Hydrothermal deamidation of 4-N-acylcytosine nucleoside derivatives: Efficient synthesis of uracil nucleoside esters

Article abstract of DOI:10.1021/ol0518378

(Chemical Equation Presented) N,O-Peracylated cytidine and 2′-deoxycytidine derivatives in superheated water/DME solutions (oil bath at 125°C) undergo hydrolytic deamidation (and/or N-deacylation). Acylated starting materials derived from arylcarboxylic acids give the corresponding uridine esters cleanly, and such derivatives crystallize selectively from the cooled reaction mixtures in high yields.

Full text of DOI:10.1021/ol0518378

ORGANIC
LETTERS
2005
Vol. 7, No. 22
4903-4905
Hydrothermal Deamidation of
4-N-Acylcytosine Nucleoside
Derivatives: Efficient Synthesis of
Uracil Nucleoside Esters†
Ireneusz Nowak and Morris J. Robins*
Department of Chemistry and Biochemistry, Brigham Young UniVersity,
ProVo, Utah 84602-5700
morris_robins@byu.edu
Received August 1, 2005
ABSTRACT
N,O-Peracylated cytidine and 2
′
-deoxycytidine derivatives in superheated water/DME solutions (oil bath at 125
°C) undergo hydrolytic deamidation
(and/or N-deacylation). Acylated starting materials derived from arylcarboxylic acids give the corresponding uridine esters cleanly, and such
derivatives crystallize selectively from the cooled reaction mixtures in high yields.
Genetic information is altered by chemical or enzymatic
modification of nucleic acid sequences. Deamination of
cytosine in DNA is the most common promutagenic change.
This can result in generation of misfolded polypeptides and
dominant-negative proteins and can be the cause of other
mutations. “DNA cytidine deaminases” provide mechanisms
for the beneficial diversification of Ig genes and inhibition
of retroviral infections.1,2
isms.2 Different families catalyze deamination of substrates
including the cytosine base, nucleosides, nucleotides, oligo-
nucleotides, DNA, and RNA.
A limited number of chemical conversions of cytosine to
uracil compounds have been described. Diazotive deamina-
tion of cytosine with nitrous acid gave uracil, but yields were
poor because of decomposition(s) of the cytosine ring.4 Nitric
oxide and its progenitors also cause DNA damage and
mutations.5 Holy noted that treatment of 4-N-2′,3′,5′-tri-O-
tetraacetyl(or tetrabenzoyl)cytidine with 80% aqueous acetic
acid at reflux resulted in rearrangement of the acyl group
from N4 to N3 of the cytosine ring. Uridine was obtained
upon treatment of the N3 isomers with NaOMe/MeOH.6
Hydrolysis pathways for 3-methyl-2′-deoxycytidine were
dependent on pH, and fairly clean amino-hydrolysis was
Cytidine deaminases (CDAs) are metalloenzymes that
employ zinc at the active site to enhance the nucleophilicity
of a sequestered water molecule. Addition of the water
oxygen at C4 of the cytosine ring followed by enzyme-
mediated departure of ammonia and concomitant tautomer-
ization give the uracil products.3 Various zinc-dependent
multisubunit CDA enzymes have been found in all organ-
† Nucleic Acid Related Compounds. 129. Paper 128: Zhong, M.; Nowak,
I.; Robins, M. J. Org. Lett. 2005, 7, 4601-4604.
(1) Cascalho, M. J. Immunology 2004, 172, 6513-6518.
(2) Pham, P.; Bransteitter, R.; Goodman, M. F. Biochemistry 2005, 44,
2703-2715.
(4) Wempen, I.; Brown, G. B.; Ueda, T.; Fox, J. J. Biochemistry 1965,
4, 54-56.
(5) Wink, D. A.; Kasprzak, K. S.; Maragos, C. M.; Elespuru, R. K.;
Misra, M.; Dunams, T. M.; Cebula, T. A.; Koch, W. H.; Andrews, A. W.;
Allen, J. S.; Keefer, L. K. Science, 1991, 254, 1001-1003.
(6) Holy, A. Collect. Czech. Chem. Commun. 1979, 44, 1819-1827.
(3) Xiang, S.; Short, S. A.; Wolfenden, R.; Carter, C. W., Jr. Biochemistry
1997, 36, 4768-4774.
10.1021/ol0518378 CCC: $30.25
© 2005 American Chemical Society
Published on Web 10/07/2005

Scheme 1. Hydrolysis of the N-Acylcytosine Derivatives 1
observed only in basic solution (pH ∼12). Amino-hydrolysis
h. This presumably resulted from the increased steric
hindrance and/or hydrophobic effect of the 4-N-pivalyl group.
The starting material and product pairs were soluble in H2O/
DME (2:3) solutions at 125 °C. However, the product esters
derived from the arylcarboxylic acids, 2a,b,d,e, precipitated
upon cooling and were isolated directly from the reaction
mixtures in high yields (entries 1, 2, 4, and 5). Both the
starting materials 1 and products 2 derived from the aliphatic
acids were soluble in H2O/DME solutions at ambient
temperature. The latter products 2 were isolated by evapora-
tion of volatiles and chromatography of the residues.
of cytosine to uracil derivatives proceeds slowly at most pH
values,7,8 and a patent report9 of hydrolytic deamination of
cytidine in 1 M NaOH/H2O at 100 °C attests to the severity
of the conditions required.
Bisulfite-mediated hydrolytic deaminations are accelerated
by prior addition of the sulfite nucleophile at C6 of the 5,6-
double bond.10 The latter method is useful for small-scale
deaminations but impractical for gram-scale synthesis.
The need arose in our laboratory to convert multigram
quantities of 2′-deoxycytidine into 2′-deoxyuridine. At-
tempted application of Holy’s method to 2′-deoxy-4-N-3′,5′-
di-O-tri(4-methylbenzoyl)cytidine (1b) resulted in formation
of complex mixtures. We then substituted a neutral organic
cosolvent (1,4-dioxane or DME) for Holy’s acetic acid
component and heated the solution in a pressure vessel at
125 °C. We were delighted that 2′-deoxy-3′,5′-di-O-(4-
methylbenzoyl)uridine (2b) was produced in high yield and
that 2b crystallized from the reaction mixture upon cooling.
We then investigated the generality of this hydrothermal
deamination with other cytosine nucleoside derivatives 1
(Scheme 1, Table 1).
Because the O-acetyl and O-propionyl esters underwent
slow hydrolysis under our standard conditions, the reaction
time was shortened from 12 to 9 h (entries 8 and 9). The
amides derived from aliphatic acids, 1f-i, underwent
hydrolytic attack at both C4 and the amide carbonyl carbon
to give uridine 2 and cytidine 3 derivatives (2/3, 3:2-2:1)
(entries 6, 7, 8, and 9). Compound 1j (entry 10) with the
strongly electron-withdrawing trifluoroacetyl group at N4
underwent rapid hydrolysis of the amide bond. A minor
amount of uridine derivative 2c (16%) was formed plus the
cytidine compound 3j (66%, as a trifluoroacetate salt).
Two sites are susceptible to nucleophilic attack by water.
Addition at C4 followed by elimination of an amide results
in formation of uridine derivatives 2 (Scheme 2, path 1).
Attack at the amide carbonyl carbon results in hydrolysis of
the amide linkage and formation of a carboxylic acid and
cytidine products 3 (path 2). The preference for path 1 or 2
depends primarily on the electronic effects of the group
attached to the carbonyl carbon. However, steric and/or
hydrophobic effects might also be involved. Conjugation of
the carbonyl group with an aromatic ring results in clean
elimination of an aryl amide from C4. By contrast, amides
derived from aliphatic acids undergo partitioning between
paths 1 and 2, and in the case of the trifluoroacetamide 1j,
pathway 2 is clearly preferred.
Table 1. Hydrolysis of 1 (H2O/DME/∆)a To Give 2 and 3
entryb
R
R′
X
h
2c
3c
1 (a)
2 (b)
3 (c)
4 (d)
5 (e)
6 (f)
7 (g)
8 (h)
9 (i)
Ph
MePh
Ph
MePh
ClPh
Ph
iPr
Et
Ph
MePh
Ph
MePh
ClPh
t-Bu
iPr
Et
Me
CF3
H
H
PhCO2
MePhCO2
ClPhCO2
PhCO2
iPrCO2
EtCO2
12
12
12
12
12
12
12
9
82
84
e
87
85
53
54
49
55
16
d
d
d
d
25
38
30
25
66
Me
Ph
MeCO2
PhCO2
9
1
10 (j)
a Reactions were performed by the general procedure (see Supporting
Information). b Starting materials in parentheses. c Isolated % yield relative
to starting nucleoside (acylated and then subjected to hydrolysis). d Traces
of 3 were detected in the polar residue from the mother liquor. e Reaction
was complete (1H NMR), but 2c and benzamide were not separated by
chromatography or recrystallization.
We also considered the possibility that compounds 3 might
undergo hydrolytic deamination under our reaction condi-
(8) (a) Frick, L.; Mac Neela, J. P.; Wolfenden, R. Bioorg. Chem. 1987,
15, 100-108. (b) Snider, M. J.; Gaunitz, S.; Ridgway, C.; Short, S. A.;
Wolfenden, R. Biochemistry 2000, 39, 9746-9753.
In almost all cases, reactions were complete (or near
complete) within 12 h. Starting material (18%) remained after
heating the 4-N-(trimethylacetyl) compound (entry 6) for 12
(9) Fujishima, T.; Uchida, K.; Yoshino, H. Jpn. Kokai Tokkyo Koho 1973;
Chem. Abst. 1974, 80, 3772.
(10) (a) Shapiro, R.; Servis, R. E.; Welcher, M. J. Am. Chem. Soc. 1970,
92, 422-424. (b) Hayatsu, H.; Wataya, Y.; Kai, K. J. Am. Chem. Soc. 1970,
92, 724-726. (c) Shapiro, R.; DiFate, V.; Welcher, M. J. Am. Chem. Soc.
1974, 96, 906-912.
(7) Garret, E. R.; Tsau, J. J. Pharm. Sci. 1972, 61, 1052-1061.
4904
Org. Lett., Vol. 7, No. 22, 2005

hydrothermal conditions provide a new mode of highly
selective reactivity at C4 of cytosine nucleosides that are
activated by N,O-peracylation with arylcarboxylic acids.
Spontaneous crystallization of the product uracil nucleoside
4-chlorobenzoyl and 4-methylbenzoyl esters upon cooling
the hydrolysis reaction mixtures provides very convenient
access to these compounds 2.
Scheme 2. Possible Pathways for Hydrolysis of 1
Idoxuridine (2′-deoxy-5-iodouridine) has been in clinical
practice as an ophthalmic antiviral agent for decades.13 We
had demonstrated that iodination of 2′-deoxy-3′,5′-di-O-(4-
methylbenzoyl)uridine (2b) (ICl/CH2Cl2) gave the 5-iodo
compoud in 98% isolated yield.14 The present methodology
produces the crystalline intermediate 2b in 84% overall yield
from 2′-deoxycytidine (without optimization).15
In conclusion, we have demonstrated that N,O-peracyl
derivatives of cytosine nucleosides 1 undergo hydrolysis in
superheated H2O/DME to produce the corresponding O-
acyluracil nucleoside derivatives 2 (and/or O-acylcytosine
analogues 3). Almost exclusive selectivity for hydrolytic
attack at C4 to give 2 is observed with derivatives of
arylcarboxylic acids, whereas attack at the amide carbonyl
carbon to give 3 is competitive with aliphatic acid derivatives.
As expected, hydrolytic deamidation is much faster than
deamination under hydrothermal conditions. Precipitation of
arylcarboxylate esters of the product uracil nucleosides from
the hydrolytic reaction mixtures improves the convenience
and practicality of this method. Cytidine deaminases employ
coordination of zinc to strengthen the nucleophilicity of water
and proton transfer to promote the leaving ability of the
4-amino group. Enhancing the nucleophilicity of water with
a hydrogen bond-accepting cosolvent and increasing the
leaving ability of the amino group as a conjugated amide
are analogous to such strategies employed in enzyme active
sites.
tions. Byproducts in the mother liquor after filtration of 2b
were analyzed. The major component was p-toluamide, and
traces of p-toluic acid were present. Subjection of purified
3d to our standard hydrolysis conditions resulted in recovery
of almost all of the unchanged cytidine derivative 3d. Only
trace amounts of 2d (<10%) were detected by 1H NMR and
TLC. This confirmed the expectation8 that an unactivated
amino group at C4 is resistant to hydrolysis in H2O/DME at
125 °C.
The essentially exclusive attack by water at C4 of the aryl
amides is in marked contrast with our recent findings on
solvolysis of N,O-peracylated cytidine derivatives in super-
heated methanol.11 In those cases, the exocyclic amide bond
was cleaved. A minor amount (8%) of a 4-methoxy
compound (formed by attack of methanol at C4) was
observed only with the 4-N-toluyl derivative 1d. Attack by
water at C4 of the aryl amide derivatives 1a-e might result
from a combination of conjugated aromatic ring-carbonyl
group and hydrophobic effects in the aqueous DME solu-
tions. Another significant factor is the favorable equilibration
of the initial 4-hydroxypyrimidin-2-one intermediates into
their more thermodynamically stable (in aqueous environ-
ments) 4-keto tautomers, which is impossible with metha-
nolysis to 4-methoxypyrimidin-2-one derivatives.
Acknowledgment. We gratefully acknowledge pharma-
ceutical company gift funds (M.J.R.) and Brigham Young
University for support of this research.
Supporting Information Available: Experimental pro-
cedures, spectral data, and 13C NMR spectra for compounds
1f, 1j, 2a-e, 2g-i, and 3g-j. This material is available free
of charge via the Internet at http://pubs.acs.org.
OL0518378
It is noteworthy that perbenzoylated 2′-deoxycytidine
undergoes selective O-debenzoylation in a basic aqueous
solution (NaOH/H2O/MeOH/THF at 0 °C) without alteration
of the amide group on the nucleobase.12 Thus, our neutral
(12) (a) Rammler, D. H.; Khorana, H. G. J. Am. Chem. Soc. 1962, 84,
3112-3122. (b) Schaller, H.; Weimann, G.; Lerch, B.; Khorana, H. G. J.
Am. Chem. Soc. 1963, 85, 3821-3827.
(13) Fischer, P. H.; Lee, J. J.; Chen, M. S.; Lin, T.-S.; Prusoff, W. H.
Biochem. Pharmacol. 1979, 28, 3483-3486.
(14) Robins, M. J.; Barr, P. J.; Giziewicz, J. Can. J. Chem. 1982, 60,
554-557.
(15) Patent application filed.
(11) Nowak, I.; Conda-Sheridan, M.; Robins, M. J. J. Org. Chem. 2005,
70, 7455-7458.
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