FLUORESCENCE REAGENTS FOR LABELLING OF BIOMOLECULES. PART I. SYNTHESIS AND SPECTRAL CHARACTERIZATION OF 2- AND 4-SUBSTITUTED 9-ISOTHIOCYANATOACRIDINES

Article abstract of DOI:10.1135/cccc19940203

9-Isothiocyanatoacridines VIII-XIV were prepared from the corresponding 9-chloroacridines I - VII.The IR, 1H NMR, 13C NMR and fluorescence spectra of the products are given.The 13C NMR chemical shifts of the C-9 ipso carbon atom exhibit a trend that is in accord with the Hammett constants of substituents bonded to the C-2 carbon.Effect of these substituents on the chemical shift of C-NCS was only small.The dependence of hydrolysis of isothiocyanates VIII - XIV on pH of the medium was studied.It was found that 9-isothiocyanatoacridines do not undergo hydrolysis at pH 7 - 10.The relative fluorescence intensities (F/F₀) of compounds VIII - XIV at pH 7.4 have been determined in comparison with that of 9-aminoacridine.No direct dependence between the fluorescence intensity and the polar character of substituents has been found.

Full text of DOI:10.1135/cccc19940203

Fluorescence Reagents for Labelling of Biomolecules
203
FLUORESCENCE REAGENTS FOR LABELLING OF BIOMOLECULES.
PART I. SYNTHESIS AND SPECTRAL CHARACTERIZATION
OF 2- AND 4-SUBSTITUTED 9-ISOTHIOCYANATOACRIDINES
a
a
a,
Dana MAZAGOVA , Danica SABOLOVA , Pavol KRISTIAN *,
a
b
b
Jan IMRICH , Marian ANTALIK and Dusan PODHRADSKY
a Department of Organic Chemistry,
P. J. Safarik University, 041 54 Kosice, The Slovak Republic
b Department of Biochemistry,
P. J. Safarik University, 041 54 Kosice, The Slovak Republic
Received December 11, 1992
Accepted March 26, 1993
9-Isothiocyanatoacridines VIII – XIV were prepared from the corresponding 9-chloroacridines I – VII.
The IR, 1H NMR, 13C NMR and fluorescence spectra of the products are given. The 13C NMR
chemical shifts of the C-9 ipso carbon atom exhibit a trend that is in accord with the Hammett con-
stants of substituents bonded to the C-2 carbon. Effect of these substituents on the chemical shift of
C−NCS was only small. The dependence of hydrolysis of isothiocyanates VIII – XIV on pH of the
medium was studied. It was found that 9-isothiocyanatoacridines do not undergo hydrolysis at pH
7 – 10. The relative fluorescence intensities (F/F0) of compounds VIII – XIV at pH 7.4 have been
determined in comparison with that of 9-aminoacridine. No direct dependence between the fluores-
cence intensity and the polar character of substituents has been found.
The high chemical reactivity of isothiocyanates has become attractive for organic
chemists as well as biochemists. The polar multiple bonds N=C=S easily enter addition
and cyclization reactions under formation of various types of organic compounds1.
From the biochemical viewpoint, isothiocyanates are utilized in the determination of
structure of proteins and nucleic acids2,3.
Recently, great attention has been paid to compounds suitable for fluorescence label-
ling of biomolecules4,5. As such fluorescent reagents may be regarded also acridine
derivatives6,7 and 9-isothiocyanatoacridine7,8, commercially available since 1992
(Serva). This derivative was for the first time prepared in our Laboratory9 as early as
1961, by heating of 9-chloroacridine with silver thiocyanate in toluene. Later on, the
preparation of several other derivatives of 9-isothiocyanatoacridine has been repor-
* The author to whom correspondence should be addressed.
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204
Mazagova, Sabolova, Kristian, Imrich, Antalik, Podhradsky
ted10 – 12. In the present communication we focussed on the study of spectral, structural
and physicochemical characteristics of 2- and 4-substituted 9-isothiocyanatoacridines
with particular stress on the effect of substituents on the relative fluorescence intensity.
EXPERIMENTAL
Preparation of 2- and 4-Substituted 9-Isothiocyanatoacridines VIII – XIV. General Procedure
A mixture of 9-chloroacridine I – III, V – VII (0.024 mol), AgSCN (0.025 mol) and dry toluene
(250 ml) was refluxed for 5 – 6 h under exclusion of air moisture. The reaction mixture was filtered
while hot to remove silver chloride and the unreacted silver thiocyanate. After evaporation of toluene
in vacuo, the crude product was extracted with light petroleum in a Soxhlet apparatus for about 14 h.
The solvent was evaporated in vacuo and the obtained product was crystallized from dry acetone or
toluene (Scheme 1).
For R see Tables II and III
SCHEME 1
2-Chloro-9-isothiocyanatoacridine (XI) was prepared by reaction of 2,9-dichloroacridine (IV) with
potassium thiocyanate in a dichloromethane–water mixture in the presence of tetrabutylammonium
iodide at room temperature12.
Purity of the compounds was checked by thin-layer chromatography on Silufol sheets (Kavalier,
Votice, The Czech Republic) in benzene–acetone (7 : 1); UV-detection at 366 nm.
Spectral and Kinetic Measurements
~
The IR spectra (ν, cm−1) of 9-isothiocyanatoacridines VIII – XIV were recorded on a double-beam
1
IR-75 spectrometer (Zeiss, Jena) in chloroform. H and 13C NMR spectra (δ, ppm) were measured on
Tesla BS 487 (80 MHz) and Tesla BS 567 (25.156 MHz) instruments at room temperature in deute-
riochloroform, except for derivative V whose 13C NMR spectrum was obtained in dideuterio-1,1,2,2-
tetrachloroethane at 105 °C and 9-aminoacridine (Serva) which was measured in hexadeuterio-
dimethyl sulfoxide. The spectra were referenced to tetramethylsilane as internal standard. Tertiary
and quaternary carbon atoms were distinguished by “off resonance” decoupling. Absorption spectra
of the isothiocyanates were obtained with a UV-3000 Shimadzu spectrophotometer (concentration
1.6 . 10−5 mol l−1) and fluorescence spectra on an RF 5000 Shimadzu spectrofluorimeter (concentra-
tion 1.6 . 10−6 mol l−1) in a mixture of HEPES buffer (50 mmol l−1, pH 7.4)–acetonitrile (7 : 3).
Fluorescence emission spectra were measured at excitation wavelength λex 395 nm, whereas the ex-
citation spectra at emission wavelength λem 460 nm. Kinetic measurements of decomposition of the
acridine isothiocyanates were carried out on a Shimadzu UV-3000 spectrophotometer in 0.1 M HCl–
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

Fluorescence Reagents for Labelling of Biomolecules
205
acetonitrile (7 : 3) mixture. The rate constants were evaluated according to first-order equation. All
the measurements were performed at 25 °C.
RESULTS AND DISCUSSION
For the study of relationships between structure and fluorescence properties of 9-iso-
thiocyanatoacridines we have chosen a series of 2-substituted derivatives and also the
4-methyl and 4-methoxy derivatives which were synthetically the best accessible. As
intermediates for the synthesis of isothiocyanates we used 9-chloroacridines I – VII,
obtained by cyclization of the corresponding 2′- and 4′-substituted diphenylamino-2-
carboxylic acids with phosphorus oxychloride13 – 15
.
1
The physicochemical characteristics, together with the IR and H NMR data, are
given in Table I. Although some of the synthesized compounds have already been de-
scribed previously10, it was necessary to determine optimal conditions of their synthesis
in an analytically pure state. Crude 9-isothiocyanates were strongly contaminated with
hydrolysis products (acridone, aminoacridine) as well as with the unreacted 9-chloro-
acridines whose physical properties were similar to those of the corresponding isothio-
cyanates, making thus isolation of the pure products difficult. Because purification by
chromatography was not successful, we purified the compounds by extraction with
light petroleum and subsequent repeated crystallization from toluene or acetone
(Table I).
All the isothiocyanates synthesized exhibited in the IR spectra strong broad complex
~
bands of νas(NCS) in the region 1 950 – 2 150 cm−1 and absorption bands due to sym-
~
metrical stretching vibrations νs(NCS) at 907 – 920 cm−1 (Table I). There was no corre-
~
lation between positions of the absorption maxima of νas(NCS) and Hammett
substituent constants. The marked electron-acceptor character of the acridine skeleton
is obvious from comparison with the corresponding phenyl isothiocyanates whose ab-
sorption bands are shifted about 50 cm−1 towards higher wavenumbers16. The structure
of the obtained compounds I – XIV was also confirmed by their 13C NMR spectra
which for isothiocyanatoacridines have not been hitherto studied. The influence of sub-
stituents on the 13C NMR chemical shifts of the carbon atoms of acridine skeleton in
compounds I – XIV was followed using the easily identifiable signal of the quaternary
carbon atom C-9 and the NCS signal (Table II and III). As model compounds we used
9-aminoacridine, substituted 9-chloroacridines and their benzene analogues.
We observed an interesting effect when comparing the ipso SCS increments of the
NCS, NH2 and Cl substituents in 9-acridinyl and phenyl derivatives (Table IV). In con-
trast to the substituents NH2 and Cl, the SCS value of NCS group for the C-9 carbon in
acridine is negative (−3.3 ppm). This fact is probably connected with different interac-
tion of the NCS group with the acridine hetero atom compared with the chlorine atom
or amino group. The electron-acceptor effect of the acridine nitrogen atom manifests
itself by an increase of chemical shifts in C−NCS derivatives VII – XIV (140.4 –
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

206
Mazagova, Sabolova, Kristian, Imrich, Antalik, Podhradsky
142.4 ppm) as compared17 with meta- and para-substituted phenyl isothiocyanates
(133.9 – 140.4 ppm). These values also show that the transfer of substitution effect to
the NCS carbon atom in phenyl isothiocyanates (∆ppm = 6.5) is greater than in
isothiocyanatoacridines (∆ppm = 2.0). The similar electronic character of the syn-
thesized chloro derivatives (Table II) and isothiocyanates (Table III) is obviously the
reason why the chemical shifts of the acridine carbon atoms differ only little, except for
the C-9 atom which in chloro derivatives I – VII resonates at 138.1 – 144.1 ppm and in
isothiocyanates VIII – XIV in the region 130.0 – 136.8 ppm. Moreover, these values
TABLE I
9-Isothiocyanatoacridines VIII – XIV
~
M.p., °C
νas(NCS)
1H NMR (CH3)a
4.05 s
Compound
R
Yield, %
67
~
(Ref.10 m.p.)
νs(NCS)
VIII
137 – 138
toluene
2 060
907
2-OCH3
(137 – 138.5)
IX
48
52
59
124 – 125
acetone
2 070
913
2.65 s
2-CH3
(123 – 125)
X
131 – 132
acetone
2 060
918
–
–
H
(131 – 132)
XI
154 – 155
acetone
2 060
914
2-Cl
(155 – 156)
XIIb
47
62
82
195 – 197
2 045
908
–
2-NO2
toluene
XIIIc
165 – 167
2 075
920
4.23 s
2.65 s
4-OCH3
toluene
XIV
135 – 137
acetone
2 075
920
4-CH3
(135 – 137)
a
b
Other signals: 7.25 – 8.4 m (acridine H). For C14H7N3O2S (281.3) calculated: 59.78% C, 2.51% H,
c
14.94% N; found: 58.92% C, 2.54% H, 14.81% N. For C15H10N2OS (266.3) calculated: 67.65% C,
3.78% H, 10.52% N; found: 67.48% C, 3.70% H, 10.41% N.
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Fluorescence Reagents for Labelling of Biomolecules
207
TABLE II
13C NMR chemical shifts (δ, ppm) of 9-chloroacridines I – VII
Compound
R
C-1aa
C-8aa
C-4ab
C-5ab
C-9
C-2 or C-4
158.2
CH
CH3
55.6
I
124.4
125.2
146.1
147.3
138.1
99.8; 129.2
124.1; 129.8
125.8; 131.5
127.0
2-OCH3
II
124.2
124.3
147.9
148.4
139.6
140.9
139.7
144.1
141.0
140.7
136.9
122.5; 129.8
124.4; 129.8
126.6; 133.2
129.5
22.0
2-CH3
III
124.2
124.2
148.9
148.9
–
124.4; 129.8
124.4; 129.8
126.7; 130.3
126.7; 130.3
–
H
IV
124.3
124.4
147.0
148.9
133.0
145.8
155.4
137.8
123.0; 130.6
124.4; 131.6
127.4; 131.6
130.0
–
2-Cl
V
122.6
124.8
149.4
151.0
122.2; 130.4
123.0; 132.2
124.7; 132.4
128.3
–
2-NO2
VI
124.6
125.4
142.2
147.9
106.9; 127.4
116.5; 130.2
124.4; 130.7
127.0
56.4
18.6
4-OCH3
VII
124.0
124.2
148.1
148.5
122.5; 129.7
124.4; 129.8
126.7; 130.4
126.7
4-CH3
a,b
Assignment may be reversed.
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208
Mazagova, Sabolova, Kristian, Imrich, Antalik, Podhradsky:
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

Fluorescence Reagents for Labelling of Biomolecules
209
show a trend that corresponds to the Hammett constants of substituents on the C-2
atom. A much weaker interaction was found between substituents in position 2 and the
NCS carbon atom; this indicates that outside the acridine skeleton there is no marked
conjugation effect.
The electronic absorption spectra of 9-isothiocyanatoacridines in the region 300 –
500 nm have a resolved vibrational structure of the p-band10 the number and intensity
of which depends on the character of the substituent and pH of the medium (Fig. 1). A
more detailed study of the position and intensity of the vibrational bands will be the
subject of a separate communication.
Protonation of the nitrogen atom in the acridine skeleton destabilizes the NCS group
which is hydrolyzed to give an amine (Fig. 2) as confirmed by the spectrum obtained
after hydrolysis of 9-isothiocyanatoacridine. On the other hand, phenyl isothiocyanate
is stable in an acid medium. The acid hydrolysis of the NCS group was also observed
with other 9-isothiocyanatoacridine derivatives. In all cases the reaction was of the first
FIG. 1
FIG. 2
Electronic absorption spectra of 9-amino-
acridine (−−−−−) and 9-isothiocyanatoacridine
(X) (− − − −), measured in 50 mM buffer
HEPES (pH 7.4)–acetonitrile (7 : 3). 9-Isothio-
Kinetics of hydrolysis of 9-isothiocyanato-
acridine (X). Spectra taken at the rate 200 nm
min−1 in 0.1 M HCl–acetonitrile (7 : 3); inter-
vals: 1 1 min, 2 5 min, 3 15 min, 4 25 min, 5
40 min, 6 60 min, 7 70 min
. . . .
cyanatoacridine (X) (
) in 0.1 M HCl–aceto-
nitrile (7 : 3). Concentration of all acridine
derivatives: 1.6 . 10−5 mol l−1
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

210
Mazagova, Sabolova, Kristian, Imrich, Antalik, Podhradsky
order. The rate constants observed for the individual derivatives are listed in Table V.
The stability of 9-isothiocyanatoacridines against hydrolysis increases with increasing
pH and in the region above pH 6 there is practically no reaction at all. Further increase
TABLE IV
Substituent chemical shifts (ppm, CDCl3) of the Cl, NH2 and NCS substituents on ipso carbon in
phenyl19,20 and acridinyl derivatives
Compound
Cl
NH2
NCS
Benzene (C = 128.5)
5.9
5.1
19.2
13.8a
2.4
Acridine (C-9 = 135.9)
−3.3
a (CD3)2SO.
TABLE V
Parameters of the absorption and fluorescence emission spectra and the observed rate constants (k) of
hydrolysis of 9-isothiocyanatoacridines VIII – XIV
λ
max, nma
log ε
k . 104
c
λmax, nmb
F/F0
s−1
Compound
VIII
399
450
0.062
0.020
0.028
0.036
0.0011
0.043
0.032
1.41
2.12
3.16
3.50
5.1
4.00
IX
395
440; 460
435; 460
435; 460
417
4.40
X
393
4.00
XI
394
3.87
XII
XIII
XIV
421
3.87
414
508
3.25
3.5
4.00
397
460
4.09
a
c
Electronic absorption. b Fluorescence emission. Relative fluorescence F/F0, where F0 = 1 for 1.6 . 10−6
mol l−1 solution of 9-aminoacridine.
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Fluorescence Reagents for Labelling of Biomolecules
211
in concentration of hydroxyl ions (pH > 10) results again in hydrolysis under formation
of the corresponding aminoacridines. The determination of the region of pH in which
the isothiocyanates VIII – XIV are stable was very important, not only for the study of
reactivity of isothiocyanates with nucleophiles18 but also from the viewpoint of charac-
terization of the fluorescence spectra because 9-aminoacridine belongs to fluorescence
reagents with a high fluorescence yield. These facts have not been taken into account
by authors of a previous study8 who compared relative fluorescence yields of various
acridine derivatives in strongly acidic media. Therefore, in the case of 9-isothiocyana-
toacridine their results were affected by the fluorescence contribution of 9-amino-
acridine. For this reason we measured the fluorescence spectra of isothiocyanato-
acridines VII – XIV at pH 7.4 (HEPES–acetonitrile, 7 : 3). Acetonitrile was used to
increase the solubility of isothiocyanates which are only sparingly soluble in water.
Figure 3 shows excitation and emission spectra of 9-isothiocyanatoacridine refer-
enced to the maximum fluorescence intensity of 9-aminoacridine. The relative fluores-
cence intensities (F/F0; referenced to 9-aminoacridine) at the emission maxima for
compounds VIII – XIV are given in Table V. As seen, all the 9-isothiocyanatoacridines
investigated have lower fluorescence yield than 9-aminoacridine, the differences in the
fluorescence intensities being much more pronounced than the differences in the
13C NMR or electronic absorption spectra. The highest fluorescence intensity was
found for the 2-OCH3 derivative VIII whereas the lowest for the 2-NO2 derivative XII.
FIG. 3
Excitation and emission spectra of 9-isothiocyanato-
acridine (X). Concentration 1.6 . 10−6 mol l−1 in 50
mM buffer HEPES (pH 7.4)–acetonitrile (7 : 3).
Excitation spectrum at λem ≈ 460 nm (−−−−−),
. . . .
emission spectrum at λex ≈ 395 nm (
); spectra
referenced to that of 9-aminoacridine measured
under the same conditions
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

212
Mazagova, Sabolova, Kristian, Imrich, Antalik, Podhradsky
The importance of isothiocyanatoacridines for the detection of amino acids or other
biomolecules containing the NH2 group consists in the fact that the fluorescence in-
tensity of the addition products (the corresponding thioureas) is comparable with that of
9-aminoacridine18.
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