as
mentioned above. These facts were considered to indi-
cate that the oxidation of a hydroxymethyl group at the
5-position of a cytosine moiety took place easily. On
the basis of this consideration, we tried to apply this
oxidation to a protected 5-(hydroxymethyl)cytidine
derivative. For this purpose, 2%,3%-O-isopropylidene-5%-
O-trityl-5-(hydroxymethyl)uridine (7) was synthesized
as a starting material 2%,3%-O-isopropylidene-5%-O-trityl-
uridine in 70% yield.13 Compound 7 was acetylated
with acetic anhydride in pyridine at room temperature
overnight to give the acetylated derivative 8 in 99%
yield, which was converted to the triazolyl derivative 9
in 98% yield by a similar method described above.14
Treatment of 9 with dioxane/NH3 and then with
MeOH/NH3 gave 10 in 95% yield.14 The oxidation of
10 with 1 M CAN at 60°C for 1 h followed by
N
N
N
O
N
O
N
CH2OH
CH2OAc
CH2OAc
1. Dioxane/NH3
HN
O
HN
O
N
O
Triazole, Et3N,
POCl3
O
O
O
N
Ac2O
2. MeOH/NH3
TrO
TrO
TrO
pyridine
99%
MeCN
98%
95%
O
O
O
O
O O
7
8
9
NH2
NH2
N
NH2
N
CHO
CH2OH
CHO
N
N
O
N
O
O
N
O
O
TrO
HO
TrO
O
1 M CAN
60% HCOOH
O
O
HO OH
O
O
75% (two-step overall)
10
11
6
Scheme 2.

A. A.-H. A. Rahman et al. / Tetrahedron Letters 42 (2001) 1061–1063
1063
deprotection with 60% formic acid at 80°C for 2 h, gave
6 in 75% yield (Scheme 2).
Yokoyama, S.; Watanabe, K. Biochemistry 1974, 33,
2234–2239.
7. Kamuya, N. M.; Kamiya, H.; Karino, N.; Ueno, H.;
Kaji, H.; Matsuda, A.; Kasai, H. Nucleic Acids Res. 1999,
27, 4385–4390.
8. Itahara, T. Chem. Lett. 1991, 1591–1594.
9. Wittenburg, E. Z. Chem. 1964, 4, 303.
In conclusion, we have demonstrated two facile meth-
ods for the synthesis of 5-formylcytidine, which plays a
central role in the realization of the higher-ordered
structure and finely controlled function of tRNA. In
comparison with the previously reported methods, the
present ones are more efficient and economical. Phys-
icochemical and biochemical studies of this modified
nucleoside are now in progress.
10. Niedballa, U.; Vorbru¨ggen, H. J. Org. Chem. 1974, 39,
3654–3660.
11. Fox., J. J.; Praag, D. V.; Wempen, I.; Doerr, I. L.;
Cheong, L.; Knoll, J. E.; Eidinoff, M. L.; Bendich, A.;
Brown, G. B. J. Am. Chem. Soc. 1959, 81, 178–187.
12. Trahanovski, W. S.; Young, B. L.; Brown, C. L. J. Org.
Chem. 1967, 32, 3865–3868.
Acknowledgements
13. (a) Fromageot, H. P. M.; Grieffin, B. E.; Reese, C. B.;
Sulton, J. E. Tetrahedron 1967, 23, 2315–2331; (b) Leven,
P. A.; Tipson, R. S. J. Biol. Chem. 1934, 105, 385–393; (c)
Scheit, K. H. Chem. Ber. 1966, 3884–3891.
14. Selected physical data: 4: colorless foam: TLC (CHCl3–
A.A.-H.A.R. thanks the Japan Society for the Promo-
tion of Science for the postdoctoral fellowship for
foreign researchers (JSPS-P98431).
1
MeOH, 8:2 v/v): Rf 0.66; H NMR (300 MHz, DMSO-
d6): l 1.09 (s, 9H, 3×CH3), 3.48 (s, 2H, CH2), 3.98 (m,
2H, 5%,5%%-H), 4.28 (m, 1H, 4%-H), 4.37 (m, 2H, 2%-H, 3%-H),
5.99 (d, J=1.7 Hz, 1H, 1%-H), 7.38–7.40 (m, 6H, Ar-H),
7.64–7.71 (m, 5H, 6-H, Ar-H). 5: pale yellow foam: TLC
(CHCl3–MeOH, 8:2 v/v): Rf 0.70; 1H NMR (300 MHz,
DMSO-d6): l 1.07 (s, 9H, 3×CH3), 4.09 (m, 2H, 5%,5%%-H),
4.21–4.29 (m, 3H, 2%-H, 3%-H, 4%-H), 5.92 (d, J=1.8 Hz,
1H, 1%-H), 7.40–7.45 (m, 6H, Ar-H), 7.63–7.68 (m, 4H,
Ar-H), 8.90 (s, 1H, 6-H), 9.13 (s, 1H, CHO). 9: colorless
foam: TLC (CHCl3–MeOH, 9.5:0.5 v/v): Rf 0.80; 1H
NMR (300 MHz, CDCl3): l 1.38 (s, 3H, CH3), 1.60 (s,
3H, CH3), 2.90 (s, 3H, Ac), 3.45 (s, 2H, CH2), 3.72–3.81
(m, 2H, 5%,5%%-H), 4.31 (m, 1H, 4%-H), 4.98–5.01 (m, 1H,
3%-H), 5.05–5.11 (m, 1H, 2%-H), 5.61 (d, J=1.6 Hz, 1H,
1%-H), 6.98 (m, 6H, Ar-H), 7.25–7.38 (m, 9H, Ar-H), 8.05
(s, 1H, 6-H), 8.30 (s, 1H, H-3 triazole), 9.30 (s, 1H, H-5
triazole). 10: pale yellow foam: TLC (CHCl3–MeOH, 9:1
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