Vol. 48, No. 4, 1972 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CHLORINATION STUDIES. I. THE REACTION OF AQUEOUS HYPOCHLOROUS ACID
WITH CYTOSINE
W. Patton, V. Bacon, A. M. Duffield, B. Halpern®, Y. Hoyano
W. Pereira and J. Lederberg

Department of Genetics, Stanford University Medical Center,
Stanford, Califormia 94305

Received July 10,1972
SUMMARY

Cytosine, 5-chloro- and 5-methylcytosine react with one equivalent of
HOC] to yield the corresponding 4-N-chloro derivatives (II, VIII and IX).

Two or three equivalents of HOCl with cytosine yield mixtures of II and
VIII accompanied by two unstable compounds identified as a di- and tri-
chlorocytosine respectively. Five equivalents of HOC1 when reacted with
cytosine produce unstable tri- and 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 for many decades on the treatment of water by
chlorine! little is known about the mechanism by which it kills bacteria. 2
The toxic effect of active chlorine might involve modification or destruction
of bacterial DNA. Accordingly we have studied the action of “hypochlorous
acid"3 on some DNA bases. The stable end products of halogenation of these
bases are well known. However, only one serious attempt has been directed
at labile intermediates which might be generated in these reactions.“ On the
basis of our own contemporaneous studies we now discuss furthur aspects of the
action of hypochlorous acid on cytosine under physiological conditions.

When cytosine (I) was reacted with one equivalent of HOC1 4-N-chloro-
cytosine (II) was obtained in 76% yield. The compound contained one active

chlorine; its mass spectrum displayed parent ions at m/e 145, 147 while

fragment ions of diagnostic utility were recorded at m/e 110 (M minus c1yt

 

*Present address: Department of Chemistry, Wollongong University College,
N.S.W. 2500, Austrat‘a.

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Vol. 48, No. 4, 1972 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and m/e 95 (M minus NHC1)+. The olefinic protons (C-5, 6) evoked NMR

doublets at 6 6.35 and 7.65 (J = 7Hz).

NHR
a
Na~* R I, R= H, R' =H V, R= H, R' =I
3 ‘ II, R= Cl, R' =H VII, R= H, R' = GH,
le Ill, R= H, R' = Cl VIII, R= R' = Cl
N IV, R= H, R' = Br IX, R= Cl, R' = GH,
H

Dilute aqueous HCl, HBr or HI converts II into III, IV and V respectively.
Protonation of the N-chloro-group of II followed by halogen ion attack would
yield cytosine plus VI. This in tum halogenates> cytosine to give a simple

direct synthesis of any of the three 5-halocytosine derivatives.

“HD -Cl _

nz
ID + HX 0 —» I+x-cl—H Worry

MI

Treatment of 5-chlorm- or 5~methylcytosine (III and IV) with one equiva-
lent of HOC] yielded VIII and IX respectively. The mass spectrum of 4-N,
5-dichlorocytosine (VIII) contained structurally significant ions corres-
ponding to Mt, (M minug Cl)+, (M minus NHCO)* and (M minus NHC1)T.

Cytosine gave, with two equivalents of aqueous HOC], a precipitate, (in
variable yield depending on the rate of addition of reagent), identified as a
mixture of II and VIII. TLC indicated two other products; these were more
abundant with three equivalents of HOCl. The residues in the ether and
water phases, following ether extraction of the reaction mixture, were
examined by mass spectrometry. Molecular ion clusters were observed (aqueous
and ether phases) corresponding to the molecular ions of X and XI. Both
products contained N-Cl groups as demonstrated by their positive reaction
with starch-KI spray on TLC. These compounds proved too unstable to isolate
as they readily decomposed to VIII and III. The mass spectrum of X contains

fons corresponding to (M minus H,0)*, (M minus Cl1)+, (M minus NHCHOH)+ and

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Vol, 48, No. 4, 1972 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(M minus HOC1)+. The “trichlorocytosine" structure is less well established;
it plausibly may be produced from X by reaction with one equivalent of HOC1.
The mass spectrum of XI contains ions equivalent to (M minus Hoc1)+ and

(M minus NC1=cHoH)T,

NHCI NHC!
NZ c nZ cI
Ay OH AY OH

HH ci”

x XI

Five equivalents of HOC] converted cytosine (I) into a mixture (molecular
ions determined by high resolution mass spectrometry) of XI (C,H,N,0,C1,)
and C,H,N,0,C1,. On standing at room temperature the tetrachloro-compound
decomposed within one hour (no molecular ion visible by mass spectrometry)
and the only stable end products isolated were III and VIII.

4-N-Chlorocytosine (II) oxidized a solution of phenylalanine to phenyl-
acetaldehyde, a known product from the reaction of the amino acid with naoc1.°
This reaction is believed to proceed through initial formation of an N-chloro
amino acid derivative? possibly formed by direct transfer of chlorine between
II and phenylalanine.’

The facile formation of N-chloro-derivatives of cytosine from its
reaction with HOC] has been demonstrated. The isolation® of III from DNA

treated with NaOCl probably involved initial formation of N-chloro compounds

which were converted into III during the hydrolytic work-up employed.

EXPERIMENTAL

Aqueous solutions of HOC1 were prepared by the method of Higuchi and
Hasegawa.” Low and high resolution mass spectra were obtained with
Finnigan 1015 Quadrupole and Varian MAT 711 instruments operating at 70 eV.
Chlorination reactions were conducted at room temperature by the dropwise
addition of HOC] (1 hour per gm equivalent) to a stirred suspension of

the base in water. UV spectra were recorded in water. TLC was carried out

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Vol. 48, No. 4, 1972 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with cellulose MN300 CM plates using water as the mobile phase. NMR spectra
were recorded by a Varian HA 100 instrument using D,0/NaOD as solvent.
4-N-Chlorocytosine (II) was obtained in 76% yield, m.p. 175-200°
(decomp.). Found C, 32.99; H, 2.85; N, 29.20; Cl, 24.14. C,H,N,0CL
requires: C, 33.00; H, 2.77; N, 28.90; Cl, 24.14%. A max 226 nm (log € =
3.88) and 276 nm (log « = 3.87). Acetic acid (100°) for 1 hour converted

II into 5-chlorocytosine (95% yield).

4-N-5-Dichlorocytosine (VIII), m.p. 210° (decomp.) (1it.4m.p. 192°
decomp.). Found C, 26.47; H, 1.79; N, 23.15; Cl, 39.20. C,HN,0C1,
requires: C, 26.63; H, 1.67; N, 23.35; Cl, 39.35%. Maa 226 nm (log « =
3.94) and 280 nm (log « = 3.94). The NMR spectrum contained a singlet at

6 = 7.87.

4-N-Chloro-5-methylcytosine (IX) m.p. 210-220° (decomp.). Found:

C, 37.87; H, 3.773; N, 26.10; Cl, 22.60. CoH,N,0C1 requires: C, 37.60;

H, 3.76; N, 26.35; Cl, 22.22%. Anax 223 nm (log ¢« = 3.92) and 271 nm

(log « = 3.97).

Conversion of 4-N-Chlorocytosine (II) to 5-Halocytosines (III, IV and V)
Water (1 ml.) plus II was treated with aqueous HX (X = Cl, Br, I) (1 ml. 1N)

for 10 minutes, made alkaline (NH , OH) and the solid collected. 5-Chloro-

cytosine (III), 60% yield, m.p. 289~292° cuit. 2°

cytosine (IV) 55% yield, m.p. 250-255° ait. m.p. 245-255%, S-iodocytosine

12

m.p. 289-292°), 5-bromo-

(V) 45% yield, mop. 224-225° (lit.”” m.p. 225-245°).

Reaction of Cytosine with 3 and 5 Equivalents of HOCl.
The aqueous phase from these reactions was partitioned between ether and the
aqueous phase lyophilized overnight and then mass spectra were immediately
recorded, X had an Re = 0.76; XI Re = 0.68.

Reaction of 4-N-Chlorocytosine with Phenylalanine.
II (130 mg.) was added to phenylalanine (140 mg.) in water (10 ml.) and the

reaction stirred for 1 hour under a N, atmosphere. Ether extraction followed

2
by gas chromatography-mass spectrometry identified phenylacetaldehyde.

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Vol. 48, No. 4, 1972 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Acknowledgment, This work was supported by the National Aeronautics

and Space Administration (Grant No. NGR-05-020-004).

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