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Chlorination studies. I. The reaction of aqueous hypochlorous acid with cytosine
BIOCHEMICAL
Vol. 48, No. 4, 1972
CHLORINATION STUDIES.
I.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE REACTION OF AQUEOUS HYPOCHLOROUS ACID WITH CYTOSINE
W. Patton,
V. Bacon, A. M. Duffield, B. Halpern*, W. Pereira and J. Lederberg
Department
of Genetics, Stanford,
Received
July
Stanford University California 94305
Y. Hoyano
Medical
Center,
lo,1972 SUMMARY
Cytosine, 5-chloroand 5-methylcytosine react with one equivalent of HOC1 to yield the corresponding 4-N-chloro derivatives (II, VIII and IX). Two or three equivalents of HOC1 with cytosine yield mixtures of II and VIII accompanied by two unstable compounds identified as a di- and trichlorocytosine respectively. Five equivalents of HOC1 when reacted with cytosine produce unstable triand tetrachloro-derivatives. Acidification of II with dilute HCl, HBr or HI affords 5-Cl, 5-Br and 5-iodocytosine respectively. An aqueous suspension of II partially converts phenylalanine into phenylacetaldehyde. Despite
our
dependence
chlorine1
little
is
The toxic
effect
of active
of bacterial acidIt
DNA.
known
are well
at labile basis
When cytosine (II)
The stable
acid (I)
was
was obtained
chlorine;
its
fragment
ions
*Present
address:
might
we have
might
with
displayed
of diagnostic
utility
Copyright @ 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.
ions
recorded
Department of Chemistry, N.S.W. 2500, Australia.
880
or destruction
of these directed 4
On the
aspects
of the
reactions.
furthur
physiological
The compound
were
bacteria.*
has been
these
in
conditions. of HOC1 4-N-chlorocontained
at --m/e 145,
one active 147 while
at --m/e 110 (M minus
Wollongong
by
of "hypochlorous
attempt
one equivalent
parent
kills
of halogenation
we now discuss
76% yield.
mass spectrum
the action
be generated
under
it
of water
modification
one serious
on cytosine
in
involve
studied
studies
reacted
by which
end products
only
own contemporaneous
of hypochlorous
cytosine
chlorine
which
on the treatment
the mechanism
However,
intermediates
of our
action
known.
many decades
about
Accordingly
on some DNA bases.
bases
for
University
Cl>+
College,
Vol. 48, No. 4, 1972
BIOCHEMICAL
and m/e 95 (M minus doublets
at 6 6.35
NHCl)+.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The olefinic
and 7.65
protons
(C-5,
6) evoked
NMR
(.I = 7Hz).
NHR I , II , III IV' ,
R=H R'=H R = C; R' = H R = H 'R' = Cl R = H', R' = Br
V, VII VIII: IX ,
H Dilute
aqueous
Protonation yield
HBr or HI converts
of the N-chloro-group
cytosine
direct
HCl,
plus
synthesis
VI.
of II
This
in
turn
II
into
followed
III,
of any of the three
I
lent
of HOC1 yielded
5-dichlorocytosine ponding
to M+, (M minus Cytosine
variable
yield
mixture
of II
abundant water
phases,
examined and ether products with
contained Cl)+,
with
three following
ether
by mass spectrometry. phases)
readily
corresponding
N-Cl
spray
of addition
groups
on TLC.
decomposed
of HOCl. extraction
to (M minus
ion
clusters
compounds and III.
H20)+,
(M minus
881
ions by their proved
corres-
NHCl)+.
these
in
(in
identified
mixture, were
were
of X and XI.
(aqueous Both
reaction
too unstable
(M minus
and
observed
positive
as a
were more
the ether
The mass spectrum Cl)+,
of 4-N,
a precipitate,
products;
of the reaction
as demonstrated
to VIII
HOCl,
The residues
to the molecular
These
one equiva-
ions
of reagent),
two other
Molecular
corresponding
contained
rate
of aqueous
TLC indicated
equivalents
significant
(M minus NHCO)+ and (M minus
on the
and VIII.
and IV) with
The mass spectrum
structurally
two equivalents
depending
starch-K1
as they ions
with
gave,
(III
and IX respectively.
(VIII)
a simple
ml
or 5-methylcytosine
VIII
to give
would
+X-CI~llIlYorY
H of 5-chlorn-
attack
derivatives.
d
Treatment
ion
cytosine
5-halocytosine
JI+HX+
IV and V respectively.
by halogen
halogenates5
R=H , R'=I R=H R'=CH R = R' = Cl 3 R = Cl , R' = CH 3
to isolate of X contains
NHCHOH)+ and
Vol. 48, No. 4, 1972
BIOCHEMICAL
HOCl) + .
The "trichlorocytosine"
(M minus it
plausibly
may be produced
The mass spectrum (M minus
from
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
structure X by reaction
of XI contains
ions
is
with
equivalent
less
to (M minus
equivalents
and
xc
determined
of HOC1 converted
by high
resolution
and C4H3N302C14.
On standing
decomposed
one hour
within
and the only
stable
(no molecular
(II)
This
is believed
The facile with with were
from
possibly
into
III
formed
(molecular
(C4H4N302C13)
by mass spectrometry) and VIII.
of phenylalanine
the reaction through
of XI
visible
were
a mixture
the tetrachloro-compound
a solution
to proceed
of the initial
by direct
amino
to phenylacid
formation transfer
NaOCl. 6
with
of sn N-chloro
of chlorine
between
7
formation
of N-chloro-derivatives
HOC1 has been
demonstrated.
NaOCl probably converted
ion
isolated
oxidized
a known product
amino acid derivative6 . II and phenylalanine.
(I)
at room temperature
acetaldehyde, reaction
cytosine
mass spectrometry)
end products
4-N-Chlorocytosine
which
HOCl)+
NHCI
X
treated
of HOCl.
NCl=CHOH)+.
Five
reaction
established:
one equivalent
NHCI
ions
well
into
involved III
The isolation*
initial
during
of cytosine
formation
the hydrolytic
from
of III
its from DNA
of N-chloro work-up
compounds
employed.
EXPERIMENTAL Aqueous Hasegawa.' Finnigan Chlorination
solutions
of HOC1 were
Low and high 1015 Quadrupole reactions of HOC1 (1 hour
the base
in water.
by the method
resolution
mass spectra
and Varian
MAT 711 instruments
were
addition
prepared
per
UV spectra
conducted
obtained
at room temperature
gm equivalent) were
were
recorded
882
to a stirred in water.
of Higuchi
and
with
operating
at 70 eV.
by the dropwise suspension
of
TLC was carried
out
BIOCHEMICAL
Vol. 48, No. 4, 1972
with
cellulose
were
recorded
MN300 CM plates by a Varian
4-N-Chlorocytosine (decomp.).
3.88) II
(II)
C, 33.00;
H, 2.85;
.
3.94)
N, 29.20; Cl,
Acetic
(VIII),
Found C, 26.47;
requires:
using
phase.
NMR spectra
D20iNaOD as solvent.
Cl,
m.p.
24.14.
24.14%.
175-200" C4H4N30C1
Xmax 226 nm (log
acid
(100')
for
210"
(decomp.)
1 hour
E =
converted
(95% yield).
4-N-5-Dichlorocytosine decomp.)
as the mobile
in 76% yield,
N, 28.90;
E = 3.87).
5-chlorocytosine
water
was obtained
H, 2.77;
and 276 nm (log
into
using
HA 100 instrument
Found C, 32.99;
requires:
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
C, 26.63;
H, 1.79;
H, 1.67;
and 280 nm (log
m.p.
N, 23.15;
N, 23.35;
E = 3.94).
Cl,
Cl,
(lit.4m.p.
39.20.
39.35%.
192"
C4H3N30C12
X max 226 nm (log
The NMR spectrum
contained
E =
a singlet
at
6 = 7.87. 4-N-Chloro-5-methvlcvtosine C, 37.87;
H, 3.77;
H, 3.76;
N, 26.35;
(log
3.97).
E =
Conversion Water for
(1 ml.)
(III),
cytosine
(IV)
22.60.
22.22%.
(II)
m.p.
phase
phase
224-225"
with
aqueous
Found: C, 37.60;
E = 3.92)
to 5-Halocytosines
from
(lit.12 with
and 271 nm
(III,
HX (X = Cl,
these
lyophilized
Reaction
m.p.
Br,
IV and V) I)
(1 ml.
1N)
reactions
overnight
stirred
for
by gas chromatography-mass
between
mass spectra
were
ether
and the
immediately
XI Rf = 0.68. with
to phenylalanine
lhour
of HOCl.
was partitioned and then
of 4-N-Chlorocytosine was added
225-245").
3 and 5 Equivalents
X had an Rf = 0.76;
(130 mg.)
(decomp.).
C5H6N30C1 requires:
hmax 223 nm (log
was treated
of Cytosine
The aqueous
recorded.
II
210-220"
(NH OH) and the solid collected. 5-Chloro4 10 60% yield, m.p. 289-292" (lit. m.p. 289-292*), 5-bromo11 55% yield, m.p. 250-255' (lit. m.p. 245-2554, 5-iodocytosine
45% yield,
reaction
Cl,
m.p.
made alkaline
Reaction
II
Cl,
plus
10 minutes,
aqueous
N, 26.10;
of 4-N-Chlorocytosine
cytosine
(V)
(IX)
under
Phenylalanine. (140 mg.)
in water
(10 ml.)
a N2 atmosphere.
Ether
extraction
spectrometry
883
identified
phenylacetaldehyde.
and the followed
Vol. 48, No. 4, 1972
Acknowledgment. and Space
Administration
BIOCHEMICAL
This
work (Grant
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
was supported
by the National
Aeronautics
No. NGR-05-020-004). REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
E. J. Laubusch in Chlorine its Manufacture, Properties and Uses, Ed. J. S. Sconce, American Chemical Society Monograph Series, New York, 1962, pp. 457-458. M. A. Benarde, W. B. Snow, V. P. Olivieri and B. Davidson, m. Microbial., Is, 257 (1967); J. Lederberg, Current Contents, Life Sci., 14M: 1 (1971). G. Holst, Chem. Rev.,-&, 186 (1954). H. Hayatsu, S. K. Pan and T. Dkita, Chem. Pharm. Bull. Japan, 2, --2189 (1971). D. J. Brown, J. Sot. Chem. I&., 69, 353 (1950). K. Langheld, Chezs., 42, 392, 2360 (1909). T. H. Higuchi, A. Hurwitz and I. H. Pitman, 2. -Pharm. -Sci. A. Hussain, 3, 371 (1972). R. Prat, Cl. Nofre and A. Cier, --Ann. Inst. Past., -,114 595 (1968). T. Higuchi and J. Hasegawa, 2. Phys. C&em., 3, 796 (1965). I. Wempen and J. J. Fox, 2. -Med. w., -,6 688 (1963). G. E. Hilbert and E. F. Jansen, J. --Amer. Chem. z., x, 134 (1934). T. B. Johnson and C. 0. Johns, 2. --Biol. Chem., 1, 305 (1906).
884
Vol. 48, No. 4, 1972
CHLORINATION STUDIES.
I.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE REACTION OF AQUEOUS HYPOCHLOROUS ACID WITH CYTOSINE
W. Patton,
V. Bacon, A. M. Duffield, B. Halpern*, W. Pereira and J. Lederberg
Department
of Genetics, Stanford,
Received
July
Stanford University California 94305
Y. Hoyano
Medical
Center,
lo,1972 SUMMARY
Cytosine, 5-chloroand 5-methylcytosine react with one equivalent of HOC1 to yield the corresponding 4-N-chloro derivatives (II, VIII and IX). Two or three equivalents of HOC1 with cytosine yield mixtures of II and VIII accompanied by two unstable compounds identified as a di- and trichlorocytosine respectively. Five equivalents of HOC1 when reacted with cytosine produce unstable triand tetrachloro-derivatives. Acidification of II with dilute HCl, HBr or HI affords 5-Cl, 5-Br and 5-iodocytosine respectively. An aqueous suspension of II partially converts phenylalanine into phenylacetaldehyde. Despite
our
dependence
chlorine1
little
is
The toxic
effect
of active
of bacterial acidIt
DNA.
known
are well
at labile basis
When cytosine (II)
The stable
acid (I)
was
was obtained
chlorine;
its
fragment
ions
*Present
address:
might
we have
might
with
displayed
of diagnostic
utility
Copyright @ 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.
ions
recorded
Department of Chemistry, N.S.W. 2500, Australia.
880
or destruction
of these directed 4
On the
aspects
of the
reactions.
furthur
physiological
The compound
were
bacteria.*
has been
these
in
conditions. of HOC1 4-N-chlorocontained
at --m/e 145,
one active 147 while
at --m/e 110 (M minus
Wollongong
by
of "hypochlorous
attempt
one equivalent
parent
kills
of halogenation
we now discuss
76% yield.
mass spectrum
the action
be generated
under
it
of water
modification
one serious
on cytosine
in
involve
studied
studies
reacted
by which
end products
only
own contemporaneous
of hypochlorous
cytosine
chlorine
which
on the treatment
the mechanism
However,
intermediates
of our
action
known.
many decades
about
Accordingly
on some DNA bases.
bases
for
University
Cl>+
College,
Vol. 48, No. 4, 1972
BIOCHEMICAL
and m/e 95 (M minus doublets
at 6 6.35
NHCl)+.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The olefinic
and 7.65
protons
(C-5,
6) evoked
NMR
(.I = 7Hz).
NHR I , II , III IV' ,
R=H R'=H R = C; R' = H R = H 'R' = Cl R = H', R' = Br
V, VII VIII: IX ,
H Dilute
aqueous
Protonation yield
HBr or HI converts
of the N-chloro-group
cytosine
direct
HCl,
plus
synthesis
VI.
of II
This
in
turn
II
into
followed
III,
of any of the three
I
lent
of HOC1 yielded
5-dichlorocytosine ponding
to M+, (M minus Cytosine
variable
yield
mixture
of II
abundant water
phases,
examined and ether products with
contained Cl)+,
with
three following
ether
by mass spectrometry. phases)
readily
corresponding
N-Cl
spray
of addition
groups
on TLC.
decomposed
of HOCl. extraction
to (M minus
ion
clusters
compounds and III.
H20)+,
(M minus
881
ions by their proved
corres-
NHCl)+.
these
in
(in
identified
mixture, were
were
of X and XI.
(aqueous Both
reaction
too unstable
(M minus
and
observed
positive
as a
were more
the ether
The mass spectrum Cl)+,
of 4-N,
a precipitate,
products;
of the reaction
as demonstrated
to VIII
HOCl,
The residues
to the molecular
These
one equiva-
ions
of reagent),
two other
Molecular
corresponding
contained
rate
of aqueous
TLC indicated
equivalents
significant
(M minus NHCO)+ and (M minus
on the
and VIII.
and IV) with
The mass spectrum
structurally
two equivalents
depending
starch-K1
as they ions
with
gave,
(III
and IX respectively.
(VIII)
a simple
ml
or 5-methylcytosine
VIII
to give
would
+X-CI~llIlYorY
H of 5-chlorn-
attack
derivatives.
d
Treatment
ion
cytosine
5-halocytosine
JI+HX+
IV and V respectively.
by halogen
halogenates5
R=H , R'=I R=H R'=CH R = R' = Cl 3 R = Cl , R' = CH 3
to isolate of X contains
NHCHOH)+ and
Vol. 48, No. 4, 1972
BIOCHEMICAL
HOCl) + .
The "trichlorocytosine"
(M minus it
plausibly
may be produced
The mass spectrum (M minus
from
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
structure X by reaction
of XI contains
ions
is
with
equivalent
less
to (M minus
equivalents
and
xc
determined
of HOC1 converted
by high
resolution
and C4H3N302C14.
On standing
decomposed
one hour
within
and the only
stable
(no molecular
(II)
This
is believed
The facile with with were
from
possibly
into
III
formed
(molecular
(C4H4N302C13)
by mass spectrometry) and VIII.
of phenylalanine
the reaction through
of XI
visible
were
a mixture
the tetrachloro-compound
a solution
to proceed
of the initial
by direct
amino
to phenylacid
formation transfer
NaOCl. 6
with
of sn N-chloro
of chlorine
between
7
formation
of N-chloro-derivatives
HOC1 has been
demonstrated.
NaOCl probably converted
ion
isolated
oxidized
a known product
amino acid derivative6 . II and phenylalanine.
(I)
at room temperature
acetaldehyde, reaction
cytosine
mass spectrometry)
end products
4-N-Chlorocytosine
which
HOCl)+
NHCI
X
treated
of HOCl.
NCl=CHOH)+.
Five
reaction
established:
one equivalent
NHCI
ions
well
into
involved III
The isolation*
initial
during
of cytosine
formation
the hydrolytic
from
of III
its from DNA
of N-chloro work-up
compounds
employed.
EXPERIMENTAL Aqueous Hasegawa.' Finnigan Chlorination
solutions
of HOC1 were
Low and high 1015 Quadrupole reactions of HOC1 (1 hour
the base
in water.
by the method
resolution
mass spectra
and Varian
MAT 711 instruments
were
addition
prepared
per
UV spectra
conducted
obtained
at room temperature
gm equivalent) were
were
recorded
882
to a stirred in water.
of Higuchi
and
with
operating
at 70 eV.
by the dropwise suspension
of
TLC was carried
out
BIOCHEMICAL
Vol. 48, No. 4, 1972
with
cellulose
were
recorded
MN300 CM plates by a Varian
4-N-Chlorocytosine (decomp.).
3.88) II
(II)
C, 33.00;
H, 2.85;
.
3.94)
N, 29.20; Cl,
Acetic
(VIII),
Found C, 26.47;
requires:
using
phase.
NMR spectra
D20iNaOD as solvent.
Cl,
m.p.
24.14.
24.14%.
175-200" C4H4N30C1
Xmax 226 nm (log
acid
(100')
for
210"
(decomp.)
1 hour
E =
converted
(95% yield).
4-N-5-Dichlorocytosine decomp.)
as the mobile
in 76% yield,
N, 28.90;
E = 3.87).
5-chlorocytosine
water
was obtained
H, 2.77;
and 276 nm (log
into
using
HA 100 instrument
Found C, 32.99;
requires:
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
C, 26.63;
H, 1.79;
H, 1.67;
and 280 nm (log
m.p.
N, 23.15;
N, 23.35;
E = 3.94).
Cl,
Cl,
(lit.4m.p.
39.20.
39.35%.
192"
C4H3N30C12
X max 226 nm (log
The NMR spectrum
contained
E =
a singlet
at
6 = 7.87. 4-N-Chloro-5-methvlcvtosine C, 37.87;
H, 3.77;
H, 3.76;
N, 26.35;
(log
3.97).
E =
Conversion Water for
(1 ml.)
(III),
cytosine
(IV)
22.60.
22.22%.
(II)
m.p.
phase
phase
224-225"
with
aqueous
Found: C, 37.60;
E = 3.92)
to 5-Halocytosines
from
(lit.12 with
and 271 nm
(III,
HX (X = Cl,
these
lyophilized
Reaction
m.p.
Br,
IV and V) I)
(1 ml.
1N)
reactions
overnight
stirred
for
by gas chromatography-mass
between
mass spectra
were
ether
and the
immediately
XI Rf = 0.68. with
to phenylalanine
lhour
of HOCl.
was partitioned and then
of 4-N-Chlorocytosine was added
225-245").
3 and 5 Equivalents
X had an Rf = 0.76;
(130 mg.)
(decomp.).
C5H6N30C1 requires:
hmax 223 nm (log
was treated
of Cytosine
The aqueous
recorded.
II
210-220"
(NH OH) and the solid collected. 5-Chloro4 10 60% yield, m.p. 289-292" (lit. m.p. 289-292*), 5-bromo11 55% yield, m.p. 250-255' (lit. m.p. 245-2554, 5-iodocytosine
45% yield,
reaction
Cl,
m.p.
made alkaline
Reaction
II
Cl,
plus
10 minutes,
aqueous
N, 26.10;
of 4-N-Chlorocytosine
cytosine
(V)
(IX)
under
Phenylalanine. (140 mg.)
in water
(10 ml.)
a N2 atmosphere.
Ether
extraction
spectrometry
883
identified
phenylacetaldehyde.
and the followed
Vol. 48, No. 4, 1972
Acknowledgment. and Space
Administration
BIOCHEMICAL
This
work (Grant
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
was supported
by the National
Aeronautics
No. NGR-05-020-004). REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
E. J. Laubusch in Chlorine its Manufacture, Properties and Uses, Ed. J. S. Sconce, American Chemical Society Monograph Series, New York, 1962, pp. 457-458. M. A. Benarde, W. B. Snow, V. P. Olivieri and B. Davidson, m. Microbial., Is, 257 (1967); J. Lederberg, Current Contents, Life Sci., 14M: 1 (1971). G. Holst, Chem. Rev.,-&, 186 (1954). H. Hayatsu, S. K. Pan and T. Dkita, Chem. Pharm. Bull. Japan, 2, --2189 (1971). D. J. Brown, J. Sot. Chem. I&., 69, 353 (1950). K. Langheld, Chezs., 42, 392, 2360 (1909). T. H. Higuchi, A. Hurwitz and I. H. Pitman, 2. -Pharm. -Sci. A. Hussain, 3, 371 (1972). R. Prat, Cl. Nofre and A. Cier, --Ann. Inst. Past., -,114 595 (1968). T. Higuchi and J. Hasegawa, 2. Phys. C&em., 3, 796 (1965). I. Wempen and J. J. Fox, 2. -Med. w., -,6 688 (1963). G. E. Hilbert and E. F. Jansen, J. --Amer. Chem. z., x, 134 (1934). T. B. Johnson and C. 0. Johns, 2. --Biol. Chem., 1, 305 (1906).
884