Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
A FLUORESCENT PHOTO-AFFINITY LABEL FOR
CYCLIC AMP BINDING PROTEINS Elaine K. Keeler and P e t e r Campbell* Department of Chemistry New York University, New York, N. Y. Received
July
15,
i0003
1976
Summary A f l u o r e s c e n t a n a l o g o f c y c l i c AMP which c o n t a i n s t h e p h o t o r e a c t i v e a z i d e group has been s y n t h e s i z e d and c h a r a c t e r i z e d . S i n c e upon p h o t o l y s i s t h e m o l e c u l e becomes i r r e v e r s i b l y bound t o i t s a c c e p t o r s i t e i t should prove u s e f u l as a f l u o r e s c e n t probe f o r c y c l i c AMP b i n d i n g s i t e s . I n o r d e r t o develop a f l u o r e s c e n t l a b e l f o r t h e a c t i v e s i t e s of p r o t e i n s which b i n d c y c l i c AMP, we are engaged i n an i n v e s t i g a t i o n o f t h e a z i d e d e r i v a t i v e of l , N 6 - e t h e n o a d e n o s i n e 3 ' : 5 ' have r e c e n t l y succeeded i n
c y c l i c monophosphate.
(1-3)
u s i n g t h e a z i d e d e r i v a t i v e s of ATP and c y c l i c AMP
t o label the binding s i t e s of p r o t e i n s . sensitive activity
Haley e t a l .
The p o s s i b i l i t y
of combining t h e p h o t o -
of t h e a z i d e group with t h e f l u o r e s c e n t p r o p e r t i e s o f t h e
e t h e n o a d e n o s i n e d e r i v a t i v e s has l e d t o the s y n t h e s i s o f t h e molecule d e s c r i b e d here.
The f l u o r e s c e n c e of t h e e t h e n o a d e n o s i n e d e r i v a t i v e s has been s t u d i e d
and d e s c r i b e d e x t e n s i v e l y by Leonard and coworkers (4-7)°
We propose t o u s e
t h e s e f l u o r e s c e n t p r o p e r t i e s t o probe t h e a c t i v e s i t e s of p r o t e i n s which b i n d cyclic AMP. Materials and Methods Adenosine 3':S'-cyclic monophosphoric acid was obtained from Sigma Chemical Co. and chloroacetaldehyde
diethyl acetal from Aldrich Chemical Co.
Thin-layer chromatography was performed on Eastman cellulose sheets [13255) and developed in either solvent system A (n__-butanol-acetic acid - H20, 5:2:3, v/v), B (isopropanol - NH3(aq)-H20 , 7:1:2, v/v) or C (isobutyric acid-
*To whom correspondence Should be addressed
Copyright ® 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
575
Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
NH3(aq)-H20 , 75:1:24, v/v).
Ultraviolet spectra were determined on a Perkin
Elmer - Coleman Model 124D double - beam spectrophotometer and infrared spectra on a Perkin Elmer Model700
spectrophotometer.
Fluorescence measurements were
made on a Hitachi-Perkin Elmer MPF-2A fluorescence spectrophotometer.
Proton
magnetic resonance spectra were obtained on a Varian XLI00 spectrometer.
Water
was redistilled in glass vessels.
Dimethylformamide was distilled under vacuum
and stored over molecular sieves.
Other materials were of reagent grade and
used without luther purification. 8-BromoadenoSine 3':S'-cyclic monophosphate
(I).
8-Br-cAMP* was prepared according to the procedure of Muneyama (8) as follows: 0.2510 g (0.76 mmoles) of cyclic AMP was dissolved in 0.4 ml of 2M Na0H and 0.50 ml of IM acetate buffer was added.
0.1736 g (2 mmoles) of bromine was
dissolved in 6.0 ml of IM acetate buffer and added to the cyclic AMP solution. The reaction was allowed to stir at room temperature overnight. precipitate was filtered and washed with water,
The resulting
yield 0.2193 g (70.2%)
8-Azidoadenosine 3':S'-cyclic monophosphate
(2).
8-N3-cAMP was prepared according to the procedure of Muneyama (8) as follows: 0.1199 g (0.29 mmoles)
of 8-Br-cAMP and 0.0422 g (0.65 mmoles) of sodium azide
was dissolved in 12 ml of dry dimethylformamide
and stirred overnight at 75°C.
The solvent was removed on a rotary evaporator.
The residue was dissolved in
a minimal amount of IMNHs and the pH adjusted to 99%).
Results and Discussion Thin-layer chromatography in three different solvent systems (table I) was used to monitor the reactions and detect impurities of the nucleotide derivatives. In all cases products appeared as a single spot and were considered to be free of trace impurities.
Both e-cAMP and 8-N3-e-cAMP are intensely fluorescent and
can be easily distinguished from the other cyclic AMP derivatives,
e-cAMP ap-
pears as a bluish fluorescent spot while 8-N3-e-cAMP fluoresces with a greenblue color. The infrared spectrum of 8-N3-~-cAMP shows the characteristic bands for -N 3 stretching vibrations at 2171 cm -I (s) and 1280 cm
-I
(w).
The proton magnetic resonance spectra of cyclic AMP,e-cAMP and 8-N3-ecAMP are summarized in table II.
The absence of an absorbance for the hydrogen
on C 8 indicates substitution by the azide group at this position.
Introduction
of the etheno bridge is shown by the two doublets at 7.94 ~ and 8.37 ~. A comparison (table III) of the UV spectra of 8-N3-cAMP and 8-N3-e-cA~ shows a shift of the absorbance maximum from 281 nm to 290 nm and a decrease in the molar extinction coefficient. adenosine derivatives (6).
This decrease is characteristic of etheno-
A plot of extinction coefficient at 290 nm vs, pH
577
Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
TABLE I Rf Values for Cyclic AMP and Derivatives Compound
Solvent A a
Solvent Ca
Solvent B a
Rf
Re
cyclic AMP
O. 47
O. 48
O. 69
8-Br-cAMP
0.62
O. 60
0.68
8 -N 3- cAMP
0.63
0.61
0.67
8-N3- e- cAMpb
0.62
O. 65
O. 65
c-cAMP b
0.42
0.53
0.56
Rf~
a
see text for composition of solvents bfluorescent spots
TABLE I I Proton Magnetic Resonance Spectra Chemical shifts (8) Compound
C2-H
Cs-H
C10-H
8.24 s(2)
cyclic AMP a
Cll-"
Cl,-H 6.16 s
-
(I)
E-cAMP b
9.21 s(1)
8.43 s(1) 8.06 d, J=lHz(1)
7.68 d,J=iHz(1) 6.32 s(1)
8-N3-s-cAMpb
9.37 s(1)
-
7.94 d,J=IHz(1) 6.26 s(1)
a8 values r e l a t i v e b8 values r e l a t i v e
8.37 d, J=IHz(1)
t o i n t e r n a l DSS. to e x t e r n a l DSS.
TABLE III Ultraviolet AbsorptionData
~: x 10-3(M-lem -1)
C omp oun d
pH
Xmax (nm)
8-Br-cAMP
6.0
264
35
8-N3-cAMP
6.0
281
13
8 -N 3- c- cAMP
6.0
290
3.5
2.0
290
5.5
578
Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
-o--.-.O~o~,,~ 5.0
o
'E o 4,5 i
~E ,? 4 . 0 o u
35
30
I
~
I
I
;
J
2
:5
4
5
6
7
pH
Figure
1.
Cyclic AMP Drivatives
(figure 2) places the pK a of 8-N3-e-cAMP at 4.17, slightly higher than that of e-c~4P
(6).
The f l u o r e s c e n c e solved band with The e x c i t a t i o n
emission
spectrum of(Z)
a t pH 5 . 5 shows a s i n g l e
a maximum a t 402 nm when t h e s o l u t i o n
spectrum,
monitored
is
excited
unre-
a t 300 nm.
a t 400 nm, shows maxima a t 280 nm, 310 nm
a n d 560 nm. The p h o t o r e a c t i v i t y bind
irreversibly
o f t h e m o l e c u l e was d e m o n s t r a t e d
to celluiose
The 8-N3-e-cAHP f l u o r e s c e n t after
irradiation,
every
system. The good y i e l d s
as the this
fluorescent
molecule
AMP b i n d i n g
whereas
plates
spot remained at the a nonirradiated
obtained
control
of the product, bound fluorescent
a r e now i n p r o g r e s s
579
in these
ability
when i r r a d i a t e d origin
in all
to
a t 2 5 3 . 7 rim. solvent
systems
s p o t moved s i g n i f i c a n t l y
and the ease of purification
characteristics
a s an i r r e v e r s i b l y
proteins
thin-layer
by its
of products,
makes a t t r a c t i v e probe.
as w e l l
the use of
Such s t u d i e s
laboratories.
in
of cyclic
Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAl. RESEARCH COMMUNICATIONS
NH2 I[I~'~--B
NH2 NJ
r
N
CH2
o//~.---.-o
o~o
OH
OH
/ OH
OH
(2)
(t)
~ N ~N
o~/~o
N
OH
OH
(3) Figure 2. Spectrophotometric titration of 3 at 290 nm. The solid line is the theoretical curve for PKa=4.17, ~(low pH) =--5580 M-icm-l, ~(high pH) = 3000 M -I cm -I. Points are experimental values. Acknowledgement Acknowledgement is made to the Donors of the Petroleum Research Fund, administered by the American Chemical Society, for the support of this research. We thank Mr. Charles Strom for the determination of the proton magnetic resonance spectra.
References i. 2. 3. 4. S. 6. 7. 8.
Haley, B. E. and Hoffman, J. F. (1974) Prec. Nat. Acad. Sci. U.S.A., 71, 3367-3377. Haley, B. E. (1976) Biochemistry, 14, 3852-3857. Pomerantz, A. H., Rudolf, S. A., Haley, B. E. and Greengard, P. (1976) Biochemistry, 14, 3858-3862. Secrist, J. A. III, Barrio, J. R., Leonard, N. J., Villar-Polasi, C., and Gilman, A. G. (1972) Science, 176, 279-280. Barrio, J. R., Secrist, J. A., III and Leonard, N. J. (1972) Biochem. Biophys. Res. Commun., 46, 597-604. Secrist, J. A. III, Barrio, J. R., Leonard, N. J. and Weber, G. (1972) Biochemistry, ii, 3499-3506. Muneyama, K.,Bauer, R. J., Shuman, D. A., Robins, R. K., and Simon, L. N. (1971) Biochemistry, i0, 2390-2395. Roberts, J. E., Aizono, Y., Sonenberg, M., and Swislacki, N. I. (1975) Bioorganic Chem., 4, 181-187.
580
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
A FLUORESCENT PHOTO-AFFINITY LABEL FOR
CYCLIC AMP BINDING PROTEINS Elaine K. Keeler and P e t e r Campbell* Department of Chemistry New York University, New York, N. Y. Received
July
15,
i0003
1976
Summary A f l u o r e s c e n t a n a l o g o f c y c l i c AMP which c o n t a i n s t h e p h o t o r e a c t i v e a z i d e group has been s y n t h e s i z e d and c h a r a c t e r i z e d . S i n c e upon p h o t o l y s i s t h e m o l e c u l e becomes i r r e v e r s i b l y bound t o i t s a c c e p t o r s i t e i t should prove u s e f u l as a f l u o r e s c e n t probe f o r c y c l i c AMP b i n d i n g s i t e s . I n o r d e r t o develop a f l u o r e s c e n t l a b e l f o r t h e a c t i v e s i t e s of p r o t e i n s which b i n d c y c l i c AMP, we are engaged i n an i n v e s t i g a t i o n o f t h e a z i d e d e r i v a t i v e of l , N 6 - e t h e n o a d e n o s i n e 3 ' : 5 ' have r e c e n t l y succeeded i n
c y c l i c monophosphate.
(1-3)
u s i n g t h e a z i d e d e r i v a t i v e s of ATP and c y c l i c AMP
t o label the binding s i t e s of p r o t e i n s . sensitive activity
Haley e t a l .
The p o s s i b i l i t y
of combining t h e p h o t o -
of t h e a z i d e group with t h e f l u o r e s c e n t p r o p e r t i e s o f t h e
e t h e n o a d e n o s i n e d e r i v a t i v e s has l e d t o the s y n t h e s i s o f t h e molecule d e s c r i b e d here.
The f l u o r e s c e n c e of t h e e t h e n o a d e n o s i n e d e r i v a t i v e s has been s t u d i e d
and d e s c r i b e d e x t e n s i v e l y by Leonard and coworkers (4-7)°
We propose t o u s e
t h e s e f l u o r e s c e n t p r o p e r t i e s t o probe t h e a c t i v e s i t e s of p r o t e i n s which b i n d cyclic AMP. Materials and Methods Adenosine 3':S'-cyclic monophosphoric acid was obtained from Sigma Chemical Co. and chloroacetaldehyde
diethyl acetal from Aldrich Chemical Co.
Thin-layer chromatography was performed on Eastman cellulose sheets [13255) and developed in either solvent system A (n__-butanol-acetic acid - H20, 5:2:3, v/v), B (isopropanol - NH3(aq)-H20 , 7:1:2, v/v) or C (isobutyric acid-
*To whom correspondence Should be addressed
Copyright ® 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
575
Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
NH3(aq)-H20 , 75:1:24, v/v).
Ultraviolet spectra were determined on a Perkin
Elmer - Coleman Model 124D double - beam spectrophotometer and infrared spectra on a Perkin Elmer Model700
spectrophotometer.
Fluorescence measurements were
made on a Hitachi-Perkin Elmer MPF-2A fluorescence spectrophotometer.
Proton
magnetic resonance spectra were obtained on a Varian XLI00 spectrometer.
Water
was redistilled in glass vessels.
Dimethylformamide was distilled under vacuum
and stored over molecular sieves.
Other materials were of reagent grade and
used without luther purification. 8-BromoadenoSine 3':S'-cyclic monophosphate
(I).
8-Br-cAMP* was prepared according to the procedure of Muneyama (8) as follows: 0.2510 g (0.76 mmoles) of cyclic AMP was dissolved in 0.4 ml of 2M Na0H and 0.50 ml of IM acetate buffer was added.
0.1736 g (2 mmoles) of bromine was
dissolved in 6.0 ml of IM acetate buffer and added to the cyclic AMP solution. The reaction was allowed to stir at room temperature overnight. precipitate was filtered and washed with water,
The resulting
yield 0.2193 g (70.2%)
8-Azidoadenosine 3':S'-cyclic monophosphate
(2).
8-N3-cAMP was prepared according to the procedure of Muneyama (8) as follows: 0.1199 g (0.29 mmoles)
of 8-Br-cAMP and 0.0422 g (0.65 mmoles) of sodium azide
was dissolved in 12 ml of dry dimethylformamide
and stirred overnight at 75°C.
The solvent was removed on a rotary evaporator.
The residue was dissolved in
a minimal amount of IMNHs and the pH adjusted to 99%).
Results and Discussion Thin-layer chromatography in three different solvent systems (table I) was used to monitor the reactions and detect impurities of the nucleotide derivatives. In all cases products appeared as a single spot and were considered to be free of trace impurities.
Both e-cAMP and 8-N3-e-cAMP are intensely fluorescent and
can be easily distinguished from the other cyclic AMP derivatives,
e-cAMP ap-
pears as a bluish fluorescent spot while 8-N3-e-cAMP fluoresces with a greenblue color. The infrared spectrum of 8-N3-~-cAMP shows the characteristic bands for -N 3 stretching vibrations at 2171 cm -I (s) and 1280 cm
-I
(w).
The proton magnetic resonance spectra of cyclic AMP,e-cAMP and 8-N3-ecAMP are summarized in table II.
The absence of an absorbance for the hydrogen
on C 8 indicates substitution by the azide group at this position.
Introduction
of the etheno bridge is shown by the two doublets at 7.94 ~ and 8.37 ~. A comparison (table III) of the UV spectra of 8-N3-cAMP and 8-N3-e-cA~ shows a shift of the absorbance maximum from 281 nm to 290 nm and a decrease in the molar extinction coefficient. adenosine derivatives (6).
This decrease is characteristic of etheno-
A plot of extinction coefficient at 290 nm vs, pH
577
Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
TABLE I Rf Values for Cyclic AMP and Derivatives Compound
Solvent A a
Solvent Ca
Solvent B a
Rf
Re
cyclic AMP
O. 47
O. 48
O. 69
8-Br-cAMP
0.62
O. 60
0.68
8 -N 3- cAMP
0.63
0.61
0.67
8-N3- e- cAMpb
0.62
O. 65
O. 65
c-cAMP b
0.42
0.53
0.56
Rf~
a
see text for composition of solvents bfluorescent spots
TABLE I I Proton Magnetic Resonance Spectra Chemical shifts (8) Compound
C2-H
Cs-H
C10-H
8.24 s(2)
cyclic AMP a
Cll-"
Cl,-H 6.16 s
-
(I)
E-cAMP b
9.21 s(1)
8.43 s(1) 8.06 d, J=lHz(1)
7.68 d,J=iHz(1) 6.32 s(1)
8-N3-s-cAMpb
9.37 s(1)
-
7.94 d,J=IHz(1) 6.26 s(1)
a8 values r e l a t i v e b8 values r e l a t i v e
8.37 d, J=IHz(1)
t o i n t e r n a l DSS. to e x t e r n a l DSS.
TABLE III Ultraviolet AbsorptionData
~: x 10-3(M-lem -1)
C omp oun d
pH
Xmax (nm)
8-Br-cAMP
6.0
264
35
8-N3-cAMP
6.0
281
13
8 -N 3- c- cAMP
6.0
290
3.5
2.0
290
5.5
578
Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
-o--.-.O~o~,,~ 5.0
o
'E o 4,5 i
~E ,? 4 . 0 o u
35
30
I
~
I
I
;
J
2
:5
4
5
6
7
pH
Figure
1.
Cyclic AMP Drivatives
(figure 2) places the pK a of 8-N3-e-cAMP at 4.17, slightly higher than that of e-c~4P
(6).
The f l u o r e s c e n c e solved band with The e x c i t a t i o n
emission
spectrum of(Z)
a t pH 5 . 5 shows a s i n g l e
a maximum a t 402 nm when t h e s o l u t i o n
spectrum,
monitored
is
excited
unre-
a t 300 nm.
a t 400 nm, shows maxima a t 280 nm, 310 nm
a n d 560 nm. The p h o t o r e a c t i v i t y bind
irreversibly
o f t h e m o l e c u l e was d e m o n s t r a t e d
to celluiose
The 8-N3-e-cAHP f l u o r e s c e n t after
irradiation,
every
system. The good y i e l d s
as the this
fluorescent
molecule
AMP b i n d i n g
whereas
plates
spot remained at the a nonirradiated
obtained
control
of the product, bound fluorescent
a r e now i n p r o g r e s s
579
in these
ability
when i r r a d i a t e d origin
in all
to
a t 2 5 3 . 7 rim. solvent
systems
s p o t moved s i g n i f i c a n t l y
and the ease of purification
characteristics
a s an i r r e v e r s i b l y
proteins
thin-layer
by its
of products,
makes a t t r a c t i v e probe.
as w e l l
the use of
Such s t u d i e s
laboratories.
in
of cyclic
Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAl. RESEARCH COMMUNICATIONS
NH2 I[I~'~--B
NH2 NJ
r
N
CH2
o//~.---.-o
o~o
OH
OH
/ OH
OH
(2)
(t)
~ N ~N
o~/~o
N
OH
OH
(3) Figure 2. Spectrophotometric titration of 3 at 290 nm. The solid line is the theoretical curve for PKa=4.17, ~(low pH) =--5580 M-icm-l, ~(high pH) = 3000 M -I cm -I. Points are experimental values. Acknowledgement Acknowledgement is made to the Donors of the Petroleum Research Fund, administered by the American Chemical Society, for the support of this research. We thank Mr. Charles Strom for the determination of the proton magnetic resonance spectra.
References i. 2. 3. 4. S. 6. 7. 8.
Haley, B. E. and Hoffman, J. F. (1974) Prec. Nat. Acad. Sci. U.S.A., 71, 3367-3377. Haley, B. E. (1976) Biochemistry, 14, 3852-3857. Pomerantz, A. H., Rudolf, S. A., Haley, B. E. and Greengard, P. (1976) Biochemistry, 14, 3858-3862. Secrist, J. A. III, Barrio, J. R., Leonard, N. J., Villar-Polasi, C., and Gilman, A. G. (1972) Science, 176, 279-280. Barrio, J. R., Secrist, J. A., III and Leonard, N. J. (1972) Biochem. Biophys. Res. Commun., 46, 597-604. Secrist, J. A. III, Barrio, J. R., Leonard, N. J. and Weber, G. (1972) Biochemistry, ii, 3499-3506. Muneyama, K.,Bauer, R. J., Shuman, D. A., Robins, R. K., and Simon, L. N. (1971) Biochemistry, i0, 2390-2395. Roberts, J. E., Aizono, Y., Sonenberg, M., and Swislacki, N. I. (1975) Bioorganic Chem., 4, 181-187.
580
