- Email: [email protected]
Bioluminescent properties of fluorinated semi-synthetic aequorins
TETRAHEDRON LETTERS
Tetrahedron Letters 39 (19981 5541-5544
Pergamon
Bioluminescent Properties of Fluorinated Semi-synthetic Aequorins Takashi Hirano,* Yoshihiro Ohmiya,t Shojiro Maki, Haruki Niwa, and Mamoru Ohashitt Department of Applied Physics and Chemisto', The University of Electro-Comnumications, Chofu, Tokyo 182, Japan. ?Department of Biochemisto,, FaculG of Education, Shizuoka Universi~', Shizuoka 422. Japan. ~('~Department of Materials Science, Kanagawa UniversiO,, Hiratsuka, Kanagawa 259-12. Japan.
Received 5 March 1998; accepted 25 May 1998
Abstract Bioluminescent properties of semi-synthetic aequorins containing coelenterazine analogues possessing fluoro group(s) on the 6-(4-hydroxyphenyl) group match the fluorescent behavior of the phenolate anions of the corresponding fluorinated coelenteramide analogues, indicating that the phenolate anion of coelenteramide is the light-emitter in aequorin bioluminescence. © 1998 ElsevierScience Ltd. All rightsreserved. Kevwords: Bioluminescence,Chemiluminescence,Fluorescence
The photoprotein aequorin (AQ) isolated from the jellyfish Aequorea victoria consists of apoaequorin (apoAQ, apoprotein), coelenterazine 1, and 0 2 [1,2]. AQ shows bioluminescence by binding calcium ions, yielding a blue fluorescent protein (BFP), CO 2, and light. BFP made up of coelenteramide 2 bound to apoAQ is the light emitter [3]. AQ is obtained by incubating 1 with recombinant apoAQ under air (Scheme 1) [4]. Similarly, semisynthetic AQs are obtained by using chemically modified coelenterazine analogues [5]. The methodology using semi-synthetic AQs is useful for investigating the bioluminescent properties of AQ [5,6]. Coelenterazine 1 has three appendages on the 3,7-dihydroimidazo[1,2-a]pyrazin-3-one ring. Because the 4-hydroxyphenyl group at C6 directly conjugates to the imidazopyrazinone ring, the modification of the 6-(4-hydroxyphenyl) group changes the electronic property of 1. Of particular interest would be introduction of fluorine atoms aequorin
(AQ)
Ca2+ ,
apoaequorin (apoAQ) + coelenteramide
t
I 02 + coetenterazine1 0,~--~
y
2
+ 002 + hv Scheme 1
OH
O.~~OH
/N../,N
y 1FI (X= F, Y= H) 1F2 (X= Y= F)
/N_y.NH
HO" ~
* e-mail: [email protected]
0040-4039/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved. PII: S0040-4039(98)01115-0
~..~
2FI (X= F, Y= H) 2F2 (X= Y= F)
5542
characterized by the largest electronegativity (4.0) and the small covalent radius (0.64 A). In addition, the introduced fluorine atom is a useful probe for NMR study [7]. Therefore, we designed coelenterazine analogues 1F1 and 1F2 possessing fluoro group(s) on the 6-(4hydroxyphenyl) group for investigating electronic substituent effect on the bioluminescent properties of AQ and for 19F-NMR study [8]. This paper reports the syntheses and the bioand chemiluminescent properties of 1F1 and 1F2, and the fluorescent properties of the corresponding coelenteramide analogues 2F1 and 2F2. The result of this study shows an evidence to elucidate the ionic structure of the light-emitter during AQ bioluminescence [9]. Y X. ~
~/ N..~ L NH2
B(OH)2 +
BnO- ~ 3F1 (X= F, Y= H) 3F2 (X= Y= F) TBDMSO~CH3
Br
N 4 iii, iv
CH2Ph
Y
/N<.~NH2
x ,c-Lc.2p, y
BnO" '¢" 5F1 (X= F, Y= H; y= 92%) 5F2 (X= Y= F; y= 93%)
/N~-y.NH2
HO- " ~
6F~ (X= F, Y= H; y= 80*) 6F2 (X=Y= F; y= 83%)
," T B D M S O CH2COCH(OC2H5)2 6F1 or 6F2 7 (y= 21%)
1F1 (y= 57%) 1F2 (y= 53%)
Scheme 2 R e a g e n t s a n d conditions: i. [Pd(PPh3)4], dioxane / Na2CO3aq: ii. H 2, Pd/C: iii. NBS, CCI 4, benzoylperoxide: iv, (1) Mg, THF, (2) CsHIoNCOCH(OEt) 2, (3) FI30+; v, HCl-dioxane," 110 °C.
Analogues 1F1 and 1F2 were prepared according to a modification of the reported procedure as shown in Scheme 2 [10-12]. A simple method for preparing keto acetal 7 by two steps from t-butyldimethylsilylated p-cresol was developed [6b]. Coelenteramide analogues 2F1 and 2F2 were prepared by the acylation of 6F1 and 6F2, respectively. Semi-synthetic AQs containing 1FI and 1F2 (AQ-1F1 and AQ-1F2) and wild type AQ were prepared by incubating 1F1, 1F2, and 1 (2.1 x 10 -3 M in MeOH, 20 ktl) with recombinant apoAQ [13] (10 I.tg) in Tris-HC1 buffer (0.03 M, pH 7.6, 1.0 ml) containing dithiothreitol (0.002 M) and EDTA (0.01 M). After incubation for 20 h at 0 °C, the bioluminescence activity of the semi-synthetic AQs was assayed by mixing the incubation mixture (10 ktl) with a 0.4 ml of Tris-HCl buffer (pH 7.6) containing CaC12 (0.03 M). The amounts of total light emitted by bioluminescence of AQ-1F1 and AQ-1F2 were 67% and 1%, respectively, relative to that of wild type AQ (100%). This result indicates that 3-fluoro group of the 6-(4-hydroxyphenyl) group does not prevent the regeneration of the semisynthetic AQ, and that 1F1 is useful for NMR study. ...........i ..................a ..............;............................................... The bioluminescence emission spectra of AQ! i + 1F1, AQ-1F2, and wild type AQ are shown in Fig. 1. The spectrum (~'max 460 n m ) o f AQ-1F1 coincides with that of wild type AQ, while AQ-1F2 ....... +.~:/+.............,~.........+..'t+~...........~................+................ showed a blue-sifted emission maximum at 440 nm. ++ ',+ +~+_ + To evaluate these spectral characteristics, the E +.Ji + \ '-~-..++..... chemiluminescent properties of 1F1 and 1F2, and j++. :~ + + + + . + . + . + +++, . + .+..~'.+~.,,+++.+;, .... ; the fluorescent properties of the corresponding i I I 350 400 450 500 550 600 650 coeleneteramide analogues 2F1 and 2F2 w e r e wavelength/ nm investigated. Chemiluminescence reactions of 1F1 Fig. 1 Bioluminescence spectra of wild type AQ (a), AQ-1F1 (b), and AQ-1F2 (e). and 1F2 in DMSO under air showed the similar ......
"/+ ...........................
+,+'u :, ....................
+~...........
+
5543
I .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
a .......i
/' b ~".,
I
:~ .........
+......
//c
~1
/~'
~,.~ i',,
i ...................... i .............
L, ~', '-.
/1'
350
C
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
:
",,
"~',..
400
450 500 550 600 650 wavelength / nm Fig. 2 Chemiluminescence spectra of ! (a), IF! (b), and 1172 (c) (4.8 x 10-5 M) in DMSO under air.
350
400
450 500 550 wavelength / nm
600
650
Fig. 3 Fluorescence spectra of 2 (a), 2FI (b), and 2F2 (c) (1.1 x 10-6 M) in benzene containing n-butylamine (0.01 M). The asterisk (*) shows a fluorescence peak of the neutral form of 2.
emission maxima at 470 and 465 nm, respectively, to that (470 nm) of wild type 1 (Fig. 2). The relative chemiluminescence efficiency of 1F1 and 1F2 to that of 1 are 1.6 and 1.8, respectively. The light emitters of these chemiluminescence reactions have been assigned as the singlet excited states of the amide anions (I) of 2, 2F1, and 2F2 [14]. Little change of the chemiluminescence maxima induced by fluorination differs from the large shift of bioluminescence maxima, suggesting that the excited amide anion (I) is not the light-emitter in AQ bioluminescence. The phenolate anion (II) has also been proposed as the light-emitting structure of 2 in BFP during AQ bioluminescence [9]. We found the condition for observing a fluorescence of the phenolate anion in this work, although the phenolate anion of 2 produced in DMSO was non-fluorescent [15]. Excitation of the hydrogen-bonded complex (III) of 2 with n-butylamine in a non-polar solvent, such as benzene and CHC13, gave the singlet excited state of II in a similar manner of the naphthol-amine complex [ 16,17]. Coelenteramide 2 and its analogues 2F1 and 2F2 in benzene containing n-butylamine showed the fluorescence emissions from the phenolate anions (II) as shown in Fig. 3. The phenolate anion of 2F2 had the blue-shifted maximum (442 nm) relative to those of 2F1 (467 nm) and 2 (472 nm). The blue-shifted fluorescence of the phenolate anion of 2F2 matches the blue-shifted bioluminescence of AQ-1F2, strongly supporting that the ionic structure of 2 during AQ bioluminescence is assigned as the phenolate anion (II) [9]. The reason of the fluorescence OH
y
x..J...
OvCH2C6H4
,.-N~yN®
L
ZL
HO" ~
I
0.20°.°
O.~CH2C6H4OH
O.~CH2C6H4OH y
x..J.. - v
.~N~......NH
L
1I
Y
x. J.. BuNH2'""HO" ~
~-N.-v.NH
S.
III
shift of II induced by fluorination is explained by the charge transfer (CT) character of the singlet excited state. As reported previously, the singlet excited coelenteramide 2 has a CT character, which is originated by the combination of an electron-accepting pyrazine ring with an electron-donating 5-(4-hydroxyphenyl) group [18]. The 5-(4-oxidophenyl) group of II works as a good electron-donating group, resulting in the CT character of the excited state.
5544
The fluoro group of the phenolate anion of 2F1, which is the meta position from the pyrazinyl group on the 5-(4-oxidophenyl) group, gave a negligible change of the CT character of the excited state. On the other hand, the phenolate anion of 2F2 has an additional fluoro group at the ortho position. This fluoro group may decrease the electron donating property of the 4oxidophenyl group in the singlet excited state, resulting in the blue-shifted CT fluorescence. In conclusion, the fluorescence behavior of the phenolate anions of 2, 2F1, and 2F2 matches the bioluminescent spectral characteristics of the wild type AQ, AQ-1F1, and AQIF2, supporting that the singlet excited state of the phenolate anion (II) of 2 in BFP is the light-emitter in aequorin bioluminescence [9]. An NMR study of fluorinated semi-synthetic AQs is now in progress. Acknowledgments: The author (TH) gratefully acknowledge a financial support for this research awarded by the Mitsubishi-Yuka foundation. References and Notes [1] a) Shimomura, O.; Johnson, F. H.; Saiga, Y. J. Cell Comp. Physiol. 1962, 59, 223 (1962); b) Johnson, F. H.; Shimomura, O. Methods Enzymol. 1978, 57, 71. [2] Review: Ohmiya, Y.; Hirano, T. Chem. & Biol. 1996, 3, 337. [3] Shimomura, O.; Johnson, F. H. Tetrahedron Lett. 1973, 2963. [4] Shimomura, O.; Johnson, F. H. Nature (London) 1975, 256, 236 (1975). [5] a) Shimomura, O.; Musicki, B.; Kishi, Y. Biochem. J. 1988, 251,405; b) Shimomura, O.; Musicki, B.; Kishi, Y. Biochem. J. 1989, 261,913; c) Shimomura, O.; Inouye, S.; Musicki, B.; Kishi, Y. Biochem. J. 1990, 270, 309; d) Shimomura, O.; Kishi, Y.; Inouye, S. Biochem. J. 1993, 296, 549. [6] a) Chen, F. -Q.; Gomi, Y.; Hirano, T.; Ohashi, M.; Ohmiya, Y.; Tsuji, F. I. J. Chem. Soc., Perkin Trans. 1 1992, 1607; b) Hirano, T.; Negishi, R.; Yamaguchi, M.; Chen, F. Q.; Ohmiya, Y.; Tsuji, F. I.; Ohashi, M. Tetrahedron 1997, 53. 12903. [7] 19F NMR studies of fluorinated proteins; a) visual pigment: Colmenares, L. U.; Liu, R. S. H. Tetrahedron 1996, 52, 109; b) flavoprotein: Murthy, Y. V. S. N.; Massey, V. J. BioL Chem. 1996, 271, 19915. [8] The pioneering NMR study of aequorin using 13C labeled coelenterazines: Musicki, B.; Kishi, Y.; Shimomura, O. J. Chem. Soc.. Chem. Commun. 1986, 1566. [9] Hirano, T.; Mizoguchi, I.; Yamaguchi, M.; Chen, F. -Q.; Ohashi, M.; Ohmiya, Y.; Tsuji, F. I. J. Chem. Soc., Chem. Commun. 1994, 165. [10] a) Nakamura, H.; Takeuchi, D.; Murai, A. Synlett. 1995 1227; b) Nakamura, H.; Wu, C.; Murai, A.; Inouye, S.; Shimomura, 0. Tetrahedron Lett. 1997, 38. 1227. [11] Jones, K.; Keenan, M.; Hibbert, F. Synlett. 1996, 509. [12] 1FI: yellow powder: mp 154-158 °C (dec.); IH NMR (270 MHz, CD3OD) 67.72 (IH, br s), 7.50-7.07 (9H, m), 7.00 (1H, t, J= 8 Hz), 6.69 (2H, AA'BB'), 4.40 (2H, s), 4.07 (2H, s); 19F NMR (470 MHz, CD3OD ) ~-168.0; UV-vis ~'max (CH3OH) 413 (e= 5,300), 347 (4,900), 271 (19,000) nm; ElMS nJz 441 (M+, 26), 439 (48), 279 (100); 1F2: yellow powder: mp 158-160 °C (dec.); IH NMR (270 MHz, CD3OD) ~7.65 (IH, br s), 7.44-7.15 (6H, m), 7.14 (2H, AA'BB'), 6.80 (IH, br td, J= 9, 2.0 Hz), 6.69 (2H, AA'BB'), 4.36 (2H, s), 4.07 (2H, s); 19F NMR (470 MHz, CD3OD) S-163.2, -141.1; UV-vis ~'max (CH3OH) 413 (e= 5,000), 346 (4,3007, 263 (17,000) nm; ElMS m/z 459 (M+, 35), 457 (78), 297 (100); 2Fl: colorless powder: mp 220-221 °C; IR (KBr) v 1672, 1495 cm- I ; 1H NMR (270 MHz, CD3OD) S 8.70 (1 H, s), 7.79 (IH, dd, J= 12.5, 2 Hz), 7.71 (br dd, J= 8.5, 2 Hz), 7.30-6.92 (8H, m), 6.76 (2H, AA'BB'), 4.10 (2H, s), 3J7 (2H, s); 19F NMR (470 MHz, CD3OD) ~ -138.33; EIMS m/z 429 (M +, 64), 295 (100); 2172:colorless fibers: mp 178-180 C; IR (KBr) v 1663, 1491 cm-l; 1H N'MR (270 MHz, CD3OD) t~ 8.67 (1H, s), 7.62 (IH, ddd, J= 9, 9, 2.3 Hz), 7.24-7.10 (5H, m), 7.00 (2H, m), 6.84 (IH, ddd, J= 8, 8, 2.0 Hz), 6.77 (2H, AA'BB'), 4.12 (2H, s), 3.58 (2H, s); 19F NMR (470 MHz, CD3OD) 6-141.97, -163.98; EIMS m/z 447 (M +, 43), 313 (100). [13] a) Inouye, S.; Noguchi, M.; Sasaki, Y.; Takagi, Y.; Miyata, T.; Imanaga, S.; Miyata, T.; Tsuji, F. I. Proc. Natl. Acad. Sci. USA 1985, 82, 3154; b) Tsuji, F. I.; Inouye, S.; Goto, T.; Sakaki, Y. Proc. Natl. Acad. Sci. USA 1986, 83, 8107. [14] Hirano, T.; Gomi, Y.; Takahashi, T.; Kitahara, K.; Chen, F. -Q.; Mizoguchi, I.; Kyushin, S.; Ohashi, M. Tetrahedron Left. 1992, 33, 5771, and references therein. [15] Ohashi, M. In Dynamic Aspects of Natural Products Chemisoy - Molecular Biological Approaches; Ogura, K., Sankawa, U. Eds.; Kodansha: Tokyo, 1997; p 269. [16] a) Tolbert, L. M.; Nesselroth, S. M. J. Phys. Chem. 1991, 95, 10331; b) Willis, K. J.; Szabo, A. G. J. Phys. Chem., 1991, 95, 1585. [17] Fluorescence spectra of coelenteramide analogues, whose hydroxy group was protected by methylation, were not affected by the addition of n-butylamine, indicating that the 5-(4-hydroxyphenyl) group of 2 is essential for generating the excited phenolate anion; Shibata, T.; Hirano, T. unpublished result. [18] Saito, R.; Hirano, T.; Niwa, H.; Ohashi, M. J. Chem. Soc., Perkin Trans. 2 1997, 171 I.
Tetrahedron Letters 39 (19981 5541-5544
Pergamon
Bioluminescent Properties of Fluorinated Semi-synthetic Aequorins Takashi Hirano,* Yoshihiro Ohmiya,t Shojiro Maki, Haruki Niwa, and Mamoru Ohashitt Department of Applied Physics and Chemisto', The University of Electro-Comnumications, Chofu, Tokyo 182, Japan. ?Department of Biochemisto,, FaculG of Education, Shizuoka Universi~', Shizuoka 422. Japan. ~('~Department of Materials Science, Kanagawa UniversiO,, Hiratsuka, Kanagawa 259-12. Japan.
Received 5 March 1998; accepted 25 May 1998
Abstract Bioluminescent properties of semi-synthetic aequorins containing coelenterazine analogues possessing fluoro group(s) on the 6-(4-hydroxyphenyl) group match the fluorescent behavior of the phenolate anions of the corresponding fluorinated coelenteramide analogues, indicating that the phenolate anion of coelenteramide is the light-emitter in aequorin bioluminescence. © 1998 ElsevierScience Ltd. All rightsreserved. Kevwords: Bioluminescence,Chemiluminescence,Fluorescence
The photoprotein aequorin (AQ) isolated from the jellyfish Aequorea victoria consists of apoaequorin (apoAQ, apoprotein), coelenterazine 1, and 0 2 [1,2]. AQ shows bioluminescence by binding calcium ions, yielding a blue fluorescent protein (BFP), CO 2, and light. BFP made up of coelenteramide 2 bound to apoAQ is the light emitter [3]. AQ is obtained by incubating 1 with recombinant apoAQ under air (Scheme 1) [4]. Similarly, semisynthetic AQs are obtained by using chemically modified coelenterazine analogues [5]. The methodology using semi-synthetic AQs is useful for investigating the bioluminescent properties of AQ [5,6]. Coelenterazine 1 has three appendages on the 3,7-dihydroimidazo[1,2-a]pyrazin-3-one ring. Because the 4-hydroxyphenyl group at C6 directly conjugates to the imidazopyrazinone ring, the modification of the 6-(4-hydroxyphenyl) group changes the electronic property of 1. Of particular interest would be introduction of fluorine atoms aequorin
(AQ)
Ca2+ ,
apoaequorin (apoAQ) + coelenteramide
t
I 02 + coetenterazine1 0,~--~
y
2
+ 002 + hv Scheme 1
OH
O.~~OH
/N../,N
y 1FI (X= F, Y= H) 1F2 (X= Y= F)
/N_y.NH
HO" ~
* e-mail: [email protected]
0040-4039/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved. PII: S0040-4039(98)01115-0
~..~
2FI (X= F, Y= H) 2F2 (X= Y= F)
5542
characterized by the largest electronegativity (4.0) and the small covalent radius (0.64 A). In addition, the introduced fluorine atom is a useful probe for NMR study [7]. Therefore, we designed coelenterazine analogues 1F1 and 1F2 possessing fluoro group(s) on the 6-(4hydroxyphenyl) group for investigating electronic substituent effect on the bioluminescent properties of AQ and for 19F-NMR study [8]. This paper reports the syntheses and the bioand chemiluminescent properties of 1F1 and 1F2, and the fluorescent properties of the corresponding coelenteramide analogues 2F1 and 2F2. The result of this study shows an evidence to elucidate the ionic structure of the light-emitter during AQ bioluminescence [9]. Y X. ~
~/ N..~ L NH2
B(OH)2 +
BnO- ~ 3F1 (X= F, Y= H) 3F2 (X= Y= F) TBDMSO~CH3
Br
N 4 iii, iv
CH2Ph
Y
/N<.~NH2
x ,c-Lc.2p, y
BnO" '¢" 5F1 (X= F, Y= H; y= 92%) 5F2 (X= Y= F; y= 93%)
/N~-y.NH2
HO- " ~
6F~ (X= F, Y= H; y= 80*) 6F2 (X=Y= F; y= 83%)
," T B D M S O CH2COCH(OC2H5)2 6F1 or 6F2 7 (y= 21%)
1F1 (y= 57%) 1F2 (y= 53%)
Scheme 2 R e a g e n t s a n d conditions: i. [Pd(PPh3)4], dioxane / Na2CO3aq: ii. H 2, Pd/C: iii. NBS, CCI 4, benzoylperoxide: iv, (1) Mg, THF, (2) CsHIoNCOCH(OEt) 2, (3) FI30+; v, HCl-dioxane," 110 °C.
Analogues 1F1 and 1F2 were prepared according to a modification of the reported procedure as shown in Scheme 2 [10-12]. A simple method for preparing keto acetal 7 by two steps from t-butyldimethylsilylated p-cresol was developed [6b]. Coelenteramide analogues 2F1 and 2F2 were prepared by the acylation of 6F1 and 6F2, respectively. Semi-synthetic AQs containing 1FI and 1F2 (AQ-1F1 and AQ-1F2) and wild type AQ were prepared by incubating 1F1, 1F2, and 1 (2.1 x 10 -3 M in MeOH, 20 ktl) with recombinant apoAQ [13] (10 I.tg) in Tris-HC1 buffer (0.03 M, pH 7.6, 1.0 ml) containing dithiothreitol (0.002 M) and EDTA (0.01 M). After incubation for 20 h at 0 °C, the bioluminescence activity of the semi-synthetic AQs was assayed by mixing the incubation mixture (10 ktl) with a 0.4 ml of Tris-HCl buffer (pH 7.6) containing CaC12 (0.03 M). The amounts of total light emitted by bioluminescence of AQ-1F1 and AQ-1F2 were 67% and 1%, respectively, relative to that of wild type AQ (100%). This result indicates that 3-fluoro group of the 6-(4-hydroxyphenyl) group does not prevent the regeneration of the semisynthetic AQ, and that 1F1 is useful for NMR study. ...........i ..................a ..............;............................................... The bioluminescence emission spectra of AQ! i + 1F1, AQ-1F2, and wild type AQ are shown in Fig. 1. The spectrum (~'max 460 n m ) o f AQ-1F1 coincides with that of wild type AQ, while AQ-1F2 ....... +.~:/+.............,~.........+..'t+~...........~................+................ showed a blue-sifted emission maximum at 440 nm. ++ ',+ +~+_ + To evaluate these spectral characteristics, the E +.Ji + \ '-~-..++..... chemiluminescent properties of 1F1 and 1F2, and j++. :~ + + + + . + . + . + +++, . + .+..~'.+~.,,+++.+;, .... ; the fluorescent properties of the corresponding i I I 350 400 450 500 550 600 650 coeleneteramide analogues 2F1 and 2F2 w e r e wavelength/ nm investigated. Chemiluminescence reactions of 1F1 Fig. 1 Bioluminescence spectra of wild type AQ (a), AQ-1F1 (b), and AQ-1F2 (e). and 1F2 in DMSO under air showed the similar ......
"/+ ...........................
+,+'u :, ....................
+~...........
+
5543
I .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
a .......i
/' b ~".,
I
:~ .........
+......
//c
~1
/~'
~,.~ i',,
i ...................... i .............
L, ~', '-.
/1'
350
C
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
:
",,
"~',..
400
450 500 550 600 650 wavelength / nm Fig. 2 Chemiluminescence spectra of ! (a), IF! (b), and 1172 (c) (4.8 x 10-5 M) in DMSO under air.
350
400
450 500 550 wavelength / nm
600
650
Fig. 3 Fluorescence spectra of 2 (a), 2FI (b), and 2F2 (c) (1.1 x 10-6 M) in benzene containing n-butylamine (0.01 M). The asterisk (*) shows a fluorescence peak of the neutral form of 2.
emission maxima at 470 and 465 nm, respectively, to that (470 nm) of wild type 1 (Fig. 2). The relative chemiluminescence efficiency of 1F1 and 1F2 to that of 1 are 1.6 and 1.8, respectively. The light emitters of these chemiluminescence reactions have been assigned as the singlet excited states of the amide anions (I) of 2, 2F1, and 2F2 [14]. Little change of the chemiluminescence maxima induced by fluorination differs from the large shift of bioluminescence maxima, suggesting that the excited amide anion (I) is not the light-emitter in AQ bioluminescence. The phenolate anion (II) has also been proposed as the light-emitting structure of 2 in BFP during AQ bioluminescence [9]. We found the condition for observing a fluorescence of the phenolate anion in this work, although the phenolate anion of 2 produced in DMSO was non-fluorescent [15]. Excitation of the hydrogen-bonded complex (III) of 2 with n-butylamine in a non-polar solvent, such as benzene and CHC13, gave the singlet excited state of II in a similar manner of the naphthol-amine complex [ 16,17]. Coelenteramide 2 and its analogues 2F1 and 2F2 in benzene containing n-butylamine showed the fluorescence emissions from the phenolate anions (II) as shown in Fig. 3. The phenolate anion of 2F2 had the blue-shifted maximum (442 nm) relative to those of 2F1 (467 nm) and 2 (472 nm). The blue-shifted fluorescence of the phenolate anion of 2F2 matches the blue-shifted bioluminescence of AQ-1F2, strongly supporting that the ionic structure of 2 during AQ bioluminescence is assigned as the phenolate anion (II) [9]. The reason of the fluorescence OH
y
x..J...
OvCH2C6H4
,.-N~yN®
L
ZL
HO" ~
I
0.20°.°
O.~CH2C6H4OH
O.~CH2C6H4OH y
x..J.. - v
.~N~......NH
L
1I
Y
x. J.. BuNH2'""HO" ~
~-N.-v.NH
S.
III
shift of II induced by fluorination is explained by the charge transfer (CT) character of the singlet excited state. As reported previously, the singlet excited coelenteramide 2 has a CT character, which is originated by the combination of an electron-accepting pyrazine ring with an electron-donating 5-(4-hydroxyphenyl) group [18]. The 5-(4-oxidophenyl) group of II works as a good electron-donating group, resulting in the CT character of the excited state.
5544
The fluoro group of the phenolate anion of 2F1, which is the meta position from the pyrazinyl group on the 5-(4-oxidophenyl) group, gave a negligible change of the CT character of the excited state. On the other hand, the phenolate anion of 2F2 has an additional fluoro group at the ortho position. This fluoro group may decrease the electron donating property of the 4oxidophenyl group in the singlet excited state, resulting in the blue-shifted CT fluorescence. In conclusion, the fluorescence behavior of the phenolate anions of 2, 2F1, and 2F2 matches the bioluminescent spectral characteristics of the wild type AQ, AQ-1F1, and AQIF2, supporting that the singlet excited state of the phenolate anion (II) of 2 in BFP is the light-emitter in aequorin bioluminescence [9]. An NMR study of fluorinated semi-synthetic AQs is now in progress. Acknowledgments: The author (TH) gratefully acknowledge a financial support for this research awarded by the Mitsubishi-Yuka foundation. References and Notes [1] a) Shimomura, O.; Johnson, F. H.; Saiga, Y. J. Cell Comp. Physiol. 1962, 59, 223 (1962); b) Johnson, F. H.; Shimomura, O. Methods Enzymol. 1978, 57, 71. [2] Review: Ohmiya, Y.; Hirano, T. Chem. & Biol. 1996, 3, 337. [3] Shimomura, O.; Johnson, F. H. Tetrahedron Lett. 1973, 2963. [4] Shimomura, O.; Johnson, F. H. Nature (London) 1975, 256, 236 (1975). [5] a) Shimomura, O.; Musicki, B.; Kishi, Y. Biochem. J. 1988, 251,405; b) Shimomura, O.; Musicki, B.; Kishi, Y. Biochem. J. 1989, 261,913; c) Shimomura, O.; Inouye, S.; Musicki, B.; Kishi, Y. Biochem. J. 1990, 270, 309; d) Shimomura, O.; Kishi, Y.; Inouye, S. Biochem. J. 1993, 296, 549. [6] a) Chen, F. -Q.; Gomi, Y.; Hirano, T.; Ohashi, M.; Ohmiya, Y.; Tsuji, F. I. J. Chem. Soc., Perkin Trans. 1 1992, 1607; b) Hirano, T.; Negishi, R.; Yamaguchi, M.; Chen, F. Q.; Ohmiya, Y.; Tsuji, F. I.; Ohashi, M. Tetrahedron 1997, 53. 12903. [7] 19F NMR studies of fluorinated proteins; a) visual pigment: Colmenares, L. U.; Liu, R. S. H. Tetrahedron 1996, 52, 109; b) flavoprotein: Murthy, Y. V. S. N.; Massey, V. J. BioL Chem. 1996, 271, 19915. [8] The pioneering NMR study of aequorin using 13C labeled coelenterazines: Musicki, B.; Kishi, Y.; Shimomura, O. J. Chem. Soc.. Chem. Commun. 1986, 1566. [9] Hirano, T.; Mizoguchi, I.; Yamaguchi, M.; Chen, F. -Q.; Ohashi, M.; Ohmiya, Y.; Tsuji, F. I. J. Chem. Soc., Chem. Commun. 1994, 165. [10] a) Nakamura, H.; Takeuchi, D.; Murai, A. Synlett. 1995 1227; b) Nakamura, H.; Wu, C.; Murai, A.; Inouye, S.; Shimomura, 0. Tetrahedron Lett. 1997, 38. 1227. [11] Jones, K.; Keenan, M.; Hibbert, F. Synlett. 1996, 509. [12] 1FI: yellow powder: mp 154-158 °C (dec.); IH NMR (270 MHz, CD3OD) 67.72 (IH, br s), 7.50-7.07 (9H, m), 7.00 (1H, t, J= 8 Hz), 6.69 (2H, AA'BB'), 4.40 (2H, s), 4.07 (2H, s); 19F NMR (470 MHz, CD3OD ) ~-168.0; UV-vis ~'max (CH3OH) 413 (e= 5,300), 347 (4,900), 271 (19,000) nm; ElMS nJz 441 (M+, 26), 439 (48), 279 (100); 1F2: yellow powder: mp 158-160 °C (dec.); IH NMR (270 MHz, CD3OD) ~7.65 (IH, br s), 7.44-7.15 (6H, m), 7.14 (2H, AA'BB'), 6.80 (IH, br td, J= 9, 2.0 Hz), 6.69 (2H, AA'BB'), 4.36 (2H, s), 4.07 (2H, s); 19F NMR (470 MHz, CD3OD) S-163.2, -141.1; UV-vis ~'max (CH3OH) 413 (e= 5,000), 346 (4,3007, 263 (17,000) nm; ElMS m/z 459 (M+, 35), 457 (78), 297 (100); 2Fl: colorless powder: mp 220-221 °C; IR (KBr) v 1672, 1495 cm- I ; 1H NMR (270 MHz, CD3OD) S 8.70 (1 H, s), 7.79 (IH, dd, J= 12.5, 2 Hz), 7.71 (br dd, J= 8.5, 2 Hz), 7.30-6.92 (8H, m), 6.76 (2H, AA'BB'), 4.10 (2H, s), 3J7 (2H, s); 19F NMR (470 MHz, CD3OD) ~ -138.33; EIMS m/z 429 (M +, 64), 295 (100); 2172:colorless fibers: mp 178-180 C; IR (KBr) v 1663, 1491 cm-l; 1H N'MR (270 MHz, CD3OD) t~ 8.67 (1H, s), 7.62 (IH, ddd, J= 9, 9, 2.3 Hz), 7.24-7.10 (5H, m), 7.00 (2H, m), 6.84 (IH, ddd, J= 8, 8, 2.0 Hz), 6.77 (2H, AA'BB'), 4.12 (2H, s), 3.58 (2H, s); 19F NMR (470 MHz, CD3OD) 6-141.97, -163.98; EIMS m/z 447 (M +, 43), 313 (100). [13] a) Inouye, S.; Noguchi, M.; Sasaki, Y.; Takagi, Y.; Miyata, T.; Imanaga, S.; Miyata, T.; Tsuji, F. I. Proc. Natl. Acad. Sci. USA 1985, 82, 3154; b) Tsuji, F. I.; Inouye, S.; Goto, T.; Sakaki, Y. Proc. Natl. Acad. Sci. USA 1986, 83, 8107. [14] Hirano, T.; Gomi, Y.; Takahashi, T.; Kitahara, K.; Chen, F. -Q.; Mizoguchi, I.; Kyushin, S.; Ohashi, M. Tetrahedron Left. 1992, 33, 5771, and references therein. [15] Ohashi, M. In Dynamic Aspects of Natural Products Chemisoy - Molecular Biological Approaches; Ogura, K., Sankawa, U. Eds.; Kodansha: Tokyo, 1997; p 269. [16] a) Tolbert, L. M.; Nesselroth, S. M. J. Phys. Chem. 1991, 95, 10331; b) Willis, K. J.; Szabo, A. G. J. Phys. Chem., 1991, 95, 1585. [17] Fluorescence spectra of coelenteramide analogues, whose hydroxy group was protected by methylation, were not affected by the addition of n-butylamine, indicating that the 5-(4-hydroxyphenyl) group of 2 is essential for generating the excited phenolate anion; Shibata, T.; Hirano, T. unpublished result. [18] Saito, R.; Hirano, T.; Niwa, H.; Ohashi, M. J. Chem. Soc., Perkin Trans. 2 1997, 171 I.