volume 10 Number 201982
Nucleic Acids Research
N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective
modification of guanine in DNA
Hikoya Hayatsu , Yasuhiro Yamashita , Seiko Yui , Yuriko Yamagata , Ken-ichi Tomita^ and
Kazuo Negishi
1
Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700, and 2Faculty
of Pharmaceutical Sciences, Osaka University, Yamada-oka, Suita, Osaka 565, Japan
Received 6 July 1982
ABSTRACT
When guanine-, adenine- and cytosine-nucleosides and nucleotides were
treated with formaldehyde and then with b i s u l f i t e , stable N-sulfomethyl compounds were formed. N^-Sulfomethyl guanine, N6-sulfomethyladenine, N 4 -sulfomethylcytosine and N°-sulfomethyl-9-6-D-arabinofuranosyladenine were isolated
as crystals and characterized. A guanine-specific sulfomethylation was
brought about by treatment of denatured single-stranded DNA with formaldehyde
and then with b i s u l f i t e at pH 7 and 4°C. Since native double-stranded DNA
was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides.
INTRODUCTION
I t is known that formaldehyde reacts with the amino groups of nucleic
acid bases to give N-methylol derivatives ( 1 , 2 ) . The N-methylols are unstable and readily regenerate the parent amino groups: the methylols can be
detected in aqueous solutions containing formaldehyde, but they are rapidly
deformylated to give the parent bases in the absence of formaldehyde. We
report here that the.N-methylols can be converted into stable N-sulfomethyl
derivatives by treatment with sodium b i s u l f i t e , and that a selective modification of the guanine residues in DNA can be achieved by this reaction.
RESULTS AND DISCUSSION
Formation of N-sulfomethyl derivatives of guanine, adenine and cytosine, and
t h e i r nucleosides and nucleotides
I t is known that amines can be sulfomethylated by treatment with formaldehyde and b i s u l f i t e (3).
We treated adenosine in an aqueous solution with
formaldehyde to form i t s N-methylol ( 1 ) , and then added sodium b i s u l f i t e to
the reaction mixture.
Paper chromatography of the mixture (see Table 1) re-
vealed a new u l t r a v i o l e t absorbing spot that had a smaller Rp value than
adenosine.
In paper electrophoresis at pH 7, the product behaved as an
© IRL Press Limited, Oxford, England.
0305-1048/82/1020-6281S 2.00/0
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Nucleic Acids Research
NHCH2SO3-
NHCH2SO3"
R
R'NH 2 + HCHO
anion.
R'NHCHjOH
HSO3= » R'NHCHjSOf
Ultraviolet absorption spectrum of the compound resembled that of
N -methyladenosine (4).
These properties suggested that the product was N -
sulfomethy1adenos i ne.
Table 1 .
Properties of N-sul fomethy derivatives in comparison with the
parent ami no-compounds
N-Sulfomethyl
derivative of
Re in paper
enromatography—
Sol vent. 1
Guanine
Adenine
Cytosine
Guanosine
Adenosine
Cytidine
Deoxyguanosine
Deoxyadenosine
Deoxycytidine
9-0-D-Arabinofuranosyladenine
GMP
AMP
CMP
dGMP
dAMP
dCMP
GDP
ADP
ATP
dGTP
dATP
dCTP
Guanosine 3 ' , 5 ' cyclic phosphate
Adenosine 3 ' , 5 ' cyclic phosphate
R
AMP i n paper
electro phoresis
at pH 7
^max
a t pH 7
(ran)
Solvent 2
0.32
0.54
0.43
0.11
0.27
0.11
0.19
0.35
0.28
0.44
(0.69)^
(0.86)
(0.80
(0.42)
(0.76)
(0.58)
(0.55)
(0.82)
(0.73)
(0.79)
0.29
0.48
0.49
0.14
0.46
0.36
0.19
0.29
0.42
0.59
(0.29)
(0.48)
(0.51)
(0.26)
(0.56)
(0.55)
(0.38)
(0.62)
(0.63)
(0.51)
0.86
0.97
1.11
0.54
0.51
0.67
0.46
0.55
0.85
0.79
(0)
(0)
(0)
(0)
(0)
(0)
(0)
276
268
271
254
268
273
255
268
273
268
0.07
0.15
0.20
0.08
0.16
0.08
0.15
0.09
0.07
0.13
0.04
0.07
0.17
(0.17)
(0.52)
(0.31)
(0.27)
(0.56)
(0.44)
(0.25)
(0.37)
(0.27)
(0.31)
(0.26)
(0.17)
0.03
0.05
0.03
0.04
0.14
0.08
0.03
0.03
0.01
0.02
0.04
0.04
(0.26)
(0.04)
(0.10)
(0.04)
(0.06)
(0.14)
(0.11)
(0.03)
(0.07)
(0.05)
(0.03)
(0.06)
(0.06)
0.11 (0.30)
1.33
1.35
1.45
1.43
1.55
1.70
1.60
1.67
1.78
1.63
1.85
1.85
1.18
(1.00)
(1.00)
(1.19)
(1.07)
(1.07)
(1.15)
(1.23)
(1.25)
(1.58)
(1.40)
(1.53)
(1.64)
(0.60)
255
268
273
255
268
275
256
268
268
255
267
272
256
0.22 (0.61)
0.30 (0.48)
1.18 (0.60)
268
(0)
(0)
0
a_) Solvent 1, isobutyric acid-0.5 N NH.OH (5 : 3 ) ; solvent 2, isopropanolammonia-water ( 7 : 1 : 2 ) . b_) In 0.05 M sodium phosphate buffer, 300 V,
1 hr. c) The values in parentheses are those for parent ami no-compounds.
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Nucleic Acids Research
We treated guanine, adenine and cytosine f i r s t with formaldehyde and
then with sodium bisulfite, and were able to isolate the products as crystals
(see Experimental Section). Structures of these products were assigned as
N -sulfomethylguanine, N -sulfomethyladenine and N -sulfomethylcytosine, respectively, on the basis of elemental analysis, NMR spectra and ultraviolet
absorption spectra (resemblance to corresponding N-alkyl bases). N -Sulfomethylcytosine (NH^ salt) formed crystals suitable for X-ray diffraction
analysis. The crystal and molecular structure was determined, and the results established the structure as assigned (Fig. 1).
Nucleosides and nucleotides of guanine, adenine and cytosine were treated with formaldehyde and bisulfite, and the reaction mixtures were analyzed
by paper chromatography. Products eluted from the paper chromatograms were
examined for their ultraviolet absorption spectra and for their mobility in
paper electrophoresis. As shown in Table 1, N -sulfomethylguanine-, N sulfomethyladenine-, and N -sulfomethylcytosine-nucleosides and nucleotides
were formed from each parent compound. A nucleoside, N -sulfomethyl-9-B-Darabinofuranosyladenine, was isolated as a crystalline material in a preparative experiment, and was chracterized.
The N-sulfomethyl groups were generally stable at pH 1-11 at room
temperature. They regenerated the parent ami no groups on treatment with
0.1 N NaOH at 100°C for 30 min or with 1 N NaOH at 25°C for 12 hrs.
Studies on the reaction conditions for modification of mononucleotides
A preliminary study showed that treatments of the amino-nucleotides with
a pre-formed mixture of formaldehyde and bisulfite did not yield the sulfomethylated derivatives. Namely, the reactions must be carried out sequenti a l l y , the methylol-formation followed by the treatment with bisulfite. I t
was also recognized that the sulfonation by treatment with bisulfite proceeded to a greater extent at lower temperatures. At higher temperatures, e.g.,
Fig. 1.
Molecular structure of N4-sulfomethylcytosine.
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Nucleic Acids Research
at 37°C, the treatment with b i s u l f i t e yielded higher extents of parent aminonucleotides than at lower temperatures, although the reaction reached completion in shorter periods.
Obviously, in this bisulfite-treatment, both the
sulfonation to form the product and the deformylation to regenerate the amino
group were taking place simultaneously.
The y i e l d of the N-sulfomethyl com-
pound, therefore, is governed by the competition between these two reactions.
A quantitative study on the effect of pH in the sulfonation step was
carried out by use of paper chromatography.
The results presented in Table
2 show that the reactions on the amino groups can take place at both pH 5.3
Table 2.
Sulfomethylation of 5'-nucleotides and nucleosides.
pH in the b i s u l f i t e treatment!)
Substrate
Reaction extent (%.substrate consumed)
pH 5. 3
pH 2.4
4 hr
28 hr
4 hr
GMP
56
87
70
89
dGMP
31
50
53
72
AMP
12
51
69
90
CMP
50^
-
58^
84^
}
-
52^
70^
25^
9
17 £)
Deoxycytidine
5-Methyldeoxycyti di ne
Effect of
4215
28 hr
a) Solutions containing 0.05 M substrate, 1 M formaldehyde and 0.05 M sodium
phosphate, the final pH being 7 . 1 , were allowed to stand at room temperature
for 16 hr. The mixtures were cooled in ice and NaHS03 was added to them at
an amount 300 mg/ml. The mixtures as such, pH 5.3, or after adjustment of
pH to 2.4 by addition of HC1, were allowed to stand at 1° for 4 hr or 28 hr.
Exact volumes of the solutions were subjected to paper chromatography
(solvent: isobutyric acid-0.5 N NH4OH, 5 : 3) to quantitatively determine
the amounts of substrates consumed during the reaction. For reactions of
cytidine 5'-phosphate and deoxycytidine, the reaction mixtures were made
alkaline (5) before submitting them to the chromatographic analysis. The
quantifications were done spectrophotometrically for duplicate samples. An
appropiate control was prepared for each reaction. Absorbances of the Nsulfomethyl derivatives on the chromatograms were also recorded, and their
molar absorption coefficients were calculated on the assumption that they
were the sole reaction products. The values thus found were, 15.0 x 103
(A256 at pHfi7) for N2-sulfomethylguanosine 5'-phosphate, 20.0 x 103 (A268 a t
pH 7) for N°-sulfomethyladenosine 5'-phosphate, and 19.5 x 103 (A2go at pH 1)
for N -sulfomethylcytidine 5'-phosphate. b) About 2 % uridine 5'-pnosphate
was formed, the rest being N-sulfomethylcytidine 5'-phosphate. c) No
deaminations were detected, 6) About 6 % deoxyuridine was formed, e) About
8 % deoxyuridine was formed.
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and pH 2.4, and that the reactions are faster at pH 2.4 than at pH 5.3.
The
products in these reactions were only the N-sulfomethyl derivatives, except
for the cytosine compounds which were slowly deaminated by the b i s u l f i t e
treatment (5).
In the modification of cytosine compounds, however, the de-
aminations can be minimized by l i m i t i n g the b i s u l f i t e treatment to shorter
periods or by fixing the pH of the treatment at lower than 3.
I t was also found that the b i s u l f i t e treatment, i f carried out at pH 7,
can produce almost an exclusive reaction for guanine nucleotide: thus, treatment of guanosine 5'-phosphate, adenosine 5'-phosphate and cytidine 5'phosphate with 1 M formaldehyde, under conditions specified in Table 2, then
with 2 M b i s u l f i t e at 4°C for 30 hrs, resulted in the formation of 72 % N 2 sulfomethylguanosine 5'-phosphate, 6 % N-suifomethyl adenosine 5'-phosphate
and 4 % cytidine 5'-phosphate, respectively.
5-Methyldeoxycytidine also gave
the N-sulfomethyl derivative, but the yields were low (Table 2).
Modification of DNA
Since formaldehyde can react specifically with the single-stranded polynucleotides (1), i t may be expected that this sulfomethylation can serve as
a probe for conformations of nucleic acids.
DNA, heat-denatured single-
stranded and native double-stranded samples, were treated with formaldehyde
then with b i s u l f i t e , both steps being carried out at pH 7 and 4°C.
The
modified DNA samples were digested enzymatically into mononucleotides and
subjected to high-performance l i q u i d chromatographic analysis.
In this
chromatography, an anion exchange column was employed and the mononucleotides,
both modified and unmodified, were separated s a t i s f a c t o r i l y .
As shown in
Fig. 2 and Table 3, the modification resulted in a selective modification
of guanine moiety.
ed DNA.
Furthermore, the reaction was specific for single-strand-
The small amount of modification that took place in the "native"
DNA (Fig. 2-b) was probably due to partial denaturation of DNA caused by
formaldehyde of a high concentration (1,2).
The results also show that the
reaction extents at the polynucleotide level were considerably lower than at
the monomer level.
Thus, under the conditions where more than 70 % modifi-
cation should take place for guanine mononucleotide (cf. Table 2), only 13 %
modification was observed for the guanine in DNA (Table 3).
By carrying out
the reaction at high concentrations of the reagents for longer periods, the
modification of guanine can be promoted to nearly 40 % (Fig. 2-a).
Resistance of N-suifomethylated nucleotides towards digestion with some
nucieases
Changes by the sulfomethylation in the susceptibility of purine nucle6285
Nucleic Acids Research
Si ngl«-(trended DNA , trtaltd
0.3
C
A T G
«.
G*
0.2
C*
A*
0.1
E
in
Doublt-strandtd ONA . t r t a t M
3
C
0.3
A T 6
G*
0.1
0
••
0
10
20
30
Rtttntion llm« ( min )
Fig. 2.
High-performance liquid chromatography of the enzymatic digest
of modified DNA.
DNA samples treated with 3 M formaldehyde at pH 7 and 4°C for 11 days
and then with 2 M NaHS03 at pH 7 and 4°C for 3 days were digested into
nucleoside 5'-phosphates and subjected to the chromatography (see text for
d e t a i l s ) . C, A, T, G, C*, G* and A* represent deoxycytidine-, deoxy„
adenosine-, thymidine-, deoxyguanpsine-, N^-sulfomethyldeoxycytidine-, N sulfomethyldeoxyguanosine-, and N°-sulfomethyldeoxyadenosine-5'-phosphate,
respectively.
otide esters to enzymatic hydrolysis were examined.
N -Sulfomethylguanylyl-
( 3 ' - 5 ' ) u r i d i n e was resistant to the treatment with ribonucleases Uo and L,
6
^
and N -sulfomethyladenylyl(3'-5')uridine was resistant to ribonuclease IL
but not to Tp. These properties should help to determine the locations of
modification on sulfomethylated RNA.
Implications of the new modification
As shown above, N-sulfomethylated guanine, adenine and cytosine derivatives can be readily prepared. The sul fomethyl groups are stable i f not
exposed to strong a l k a l i . Biological properties of the new derivatives of
nucleoside and nucleotide would be an interesting problem to be investigated.
The modification of guanine in polynucleotides can proceed to an extent
of nearly 40 %. Since the reaction is specific for single-stranded polymers,
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Nucleic Acids Research
Table 3.
Base composition of modified DNA
Treatment^.)
Mole %
Formaldehyde; Bisul fite
G
A
% Modification
G*b)
C
T
28.1
28.0
27.8
27.8
27.8
26.6
26.5
28.0
21.4
21.3
21.4
21.4
21.2
20.5
21.7
21.6
28 .4
28 .5
28 .2
28 .2
28 .1
27 .2
25
28 .2
0.9
1.8
2.8
2.8
1.5
2.3
9.3
-
4
8
13
2.5
7
9
36
-
27.3
27.4
21.0 27 .8
21.3 25
0
0.8
0
3
Denatured DNA
1 M, 50 hrs; 1 M, 7 hrs 21.0
1 M, 50 hrs; 1 M, 24 hrs 20.1
1 M, 50 hrs; 1 M, 60 hrs 19.5
1 M, 50 hrs;O.Ji M, 60 hrs 21.7
1 M, 24 hrs; 1 M, 60 hrs 20.9
l.M, 170 hrs; 1 M, 60 hrs 23.0
3 M, 11 days; 2 M, 3 days 16.7
None; None
22.2
Native DNA
1 M, 50 hrs; 2 M, 60 hrs 23.8
3 M, 11 days; 2 M, 3 days 24.6
a) All treatments were at pH 7 and 4°C. b) G represents N -sulfomethylguanine. c) These values were about 2 % lower than expected. We interpret
this as a deviation of the quantification, since there is no evidence that
thymine should give stable products in the reaction.
this modification can be used as a probe to detect guanine residues in exposed regions of polynucleotides.
Because of their l a b i l i t y , the formaldehyde adducts of nucleobases have
never been isolated, and therefore their structures are regarded as i n adequately characterized (6).
The isolation and characterization of the
sulfomethyl derivatives of nucleotides, together with the resemblance of the
u l t r a v i o l e t absorption spectra of formaldehyde adducts to those of sulfomethyl derivatives, provide strong evidence that the positions of formaldehyde addition are indeed the exocyclic amino groups and that the adducts
have the methylol rather than the schiff base structures.
EXPERIMENTAL SECTION
2
N -Sul fomethyl quam ne
A suspension of guanine (2 mmol) in water containing formaldehyde (50
mmol) was wanned at 60°C, and NaOH was added to give a solution of pH 10.5,
the volume being 33 ml.
ature for 3 hrs.
This solution was allowed to stand at room temper-
Sodium bisulfite (60 mmol) and some HC1 were added to this
solution, and the reaction was allowed to proceed at pH 2.5 and at room
temperature for 5 hrs. The pH was adjusted to 5.8 and the solution was kept
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in a refrigerator overnight.
The precipitate that formed was collected by
f i l t r a t i o n and washed with water, ethanol and ether to give a crude product
(450 mg).
This material contained N -sulfomethylguanine and guanine in a
ratio of 91 : 9, as checked by paper chromatography.
The material was dis-
solved in 0.01 M t r i e t h y l ammonium bicarbonate (pH 10), and the solution was
loaded on a column of DEAE-Sephadex A-25 (1.5 x 21 cm).
Washing with 0.01 M
triethylammonium bicarbonate (pH 9.5) eluted guanine, and subsequent elution
with 0.2 M triethylammonium bicarbonate (pH 9.5) gave N -sulfomethylguanine.
The solvent and the triethylammonium bicarbonate were evaporated under
reduced pressure, and the product was precipitated as crystals from the
acidified solution.
Yield, 56 % as the crystals.
Anal. Calcd. for C6H7N504S
(a zwitter ionic form): C, 29.39; H, 2.88; N, 28.58; S, 13.07 %. Found: C,
29.16; H, 2.85; N, 28.21; S, 12.75 %. ]H-NMR (DgO, pD 10): 6 4.67 (s, N 2 CH?), 7.83 (s, 8-H).
^
UV: \
o
(e) in aqueous solutions; at pH 1 , 252 nm
max
o
_
(15.4 x 10°); at pH 7, 249 nm (13.5 x 10J) and 276 nm (9.2 x 10 J ); at pH 13,
276.5 nm (9.1 x 10 3 ).
mp, 300°C (dec).
N -Sulfomethyladenine
Adenine (1.9 mmol) was treated with formaldehyde (33 mmol) in a 20 ml
solution at pH 9.5 and at room temperature for 4.5 hrs.
After addition of
sodium b i s u l f i t e (40 mmol), the solution was adjusted to pH 2.4 and allowed
to stand for 36 hrs at room temperature.
The precipitate that formed was
collected by f i l t r a t i o n and washed with water, ethanol and ether.
This
material weighed 425 mg and contained N -sulfomethyladenine and adenine in a
ratio of 92 : 8 (analysis by paper electrophoresis at pH 7).
Purification of
the product was achieved by chromatography of the sample through a column of
2
DEAE-Sephadex A-25. The elution was done as described for N -sulfomethylguanine and the product was obtained as crystals on acidification of i t s
aqueous solution and storage at 4°C overnight.
Yield, 54 %. Anal. Calcd.
for C6H7N503S'3/4H20: C, 29.69; H, 3.53; N, 28.85; S, 13.21 %. Found: C,
29.52; H, 3.27; N, 29.12; S, 13.67 %. V N M R : 6, 4.90 (s, N6-CH2), 8.07 ( s ,
8-H), 8.23 (s, 2-H).
UV: Xm;lv (e) in aqueous solutions; at pH 1 , 279 nm
(19.4 x 10 J ); at pH 7, 268 nm (18.0 x 10 J ); at pH 13, 274 nm (16.7 x 10 J ).
mp, 280-281°C (dec).
N -Sulfomethylcytosine
An aqueous solution (20 ml) of cytosine (2 mmol) containing formaldehyde
(33 mmol) was allowed to stand at 0°C and at pH 9 for 48 hrs.
The pH was
lowered to 3 by addition of HC1, sodium b i s u l f i t e (40 mmol) was added, and
the pH was further adjusted to 2.5 with HC1. The solution was allowed to
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Nucleic Acids Research
stand at 4°C for 36 hrs, then was adjusted to pH 9 by addition of NaOH, and
loaded on a column of activated charcoal -eel 1ite (1 to 1 mixture, 1.5 x
12 cm).
Washing with water eluted the b i s u l f i t e s a l t s , and subsequent wash-
ing with ammoniacal 50 % ethanol (pH 10) eluted a mixture of N 4 -sulfomethylcytosine, cytosine and u r a c i l .
This fraction was evaporated to dryness, and
the residue was chromatographed through a column of DEAE-Sephadex A-25.
The
column was washed f i r s t with 0.01 M ammonium formate, pH 6.5, to elute
cytosine and u r a c i l , and then with 0.2 M ammonium formate, pH 6.5, to elute
N -sulfomethylcytosine.
The solution was evaporated to dryness under reduced
pressure, and the ammonium formate was removed by repeated coevaporation with
water.
On refrigeration of an aqueous solution of the residue, crystals of
ammonium N -sulfomethylcytosine were obtained ( y i e l d , 19 %). This material
was recrystallized from water to give prisms that were s u f f i c i e n t l y large
for submitting to X-ray crystallographic analysis.
Anal. Calcd. for
C5H6N304S-NH4: N, 25.21; S, 14.42 %. Found: N, 24.98; S, 14.71 %. ]H-NMR
(D 2 0): 6, 6.05 (d, 5-H), 7.54 (d, 6-H)(the N4-CH2 proton signal overlapped
with the DOH signal near 4.70 ppm).
UV: X
(e) in aqueous solutions; at
J
pH 1, 286 nm (12.9 x 10 ); at pH 1, 271 nm (8.8 x 10 J ); at pH 13, 284 nm
(9.0 x 10 3 ).
mp, 270-273°C (dec).
N -Sulfomethy!-9-B-D-arabinofuranosy1adenine
9-B-D-Arabinofuranosyladenine (0.5 mmol) was treated with formaldehyde
(40 mmol) in an aqueous solution (25 ml) at room temperature and at pH 9.5
for 24 hrs.
Sodium b i s u l f i t e (50 mmol) was added and the solution was
adjusted to pH 2.5 with HC1. The solution was allowed to stand at 4°C for
7 days, and then passed through a column of charcoal-cellite (1 : 1 mixture,
1.5 x 12 cm).
After washing with water to remove b i s u l f i t e , the nucleosides
were eluted from the column with ammoniacal 50 % ethanol (pH 10).
The
solvent was evaporated, the residue obtained was dissolved in water, and the
solution was refrigerated overnight.
Crystals that formed were collected by
f i l t r a t i o n and washed with water, ethanol and ether.
Yield, 53 %. Anal.
Calcd. for C ^ H ^ N g O ^ ' H ^ : C, 34.83; H, 4.52; N, 18.46; S, 8.45 %. Found:
C, 34.50; H, 4.58; N, 18.22; S, 8.98 %. ]H-NMR (D 2 0): 6, 4.92 (s, N6-CH2),
3.98 (m, 5 ' - C H j , 6.45 (d, l ' - H ) , 8.32 ( s , 8-H), 8.41 ( s , 2-H).
UV: X
(e)
in aqueous solutions; at pH 1, 271 nm (17.6 x 10 ); at pH 7, 268 nm (19.0 x
10 3 );
at pH 13, 268 nm (19.1 x 10 3 ).
mp, 227-228°C (dec).
Regeneration of parent compounds from N-sulfomethyl derivatives by treatment
with alkali
2
6
4
About 1 mg each of N -sulfomethylguanine, N -sulfomethyladenine, N 6289
Nucleic Acids Research
sulfomethylcytosine and N -sulfomethyl-9-g-D-arabinofuranosyladenine was
dissolved in 0.6 ml 0.1 N NaOH and the solution was heated in boiling water.
At 10 min and 30 min of heating, 0.1 ml aliquots were removed, neutralized
with 0.1 N HC1, and subjected to TLC on cellulose using isobutyric acid-0.5 N
NH40H (5 : 3) as developing solvent.
By inspection, i t was found that, for
a l l the compounds tested, the 30 min-heating resulted in complete regeneration of the parent compound, though the 10 min-heating gave incomplete
reaction.
Similarly, 1 mg each of the N-sulfomethyl derivatives was treated in
0.1 ml 1 N NaOH at 25°C and analyzed by TLC, and was found to completely
regenerate the parent compound by 12 hr-treatment.
Modification of DNA with the formaldehyde-bisulfite reaction
A solution of calf thymus DNA (Sigma) in 0.0015 M Na-citrate-0.015 M
NaCl (pH 7)(5 mg DNA/ml) was heated at 100°C for 5 min, and rapidly cooled
in ice to prepare denatured DNA.
before heating was used.
For reaction of native DNA, the solution
The solution (3 ml) was mixed with a chilled
solution (3.6 ml) of formaldehyde in potassium phosphate buffer, and the
mixture was allowed to stand at 4°C.
The composition of this mixture was
2.3 mg DNA/ml, 1 M formaldehyde, 0.02 M phosphate and 1 M KC1, and the final
pH was 7.0.
After a desired period of reaction, 10 volume of 1.2 M sodium
b i s u l f i t e solution, which had been adjusted to pH 7 by use of KOH, was added.
Since b i s u l f i t e reacts with formaldehyde, the effective concentration of
b i s u l f i t e in this reaction mixture was approximately 1 M.
allowed to proceed at 4°C.
At a desired period
The reaction was
of reaction, an aliquot (5
ml) was taken up and submitted to extensive dialysis to remove the reagents
(the buffers used for the dialysis were 0.05 M t r i s - H C l , pH 9, containing
1 x 10~3 M EDTA, and 0.01 M tris-HCl, pH 7.5).
After adjustment of the NaCl
concentration to 0.1 M, the DNA was precipitated by addition of 2.5 volumes
of cold ethanol.
The mixture was kept overnight at - 20°C, and the DNA was
collected by centrifugation.
For analysis of the modified DNA, the DNA (20 A^gg units) was digested
f i r s t with pancreatic DNase I (Sigma; 0.1 mg enzyme, pH 8, 37°C, 18 hrs) and
then with snake venom phosphodiesterase (Worthington; 0.1 mg enzyme, pH 9.5,
37°C, 5 hrs) to give a mixture of deoxyribonucleoside 5'-phosphates.
The
progress of the digestion was monitored by use of cellulose thin layer chromatography (solvent; isobutyric acid-0.5 N NH.OH, 5 : 3 ) .
The nucleotides
were fractionated by high performance l i q u i d chromatography.
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Fractionation of nucleotides by high performance l i q u i d chromatography
The deoxyribonucleoside 5'-phosphate mixture prepared as above was heated at 100°C for 5 min to denature the enzyme proteins, and f i l t e r e d through
a Durapore f i l t e r by centrifugation.
A portion of the solution (0.25 A ? 5 4
units) was injected onto a column of anion exchanger Nucleosil 10-DMA (4.6 x
250 mm), and the column was washed f i r s t with 0.05 M ammonium phosphate, pH
3.35, which eluted the unmodified nucleotides dCMP, dAMP, dTMP and dGMP i n
this order, and then with 0.2 M ammonium phosphate, pH 3.3, to elute the Nsulfomethyl derivatives of dCMP, dGMP and dAMP in this order.
The flow rate
was 0.8 mi/min.
The nucleotides eluted were detected by recording the absorb-
ance at 254 nm.
For quantification, calibration lines for indivisual nucle-
otides were prepared.
The N-sulfomethyl derivatives of dGMP, dAMP and dCMP
were prepared by treatment of the nucleotides (0.05 M) with 1 M formaldehyde
at pH 9.5 and room temperature for 5 hrs, and then with 1 M sodium b i s u l f i t e
at pH 3.0 and 4°C for 6 hrs.
The N-sulfomethyl derivatives were separated
from the parent nucleotides by paper chromatography (solvent; isobutyric acid0.5 N NH.OH, 5 : 3 ) , and purified further by high performance l i q u i d chromatography on P a r t i s i l 10-SAX (eluent; 0.2 M ammonium phosphate, pH 3.3) and
subsequent desalting through a charcoal column (adsorption at pH 3, washing
with water, and elution with ammoniacal 50 % etnanol, pH 10).
The molar
absorption coefficients employed f o r the quantification were 15.0 x 10
(Aocg
at pH 7) for N2-sulfomethylguanine nucleotide, 19.5 x 103 (A, Qn at pH 1) for
N -sulfomethylcytosine nucleotide, and 19.0 x 10
methyladenine nucleotide.
(A 268 at pH 7) for N - s u l f o -
The former two values were found in the experiments
described in Table 2, and the value for N -sulfomethyladenine nucleotide was
the one found for N -sulfomethyl-9-B-D-arabinofuranosyl adenine (see above).
Preparation of modified dinucleoside monophosphates and their susceptibility
towards enzymatic hydrolysis
Guanylyl(3'-5')uridine (50 y l , 0.58 A 2 5 5 / u l ) was mixed with 1 M sodium
phosphate buffer, pH 8.6 (4 y l ) and formaldehyde (6 yl of 37 % solution), and
the resulting solution was allowed to stand at room temperature for 2 hrs.
Sodium b i s u l f i t e (15.1 mg) was added and the reaction was allowed to proceed
for 8 hrs.
1 M Tris-HCl, pH 9 (10 y l ) and some ammonia were added to adjust
the pH to 9, and after 3 hrs the product, Nv-sulfomethylguanylyl(3'-5')uridine
(G*pU), was separated by TLC on cellulose (solvent, n-butanol-acetic acidwater, 2 : 1 : 1 ) .
Similarly, N 6 -sulfomethyladenylyl(3'-5')uridine (A*pU)
was prepared from adenyly1(3'-5')uridine.
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Enzymatic hydrolysis of the dinucleoside monophosphates was monitored
by the TLC in a quantitative manner.
G*pU (0.72 A 26Q ) was unaffected on
treatment with RNase T2 (0.5 unit) at pH 4.7 and 37°C for 3 hrs, conditions
under which GpU (0.61 A 26Q ) was completely hydrolyzed.
^26o'
was
com
However, G*pU (0.36
e
P^ tely hydrolyzed on treatment with a large amount (10 units)
of RNase T- for 8 hrs.
A*pU (0.73 A 2 g 0 ) was completely hydrolyzed on reaction
with RNase T"2 (0.5 unit) for 1 hr.
G*pU (0.36 A 26Q ) was hydrolyzed by RNase
T1 (0.06 unit, pH 7, 37°C, 1 hr) only to 48 %, while GpU (0.77 A 26Q ) was
completely hydrolyzed (0.03 unit, pH 7, 37°C, 1 h r ) .
In the hydrolysis with
RNase U2 (pH 4.7, 37°C), both G*pU and A*pU were resistant, requiring 50-fold
larger amounts of enzyme than those for GpU and ApU, respectively, to obtain
comparable rates of hydrolysis.
Crystal and molecular structure of N -sulfomethylcytosine
A colorless prism of ammonium N -sulfomethylcytosine obtained by rec r y s t a l l i z i n g the compound from water was analyzed by X-ray d i f f r a c t i o n .
The crystal data found were as follows: chemical formula CgHgN,O.S'NH.;
molecular weight 222.2; systematic absences, 0k]_ O_ = 2^ + 1), hOJ_ (]_ = 2n_ +
1) and JikO (Jn_ + Jk_ = 2j^ + 1); space group Pccn; unit cell dimensions, a^ =
9.693(1), b = 10 598(1)
and
£ = 17.755(2) A; V. = 1823.8(6.) A 3 ; Dm = 1.608(1)
(determined by floatation of the crystal in a mixture of C,H, and CC1.) and
Dx = 1.618 Mgm"3; Z = 8; u(Cu-Ka) = 3.10 mm'1; F(000) = 928.
°-l
Intensities of 1459 reflections within sin6/X = 0.58 A were collected
on a Rigaku automatic four-circle diffractometor, the Cu-Ka radiation and
the o)-2£ scan technique being employed.
The structure was solved by direct
methods according to the program MULTAN ( 7 ) , and was refined by a f u l l - m a t r i x
least-squares method in the l i g h t of anisotropic temperature factors for a l l
the non-H atoms.
Location of a l l the H-atoms was achieved by use of a d i f f e r -
ence Fourier synthesis.
The final refinement incorporating the isotropic
temperature factors for the H-atoms reduced the R value to 0.064 (Rw = 0.059).
Figure 1 is an ORTEP (8) drawing showing 50 % probability ellipsoids.
The
l i s t s of atomic parameters, and the observed and calculated structure factors
are available on request, from the Director of the Cambridge Crystallographic
Data Centre, University Chemical Laboratory, Lensfield, Cambridge CB2 1EW,
England.
ACKNOWLEDGMENTS
This work was supported by a Grant f o r S c i e n t i f i c Research from the
M i n i s t r y o f Education, Science and C u l t u r e .
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