Synthesis and physicochemical properties of new fluorescent derivatives of cytosine

Article abstract of DOI:10.1002/jhet.5570430213

Sixteen new fluorescent N⁴-(E)-stilbenyloxyalkylcarbonyl- cytosines 9-16 and N⁴-(E)-stilbenyloxyalkylcarbonyl-1-methylcytosines 17-24 have been synthesized. The differences in ¹H and ¹³C NMR spectra in two solve

Full text of DOI:10.1002/jhet.5570430213

Mar-Apr 2006
337
Synthesis and Physicochemical Properties of New Fluorescent
Derivatives of Cytosine
Dorota Prukała
Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznań, Poland
Received May 16, 2005
4
4
Sixteen new fluorescent N -(E)-stilbenyloxyalkylcarbonyl-cytosines 9-16 and N -(E)-stilbenyloxyalkyl-
1
13
carbonyl-1-methylcytosines 17-24 have been synthesized. The differences in H and C NMR spectra in
two solvents (DMSO and TFA) have been pointed out and discussed. Assignment of the signals in the spec-
tra of the compounds 9-24 in NMR in DMSO-d solutions has been made the basis of the homonuclear
6
(COSY) and heteronuclear (HETCOR) spectra. The effect of the substituent (Cl, Br, NO ) on the stilbene
2
moiety on the fluorescence spectrum of each compound has been discussed.
J. Heterocyclic Chem., 43, 337 (2006).
Introduction.
Results and Discussion.
4
A series of sixteen new fluorescent N -[(E)-stilbeny-
The modification of nucleobases or deoxyribose moiety
in DNA by (E)-stilbene containing derivatives has been
widely reported in literature [1-5] because of their fluores-
cent properties and high quantum yield. These compounds
are potentially useful as probes in the study of the structure
and dynamics of nucleic acid and their complexes with
proteins; they may also find use as fluorescent labels for
nucleic-acid-based biomedical diagnostic methods.
4
loxyalkylcarbonyl-cytosines (9-16) and N -(E)-stilbenyloxy-
alkylcarbonyl-1-methylcytosines (17-24) have been synthe-
sized in the reaction of the corresponding (E)-4-chloro-
carbonylalkoxystilbenes (1-8) with cytosine (or 1-methylcy-
tosine) in boiling pyridine solutions, in the presence of a small
amount of DMAP (Scheme 1). Noteworthy is the fact that the
presence of the methyl substituent at the annular nitrogen
atom N-1 of the pyrimidine ring of molecules (17-24)
changes the physicochemical properties of these compounds
relative to those of 9-16. In particular the values of the melting
points of 17-24 are lower and the solubility in organic sol-
vents is higher. This fact allows making use of column chro-
matography for purification of 17, 18, 20, 22, 23, 24.
On the other hand, there is a wealth of published infor-
mation on the fluorescent compounds of cytosine and cyti-
dine [6-10], including their N-acyl derivatives [11]. This
4
fact has stimulated the preparation of a series of N -(E)-
4
stilbenyloxyalkylcarbonyl-cytosines (9-16) and N -(E)-
stilbenyloxyalkylcarbonyl-1-methylcytosines (17-24) aim-
ing at obtaining specific fluorescent modification of these
compounds. The fluorescent moieties chosen for the study
are (E)-stilbene, (E)-4'-chlorostilbene, (E)-4'-bromostil-
bene and (E)-4'-nitrostilbene.
The structures of all compounds obtained were deter-
1
13
mined by examining their UV/VIS, IR, H and C NMR
spectra as well as on the basis of elemental analyses
(Tables I-Vd).
This paper deals with the synthesis and physicochemi-
cal properties of compounds 9-24. The NMR spectra
have been obtained in two solvents to examine the influ-
This paper also presents the physicochemical properties
of four stilbenes (4, 6, 7, 8) unknown in literature (Table
II). They have been synthesized according to literature
procedure with some modifications in the preparation and
isolation procedures. The physicochemical properties of
(E)-4-chlorocarbonylalkoxystilbenes (1, 2, 3, and 5) have
been determined by us earlier [12].
1
13
ence of acid solvent (TFA) on the changes in H and
C
NMR spectra relative to those measured in standard
DMSO-d solutions. The spectral analysis (IR, UV/VIS
6
1
and H NMR) has been also performed to check the pos-
1
13
sibility of differentiation of metamers existing in those
series of compounds.
Investigation of the fluorescence spectra has been under-
taken to check the dependence of quantum yields and
The data from H and C NMR spectra of compounds
9-24 recorded in DMSO-d and CF COOD solutions are
6
3
1
given in Tables III and IVa-d. Assignments of the H and
C NMR resonances of these compounds were deduced
13
emission maxima on the substituents (H, Cl, Br, NO ) in
the (E)-stilbene moiety.
on the basis of signal multiplicities, by comparison with
literature data [13-19] and by the concerted application
2

338
D. Prukała
Vol. 43
Scheme 1
Table Ia
Chemical and Physical Data of Compounds 9-16
Comp.
Formula
(mol. weight)
M.p.
[°C]
Yield
[%]
R [a]
Elemental analysis, Calculated (Found)
f
C
H
N
9
10
11
12
13
14
15
16
C
H
N O
3 3
286-289
265-268
305-310
296-301
314-316
300-305
315-319
308-311
31
33
39
35
40
30
39
40
0.70
0.78
0.65
0.67
0.58
0.74
0.56
0.64
65.75
(65.93)
68.11
(68.15)
62.91
(62.56)
63.79
(63.80)
55.17
(55.38)
57.27
(57.10)
58.54
(58.90)
59.43
(59.96)
5.21
(5.26)
5.41
(5.48)
4.19
(4.55)
4.56
(4.65)
3.91
(3.99)
4.09
(4.45)
4.39
(4.51)
4.71
(4.98)
11.51
(11.16)
11.35
(11.58)
10.01
(10.39)
10.63
(10.54)
9.66
20 17
347 x 1H O
2
C
H N O
21 19 3 3
361 x 0,5 H O
2
C
C
C
H N O Cl
20 16 3 3
381.5
N O Cl
H
21 18
3 3
395.5
N O Br
H
20 16
3 3
426 x 0,5 H O
(9.63)
9.55
2
C
H N O Br
440
H N O
21 18 3 3
(9.61)
13.66
(13.62)
13.21
(12.99)
C
20 16
4 5
392 x 1 H O
2
C
H N O
21 18 4 5
406 x 1 H O
2
[a]- CHCl :CH OH 5:1.
3
3
involvement of the two dimensional NMR techniques
HETCOR and COSY, which clearly showed the connectiv-
ity of hydrogen and carbon atoms involved. The employ-
ment of these techniques allowed unequivocal assignment
of the spectra especially for the stilbene rings.
The (E)-configuration in the stilbene moiety was deter-
mined on the basis of their UV/VIS and IR spectra.
According to literature [20-22] (E)-stilbenes exhibit the
values of λ
values of λ
in range 290-360 nm and for (Z)-stilbenes
fall in the range 260-280 nm.
max
max

Mar-Apr 2006
Synthesis and Physicochemical Properties of New Fluorescent Derivatives of Cytosine
339
Table Ib
Chemical and Physical Data of Compounds 17-24
Comp.
Formula
(mol. weight)
M.p.
[°C]
Yield
[%]
R [a]
Elemental analysis, Calculated (Found)
f
C
H
N
17
18
19
20
21
22
23
24
C
C
H
N O
261-263
115-120
261-267
210-215
267-273
215-220
300-303
225-230
42
51
40
62
47
52
72
85
0.63
0.75
0.67
0.75
0.65
0.81
0.67
0.71
69.81
(69.49)
70.40
(70.39)
63.72
(63.52)
64.47
(64.21)
57.27
(56.99)
58.15
(58.06)
62.07
(61.88)
62.86
(62.64)
5.26
(4.97)
5.60
(5.24)
4.55
(4.50)
4.88
(4.90)
4.09
(4.05)
4.41
(4.26)
4.43
(4.21)
4.76
(4.59)
11.63
(11.64)
11.20
(11.05)
10.62
(10.25)
10.26
(9.98)
9.55
21 19
3
3
361
N O
H
22 21
3
3
375
H N O Cl
C
C
C
C
21 18
3 3
395.5
N O Cl
H
22 20
3 3
409.5
N O Br
H
21 18
3 3
440
N O Br
(9.34)
9.25
H
22 20
3 3
454
H N O
406
H N O
(9.18)
13.79
(13.54)
13.33
(13.11)
C
21 18
4
5
5
C
22 20
4
420
Table II
Chemical and Physical Data of Compounds 4, 6, 7, 8
1
Comp.
X
R
Formula
(mol. weight)
M.p.
[°C]
Yield
[%]
Elemental analysis, Calculated (Found)
C
H
N
-
4
6
7
8
Cl
CH
CH
H
C
321
H
O Cl
2
68
98
97
95
99
63.55
(63.79)
55.81
(55.88)
60.47
(60.55)
61.54
4.36
(4.35)
3.83
(3.85)
3.78
(3.77)
4.22
3
17 14
2
Br
C
H
O BrCl
75
-
3
17 14
2
365.5
NO
NO
C
H
NO Cl
109
54
4.41
(4.30)
4.22
2
16 12
4
317.5
CH
C H NO Cl
2
3
17 14 4
331.5
(61.59)
(4.23)
(4.01)
Table III
C=O). This band can be assigned to the acyclic amide
whereas the vibration in the 1663-1699 cm area can be
IR Data of 9-24
-1
-1
assigned to ν C=O (mostly) in pyrimidine ring. The IR spec-
tra confirm also the (E)-configuration in stilbene moiety
revealing the bond attributed to the out-of-plane deformation
vibration of the C-H bond of the (E)-ethylene bridge [23,24].
IR (cm )
Compd. νC=O
νC=O
Arom.skeletal δ CH=CH Ar-NO
vibrations
2
I amide in pyrimid.ring
(trans)
9
1750, 1742
1709
1750, 1744
1706
1750, 1742
1720
1749
1749
1728
1722
1728
1693
1699
1694
1699
1693
1696
1698
1696
1669
1669
1671
1668
1663
1668
1654
1653
1606, 1510
1607, 1509
1606, 1510
1606, 1510
1605, 1511
1609, 1498
1609, 1510
1605, 1510
1604, 1510
1604, 1510
1607, 1511
1603, 1498
1604, 1509
1603, 1499
1606, 1510
1604, 1508
961
965
964
966
962
962
963
967
968
964
960
970
966
970
965
967
-
-
-
-
-
-
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
The H NMR spectra are recorded in a standard DMSO-
d solution (9, 10, 11, 12, 17, 18, 20, 22, 24) and CF COOD
6
3
solution (13, 14, 15, 16, 19, 21, 23) because of low solubil-
ity of the latter in practically all organic solvents. The NMR
spectra of compounds 9 and 24 are measured in both
1339
1337
-
-
-
-
-
DMSO-d and TFA solutions for the sake of comparison.
6
The data assignment for these compounds was based on the
examination of two-dimensional HETCOR and COSY
spectra for 9, 20 and 22 (Tables IVa-IVd). The methylation
of N-1 atom in pyrimidine ring has no important influence
on hydrogen shifts in all investigated compounds, exept for
H-6'' which is shifted downfield by about 0.3 ppm.
1716, 1699
1729
1717, 1699
1724
-
1338
1338
1709
Protonation of the molecules by CF COOD, in general,
causes a shift of the resonances, particularly of 5''-H atom and
3
-1
The IR spectra, in the range 1700-1751 cm show intense
6''-H atom in cytosine moiety. The δ values of the 5''-H in the
bands corresponding to carbonyl stretching vibrations (ν

340
D. Prukała
Vol. 43
Table IVa
1
H NMR Data of 9, 11, 15
9 X=H, 11 X=Cl, 15 X=NO
2
Comp.
9
I
2
3
4
2'
3'
α
α'
5''
6''
-CH -
2
4.85s
4.99s
4.85s
4.99s
7.55d
J=7
7.51d
J=7
7.59d
J=9
7.69d
J=9
7.36t
J=7
7.35t
J=7
7.42d
J=9
8.27d
J=9
7.24t
J=7
7.26t
J=7
-
7.51d
J=9
7.55d
J=9
7.55d
J=9
7.59d
J=9
6.94d
J=9
6.95d
J=9
6.95d
J=9
7.02d
J=9
7.11d
J=16
7.12d
J=16
7.24d
J=16
7.13d
J=16
7.09d
J=16
7.05d
J=16
7.11d
J=16
7.31d
J=16
7.03d
J=7
6.95d
J=7
7.03d
J=7
7.01d
J=7
7.86d
J=7
8.25d
J=7
7.81d
J=7
8.53d
J=7
9[a]
11
15[a]
-
[a]-in TFA
Table IVb
1
H NMR Data of 10.12,14, 16
10 X=H, 12 X=Cl, 14 X=Br, 16 X=NO
2
Comp.
I
II
-CH
1.53d
J=7
1.52d
J=7
1.76d
J=7
2
3
4
2'
3'
α
α'
5''
6''
-CH-
5.08q
J=7
5.07q
J=7
5.13q
J=7
5.18q
J=7
3
10
7.56d
J=7
7.56d
J=9
7.46d
J=9
7.67d
J=9
7.36t
J=7
7.41d
J=9
7.37d
J=9
8.23d
J=9
7.24t
J=7
-
7.55d
J=9
7.54d
J=9
7.54d
J=9
7.55d
J=9
6.94d
J=9
6.90d
J=9
6.99d
J=9
7.01d
J=9
7.19d
J=16
7.21d
J=16
7.09d
J=16
7.09d
J=16
7.10d
J=16
7.09d
J=16
7.01d
J=16
7.28d
J=16
7.07d
J=7
7.05d
J=7
6.94d
J=7
6.98d
J=7
7.87d
J=7
7.86d
J=7
8.48d
J=7
8.49d
J=7
12
14[a]
16[a]
-
-
1.76d
J=7
[a]-in TFA
pyrimidine ring was observed from 7.03 to 7.07 ppm (9-16),
and from 7.08 to 7.14 ppm (17-24) in DMSO solutions, and
were upfield shifted in TFA solutions by about 0.2 ppm. The
replacement of DMSO with TFA solvent produces the most
significant changes in 6''-H assignments, for all compounds
investigated. The differences in δ values for the appropriate
protons in different solvents are the same. Noteworthy is the
DMSO shows a doublet at 7.86 ppm and at 8.25 ppm in TFA
due to 6'' proton. Compound 24 exhibits a doublet at δ 8.13 in
DMSO and at δ 8.51 in TFA, assignable to this 6'' proton.
13
The C NMR spectra have also been obtained in two
solvents, DMSO-d and deuterated TFA. Assignments of
6
13
the C NMR resonances data are given in Tables Va-Vd.
The presence of CH group at the N-1 site in 1-methylcy-
3
fact that the presence of CH substituent at the N-1 atom of
tosine derivatives caused a shift of the resonance of C-6''
atom in pyrimidine ring to higher δ values, and of C-V
atom of these compounds to lower δ values than those of
unsubstituted cytosine ring derivatives. This fact allows a
differentiation between metameric 10 and 17
3
pyrimidine ring does not influence this value. The signals
assigned to these protons are in the range 7.8-7.9 ppm (9-12)
and 8.12-8.18 ppm (17, 19, 20, 22, 24) in DMSO solutions and
are downfield shifted by more than 0.4 ppm in TFA solutions.
1
For the sake of comparison, the H NMR spectrum of 9 in
(C H N O ), 12 and 19 (C H N O Cl ), as well as 16
21 19 3 3 21 18 3 3

Mar-Apr 2006
Synthesis and Physicochemical Properties of New Fluorescent Derivatives of Cytosine
341
Table IVc
1
H NMR Data of 17, 19, 23
17 X=H, 19 X=Cl, 23 X=NO
2
Comp.
17
I
III
-CH
2
3
4
2'
3'
α
α'
5''
6''
-CH -
2
3
4.85s
4.85s
4.97s
3.22s
3.88s
3.87s
7.57d
J=7
7.59d
J=9
7.62d
J=9
7.36t
J=7
7.48d
J=9
8.23d
J=9
7.25t
J=7
-
7.56d
J=9
7.56d
J=9
7.55d
J=9
6.95d
J=9
6.95d
J=9
7.00d
J=9
7.21d
J=16
7.23d
J=16
7.09d
J=16
7.11d
J=16
7.11d
J=16
7.28d
J=16
7.08d
J=7
7.06d
J=7
6.96d
J=7
8.13d
J=7
8.12d
J=7
8.55d
J=7
19[a]
23[a]
-
[a]-in TFA
Table IVd
H NMR Data of 18, 20, 22, 24
1
18 X=H, 20 X=Cl, 22 X=Br, 24 X=NO
2
Comp
18
I
II
-CH
1.53d
J=7
1.58d
J=7
1.58d
J=7
1.53d
J=7
1.76d
J=7
III
-CH
2
3
4
2'
3'
α
α'
5''
6''
-CH-
5.32q
J=7
5.13q
J=7
5.13q
J=7
5.05q
J=7
3
3
3.19s
3.20s
3.21s
3.23s
3.85s
7.55d
J=7
7.63d
J=9
7.66d
J=9
7.81d
J=9
7.36t
J=7
7.46d
J=9
7.59d
J=9
8.21d
J=9
7.24t
J=7
-
7.54d
J=9
7.59d
J=9
7.59d
J=9
7.63d
J=9
6.90d
J=9
6.96d
J=9
6.86d
J=9
6.94d
J=9
7.19d
J=16
7.27d
J=16
7.28d
J=16
7.27d
J=16
7.07d
J=16
7.14 d
J=16
7.15d
J=16
7.14d
J=16
7.48d
J=16
7.25d
J=16
7.09 d
J=7
7.14d
J=7
7.14d
J=7
7.08d
J=7
8.12d
J=7
8.18d
J=7
8.18d
J=7
8.13d
J=7
20
22
-
-
-
24
24[a]
5.16q
J=7
7.65d
J=9
8.19d
J=9
7.52d
J=9
6.99d
J=9
6.94d
J=7
8.51d
J=7
[a]-in TFA
and 3 (C H N O ). The data of appropriate C signals are
The solvent replacement by a more acidic TFA caused
changes in the C NMR resonance signals, particularly of
21 19
4 5
4
13
as follows: 10 N -(E)-stilbenyloxyalkylcarbonyl-cytosine,
4
C-6''-147.7 ppm, C-V-162.9 ppm; 17 N -(E)-stilbeny-
carbonyl and pyrimidine ring carbons. The most important
changes appeared in the C-6'' carbon resonances. In
DMSO solutions these signals were observed in the range
147.5-147.8 ppm (for cytosine derivatives 9-16) and
151.1-151.5 ppm (for 1-methylcytosine derivatives 17-24)
and in TFA solutions they were shifted by 6 and 7 ppm
(respectively) to lower-field.
The resonances of IV C atoms (carbonyl group), in
DMSO fall in the range 168.8-169.0 ppm (9, 11, 17, 19)
and 171.8-172.9 ppm (10, 12, 18, 20, 22, 24), and were
loxyalkylcarbonyl-1-methylcytosine, C-6''-151.3 ppm, C-
4
V-161.9 ppm, C-III-37.6 ppm; 12 N -(E)-4'-chloro-
stilbenyloxyalkylcarbonyl-cytosine, C-6''-147.7 ppm, C-
4
V-162.9 ppm; 19- N -(E)-4'-chlorostilbenyloxyalkyl-
carbonyl-1-methylcytosine, C-6''-151.1 ppm, C-V 161.8
4
ppm, C-III-37.5 ppm; 16- N -(E)-4'-nitrostilbenyloxy-
4
alkylcarbonyl-cytosine, C-6''-153.6 ppm; 23- N -(E)-4'-
nitrostilbenyloxyalkylcarbonyl-1-methylcytosine, C-6''-
158.3 ppm, C-III-39.3 ppm.

342
D. Prukała
Vol. 43
Table Va
Table Vc
C NMR Shifts of 14 and 22
13
13
C NMR Shifts of 9, 10, 17, 18
Carbon
14[a]
22
Carbon
9
9[a]
10
17
18
1
2
3
4
136.1
127.2
133.6
120.9
127.9
127.4
131.4
127.4
113.1
154.8
159.6
96.1
136.6
128.0
131.6
120.1
128.8
125.3
130.1
128.2
115.1
156.9
155.7
94.8
151.5
72.7
18.3
37.6
172.1
162.0
1
2
3
4
138.3
126.2
128.7
127.2
127.9
126.5
130.3
127.8
114.7
157.4
155.9
94.6
137.0
126.0
128.4
127.5
127.8
126.8
129.9
127.7
114.7
155.4
159.2
96.0
137.3
126.2
128.6
127.3
127.9
126.6
130.4
127.8
115.0
156.7
155.9
94.6
137.3
126.2
128.7
127.3
127.9
126.5
130.3
127.8
114.7
157.4
155.6
94.8
151.4
66.6
-
37.6
168.9
161.9
137.3
126.1
128.5
127.1
127.7
126.4
130.2
127.6
114.9
156.5
155.4
94.7
151.2
72.7
18.4
37.6
172.9
161.8
α
α
α'
1'
2'
3'
4'
4''
5''
6''
I
α'
1'
2'
3'
4'
4''
5''
6''
I
153.6
75.4
15.9
147.5
66.7
-
153.8
67.4
-
147.7
72.8
18.3
II
III
IV
V
II
III
IV
V
-
-
-
-
177.2
-[b]
169.0
162.8
173.5
-[b]
172.2
162.9
[a]-in TFA; [b]-hindered upon TFA signal.
[a]-in TFA; [b]-hindered upon TFA signal.
Table Vd
Table Vb
13
C NMR Shifts of 15, 16, 24
13
C NMR Shifts of 11, 12, 19, 20
Carbon
15 [a]
16[a]
23[a]
24[a]
24
Carbon
11
12
19
20
1
2
3
4
145.9
128.9
125.8
145.9
132.8
124.5
132.5
126.9
115.2
156.8
159.7
96.5
145.5
128.6
125.4
145.6
132.3
124.1
132.2
126.5
116.0
155.7
159.7
96.3
146.2
129.1
125.9
146.2
133.0
124.7
132.7
127.2
115.4
157.0
158.5
96.3
158.3
67.5
-
39.3
174.1
-[b]
145.9
128.9
125.8
145.9
132.7
124.5
132.6
126.9
115.9
156.1
158.6
96.2
157.8
75.4
16.1
39.0
177.4
-[b]
144.2
128.6
124.3
145.7
132.6
123.9
129.5
126.8
115.0
157.3
155.4
94.7
151.3
72.7
18.4
37.6
171.8
161.8
1
2
3
4
136.3
127.9
128.7
131.5
128.8
125.2
130.1
127.9
114.8
157.6
156.0
94.6
136.3
127.9
128.6
131.5
128.7
125.2
130.1
127.8
115.1
156.9
155.9
94.6
136.3
127.9
128.6
131.4
128.8
125.1
130.1
127.8
114.8
157.5
155.6
94.7
151.1
66.7
-
37.5
168.8
161.8
136.3
128.0
128.7
131.5
128.7
126.3
130.2
127.8
115.1
156.9
155.6
94.8
151.5
72.7
18.3
37.6
172.1
161.9
α
α'
1'
2'
3'
4'
4''
5''
6''
I
α
α'
1'
2'
3'
4'
4''
5''
6''
I
151.3
67.4
-
153.6
75.1
15.7
147.6
66.7
-
147.7
72.8
18.3
II
III
IV
V
-
-
II
III
IV
V
173.8
-[b]
176.7
-[b]
-
-
169.0
162.8
172.2
162.9
[a]-in TFA; [b]-hindered upon TFA signal.

Mar-Apr 2006
Synthesis and Physicochemical Properties of New Fluorescent Derivatives of Cytosine
343
Table VI
stilbene moiety affects the fluorescent properties of the
compounds synthesized. The most suitable substituent is
UV/VIS and Fluorescence Spectra of 9-24
the –NO group (15, 16 and 23, 24).
Comp. UV/VIS
log ε
[nm]
Excitation Fluorescence Quantum
2
λ
wave
[nm]
emission wave Yield
max
Conclusions.
[nm]
Φ
The fluorescent data for 15, 16 and 23, 24 show that
these cytosine derivatives are the best fluorescence com-
pounds in our series, they show very high quantum yields.
Excitation of these compounds is possible at 410 nm,
well outside the range of absorption of proteins and
nucleic acids and indicates that these cytosine derivatives
may prove suitable for nucleic-acid-based research.
9
320, 306
320, 309
324, 309
323, 309
326, 311
326, 311
381
4.47, 4.45
4.46, 4.38
4.46, 4.45
4.45, 4.34
4.37, 4.35
4.48, 4.45
4.29
350
350
350
350
350
350
410
410
350
350
350
350
350
350
410
410
385
385
383
383
388
388
605
605
387
387
385
385
388
388
605
605
0.01
0.02
0.10
0.10
0.02
0.01
0.38
0.49
0.01
0.02
0.01
0.01
0,02
0.01
0.50
0.53
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
379
4.28
321, 305
321, 306
324, 309
324, 309
324, 310
323, 310
379
4.46, 4.45
4.46, 4.39
4.45, 4.44
4.46, 4.46
4.39, 4.37
4.46, 4.45
4.33
The use of HETCOR and COSY NMR methods allows
1
correct assignments of the resonance signals in the H and
13
especially in C NMR spectra of the stilbene moiety.
13
1
The differences in the C NMR and H NMR spectra in
two solvents (DMSO and TFA) permit assessment of the
influence of protonation of the nitrogen atom on the chem-
ical shifts.
379
4.33
1
Analysis of the H NMR, UV/VIS and IR spectra has
shifted about 5 ppm to lower-field in TFA solutions. The
upfield shifts of the C=O carbonyl signals of IV C atoms (9,
11, 17, 19) are caused by the absence of –CH (II) group in
confirmed the (E)-configuration in the stilbene parts of the
molecules of 9-24.
3
the alkyl moiety of these derivatives. The different values
of C-4'' signals are observed in DMSO and in TFA. They
fall in the same range 155.4-156.0 ppm, both for cytosine
and 1-methylcytosine derivatives and are shifted downfield
4 ppm in TFA solutions. The measured values of C-5'' shifts
were observed at 94.6-94.8 ppm in DMSO and were shifted
downfield only by about 1.5 ppm in acid solvent.
EXPERIMENTAL
Purity of all compounds studied was checked by m.p.'s, TLC and
elemental analysis. Melting points (uncorrected) were determined on
a Böetius microscope hot stage. R values refer to TLC silica gel
f
F
TLC plates (Merck) developed with CHCl /CH OH 5:1 and
254
3 3
observed under UV light (λ = 254 and 366nm). UV/VIS spectra
were recorded with a Specord UV/VIS spectrophotometer in DMF.
IR spectra were recorded with a FT-IR Bruker JFS-113 Spectrometer
It should be pointed out that the differences in the values
of the chemical shifts in different solvents for appropriate
carbon atoms are the same for both cytosine and 1-methyl-
cytosine derivatives. This suggests a strong interaction
between acidic solvent (TFA) and the compounds that are
diluted in it and demonstrates that protonation occurs in
TFA solution predominantly at the nitrogen in amide group.
The fluorescence properties of compounds 9-24 have
been also investigated. The characteristic values in
UV/VIS spectra, and the fluorescent emission maxima and
quantum yields are summarized in Table VI. All spectra
were recorded in DMF solution. Excitation spectra were
also measured at the emission maxima, and the spectra
were identical to the absorption curves for all investigated
compounds. As we can see in Table VI, compounds 9-14,
and 17-22 show medium fluorescence quantum yields, but
compounds 15, 16 and 23, 24 are strongly fluorescent.
These compounds reveal also unexpectedly great differ-
ence between absorption maxima and fluorescent emission
maxima. This fact predestinates these derivatives of cyto-
sine to further photochemical investigations. The fluores-
cence spectra of substituted cytosine 9-14 and 1-methylcy-
tosine 17-22 are similar, with almost the same value of flu-
orescence emission maxima. The type of substituent in the
1
13
in KBr pallets. H NMR and C NMR spectra were determined
with a Varian Gemini 300 (300 MHz) Spectrometer in DMSO-d
6
and deuterated TFA solutions with TMS as internal standard.
Chemical shifts are given in the δ scale (ppm) and coupling con-
stants in Hz. H NMR (300.07) spectra were recorded with spectral
width 9 kHz, acquisition time 2.0 s, pulse width 6 µs and double pre-
cision acquisition. C NMR (75.460) spectra were recorded with
1
13
spectral width 18.76 kHz, acquisition time 1.0 s, recycle delay 1.0 s
1
1
and pulse width 15 µs. Homonuclear H- H shift correlated two-
dimensional diagrams were obtained on Varian Gemini 300 spec-
trometer using the COSY pulse sequence. The spectral width was
4.97 kHz, acquisition time 0.206 s, number of increments in t 512
1
13
1
and number of scans 16. Heteronuclear 2D C - H chemical shift
correlation experiments were carried out using HETCOR spectra.
The spectra were acquired with 2K data points, 245 increments and
13
1
spectral width 19 kHz for C and 4.97 kHz for H.
Fluorescence corrected spectra were taken in DMF with a
Perkin Elmer MPF-3 instrument. Fluorescence quantum yields
2
2
were calculated by the equation Φ = (FA n Φ )/(AF n ), where
f
s
s
s 0
the subscripts refers to the standard and Φ is the quantum yield, F
the corrected, integral fluorescence; A the absorption at the exci-
tation wavelength, n the refractive index of DMF and n the
refractive index of H O. Quantum yield standards were quinine
0
2
sulfate, 10 µM in 1 N H SO (Φ=0.55) and 10 µM in 0.1 N
2
4
NaOH (Φ=0.90) [25,26].

344
D. Prukała
Vol. 43
Wirsching, W. Rettig, J. K. McCusker, D. Steve, D. P. Millar, P. G.
Schultz, R. A. Lerner, K. D. Janda, Science, 290, 307 (2000).
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Acta, 82, 2160 (1999).
[3] F. D. Lewis, Y. Wu and X. Liu, J. Am. Chem. Soc., 124,
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[4] A. Ahluwalia, D. De Rossi, G. Giusto, O. Chen, V.
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2481 (1983).
General Procedure for the Preparation of (E)-4-Chlorocarbonyl-
alkoxystilbenes 1-8.
A mixture of 0.015 mole of appropriate (E)-4-stilbenyl-
oxyalkylcarboxylic acids and thionyl chloride (6 mL, 0.075
mole) in dry benzene (20 mL) was heated under reflux for 1 hr.
Then the thionyl chloride and the solvent were evaporated in
vacuo. The solid isolated was suspended in dry benzene and fil-
tered trough silica gel (Merck 60 0.063-0.100mm). The solvent
was removed again. Compounds 1-8 were shown to be analyti-
cally pure and were used without any further purification.
4
General Procedure for the Preparation of N -(E)-stilbeny-
[7] N. K. Kochetkov, V. N. Shibaev and A. A. Kost,
Tetrahedron Letters, 1993 (1971)
loxyalkylcarbonyl-cytosines 9-16.
Cytosine (1.11 g, 0.01 mole) and DMAP (N,N-dimethyl-
aminopyridine 0.040 g 0.0003 mole) was suspended in dry pyri-
dine (20 mL) and boiled under reflux. Then the boiling mixture
was treated dropwise with the solution of 0.015 mole of appropri-
ate (E)-4-chlorocarbonylalkoxystilbene 1-8 in dry benzene (2
mL). The reaction mixture was then allowed to reflux for 6 hrs.
The solvent was then removed in vacuo and the solid was treated
with dry methanol and boiled. The precipitated solid was col-
lected by filtration from hot solvent. This procedure was repeated
three times. Solids 9-16 were collected and dried under vacuum
at room temperature. The physicochemical properties of 9-16 are
given in Table I.
[8] R. S. Hosmane and N. J. Leonard, J. Org. Chem., 46, 1457
(1981).
[9] N. J. Leonard and G. L. Tolman, Ann. N. Y. Acad. Sci.,
255, 43 (1975).
[10]
L. Brand and J. R. Gohlke, Annual Review of
Biochemistry, 41, 843 (1972).
[11] M. Müller, G. Birner and W. Dekant, Chem. Res. Toxicol.,
11, 454 (1998).
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Chem., 66, 763 (1992).
[13] K. Wüthrich , NMR of Proteins and Nucleic Acids, John
Wiley&Sons, New York, N.Y. 1986.
[14] J. N. S. Evans, Biomolecular NMR Spectroscopy; Oxford
University Press, New York, 1995.
4
General Procedure for the Preparation of N -(E)-Stilbenyloxy-
[15] C. E. Aun, T. Y. Clarkson and D. A. R. Happer, J. Chem.
Soc., Perkin Trans II, 645 (1990).
[16] E. Wyrzykiewicz J. Grzesiak and W. Prukała, Magn.
Reson. Chem., 26, 529 (1988).
[17] T. H. Fischer and P. T. Shultz, Magn. Reson. Chem., 29,
966 (1991).
[18] K. H. Galm, S. L. Eng, T. I. -Ho, and Y. -Ch. Liu, J. Chin.
Chem. Soc., 38, 591 (1991).
[19] Z. Meic, D. Vikič-Topič and H. Güsten, Org. Magn.
Reson., 22, 237 (1984).
[20] M. Calvin and H. E. Alter, J. Chem. Phys., 19, 765 (1951).
[21] E. A. Braude, J. Chem. Soc., 1902 (1949).
[22] D. F. Detar and L. A. Carpino, J. Am. Chem. Soc., 78, 475
(1945).
[23] H. W. Thompson and P. Torkington, J. Chem. Soc., 640
(1945).
alkylcarbonyl-1-methyl Cytosines 17-24.
1-Methylcytosine (1.25 g 0.01 mole) and DMAP (N,N-
dimethylaminopyridine 0.040 g, 0.0003 mole) was suspended in
dry pyridine (20 mL) and boiled under reflux. Then the boiling
mixture was treated dropwise with the solution of 0.015 mole of
appropriate (E)-4-chlorocarbonylalkoxystilbene 1-8 in dry ben-
zene (2 mL). The reaction mixture was then reflux for 5 hrs. Then
the solvent was removed under vacuum, and the solid precipi-
tated was treated with dry methanol and boiled. The solid of 17,
19, and 21 was collected by filtration from hot solvent and puri-
fied like described above. The compounds 18, 20, 22, 23, 24 were
separated by column chromatography (silica gel Merck 60 0,063-
0,100mm, CHCl :CH OH 30:1). The physicochemical properties
3
3
of 17-24 are given in Table I.
[24] M. Oki and H. Kunimoto, Spectrochimica Acta, 19, 1463
(1963)
REFERENCES AND NOTES
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(1971).
[1] A. Simeonov, M. Matsushita, E. A. Juban, E. H.
Thompson, T. Z. Hoffman, A. E. Beuscher, M. J. Taylor, P.
[26] J. Olmsted, J. Phys. Chem., 83, 2581 (1979).