- Email: [email protected]
Synthesis of Coelenterazine
308
NOTES & TIPS
cantly altered after 30 min but was significantly decreased after 60 and 120 min when compared to the 5min value. The intensity of the signal generated from the bands developed with SuperSignal ULTRA was not significantly altered throughout the 120-min protocol. Similar results were obtained for each substrate with the 10- and 5-mg bands. Summary
FIG. 2. (Top) Representative Western blot for b-actin developed 5 min after exposure to SuperSignal and SuperSignal ULTRA. Membranes were incubated with 1:100,000 dilutions of primary and secondary antibody for 30 min each. (Bottom) Comparison of signal intensity generated by SuperSignal and SuperSignal ULTRA horseradish peroxidase substrates for b-actin band in 20 mg of total rat liver protein over a 2-h period following development with the HRP substrate. * indicates significant difference (P õ 0.05) from 5-min value. † indicates significant difference (P õ 0.05) from same time point in the other group.
tions of primary and secondary antibody and exposed to SuperSignal ULTRA and SuperSignal is shown in Fig. 2 (top). Each amount of protein loaded exhibited a significantly greater intensity when developed with SuperSignal ULTRA than with SuperSignal. Five minutes after development with the HRP substrates, the densitometric values for the bands on the SuperSignal side of the membranes (N Å 7) averaged 119 { 60, 40 { 19, and 4 { 2 densitometric units while the bands on the SuperSignal ULTRA side averaged 1176 { 183, 603 { 99, and 86 { 19 units for the 20-, 10-, and 5-mg bands, respectively. The relationship between signal intensity and time after development for the 20-mg band developed with SuperSignal and SuperSignal ULTRA substrates is shown in Fig. 2 (bottom). The signal intensity generated with SuperSignal was approximately 10% of the signal generated by the corresponding bands incubated with SuperSignal ULTRA for the initial 30 min following development, and the absolute signal intensity of the two groups was statistically different throughout the 2-h protocol. Analysis of the signal intensity with time indicated that the absolute level of signal from the bands incubated with SuperSignal was not signifi-
The present data demonstrate that with equivalent amounts of protein, the chemiluminescent signal generated with SuperSignal ULTRA is greater than that generated with SuperSignal HRP substrate. In turn, the signal intensity generated by SuperSignal is greater than that by ECL. An important observation is that the intense amount of light development with SuperSignal ULTRA, and even SuperSignal, necessitates optimization of assay conditions to eliminate undesirable cross-reactivity and/or overexposure of film. In conclusion, relative to ECL, the use of more potent HRP substrates should allow the use of more dilute antibody solutions and could permit the detection of extremely low levels of protein in blotting protocols. REFERENCES 1. Kricka, L. J. (1991) Clin. Chem. 37, 1472–1481. 2. Whitehead, T. P., Kricka, L. J., Carter, T. J. N., and Thorpe, G. H. G. (1979) Clin. Chem. 25, 1531–1546.
Synthesis of Coelenterazine Guadalupe Gonzalez-Trueba,* Cristina Paradisi,*,1 and Mario Zoratti† *Dipartimento di Chimica Organica, Centro Meccanismi Reazioni Organiche del CNR, Via Marzolo 1, 35131 Padova, Italy; and †Dipartimento Scienze Biomediche, Centro Biomembrane del CNR, Via Trieste 75, 35131 Padova, Italy Received June 3, 1996
Coelenterazine, 2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)-8-benzyl-3,7-dihydroimidazo [1,2-a]pyrazin-3one, is the prosthetic group of aequorin, a coelenterate protein. The aequorin–coelenterazine complex emits light upon binding Ca2/ and has long been used as a Ca2/ indicator in biological systems (1). The method has taken added significance with the development of 1
To whom correspondence should be addressed. Fax: /39 49 8275239. ANALYTICAL BIOCHEMISTRY
240, 308–310 (1996) ARTICLE NO. 0365
0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
08-22-96 16:10:05
abnt
AP: Anal Bio
309
NOTES & TIPS
SCHEME 1
SCHEME 2
SCHEME 3
AID
AB $NAT
/
6m1d$$$nat
08-22-96 16:10:05
abnt
AP: Anal Bio
310
NOTES & TIPS
recombinant aequorins targeted to various subcellular compartments (2–6). Coelenterazine must in all cases be added to the system for the measurements to be possible. While coelenterazine is commercially available, its steep price induced us to attempt to synthesize it following published protocols (Scheme 1) (7–9). We encountered difficulties which were circumvented by introducing some modifications in the procedures, which we report here in the hope of facilitating the task of others wishing to do the same. The major problem we met concerned the synthesis of 1, one of the starting materials of Scheme 1, by the procedure of Freifelder and Hasbrouck (10) (Scheme 2). In our hands, several attempts to use this procedure, which was referred to in the already mentioned reports on the synthesis of coelenterazine (7, 9), yielded a gummy untreatable material. A successful alternative route to 1 is summarized in Scheme 3. The procedure used, which reproducibly gave pure 1 in good yields, is as follows. A 90% aqueous solution of glycinonitrile (32 mmol), freshly prepared from the commercially available hydrogen sulfate (11), was added dropwise to a stirred solution of acetophenone (26 mmol in 25 ml of toluene) in the presence of p-toluensulfonic acid (0.26 mmol) at 1007C. The mixture was kept at reflux for 6 h while the water was removed with a Dean-Stark apparatus. Dried powdered NaOH (26.3 mmol) and benzyl chloride (26 mmol) were then added at room temperature and the mixture was kept under stirring overnight. After extraction with water the organic layer was treated with 2 eq of aqueous HCl and the product (white solid) isolated from the aqueous layer by solvent removal under reduced pressure. The free amine [1H NMR (CDCl3 , TMS) d (ppm): 2.10 (broad s, NH2), 3.04 (d, J Å 5.0 Hz, CH2), 3.96 (t, J Å 5.0 Hz, CH), 7.26–7.37 (m, 5H)] was obtained as an oil by the method of Cook et al. (11). We found it convenient to use the free amine in step (a) of Scheme 1 with chloroform instead of pyridine as solvent (the yield of 2 was 68% after purification by flash chromatography, eluant CH2Cl2/AcOEt 9:1 v/v, then MeOH). Other suggestions concern the intermediates, both quite unstable, involved in steps (d) and (e). The acid chloride 3 is generally prepared in situ due to its high reactivity. We found that, once purified, it could be stored under inert atmosphere and refrigeration. It was prepared by stirring a mixture of 4-acetoxyphenylacetic acid (12) with SOCl2 (50% molar excess) and a drop of DMF (12) overnight at room temperature. The product was distilled under reduced pressure (it is essential that all unreacted SOCl2 be removed prior to heating the crude) to yield a white crystalline solid. 1H NMR (CDCl3 , TMS) d (ppm): 2.30 (s, CH3), 4.13 (s, CH2), 7.10 (dd, Jortho Å 6.5 Hz, Jmeta Å 2.1 Hz, 2H), 7.28 (dd, 2H). The bromide 4, prepared according to published procedures (13), must also be stored under inert atmosphere.
An alternative way to carry out step (d) involved conversion of 3 to 1-[4-(acetoxy)phenyl]-3-chloro-2-propanone by the addition of diazomethane to an ether solution of 3 [rather than the inverse addition to arrive at the diazoketone (8)] followed by halogen exchange with nBu4Br in 1-bromopropane (14). Finally, we found it convenient to purify coelenterazine by flash chromatography under nitrogen using CH2Cl2/MeOH 9:1 v/v as eluant. By improving the procedures as described, the synthesis of milligram quantities of coelenterazine could reproducibly be accomplished in a few weeks. REFERENCES 1. Blinks, J. R., Mattingly, P. H., Jewell, B. R., van Leeuwen, M., Harrer, G. C., and Allen, D. G. (1978) Methods Enzymol. 57, 292–328. 2. Knight, M. R., Campbell, A. K., Smith, S. M., and Trewavas, A. J. (1991) Nature 352, 524–526. 3. Brini, M., Marsault, R., Bastianutto, C., Alvarez, J., Pozzan, T., and Rizzuto, R. (1995) J. Biol. Chem. 270, 9896–9903. 4. Rizzuto, R., Simpson, A. W. M., Brini, M., and Pozzan, T. (1992) Nature 358, 325–327. 5. Montero, M., Brini, M., Marsault, R., Alvarez, J., Sitia, R., Pozzan, T., and Rizzuto, R. (1995) EMBO J. 14, 5467–5475. 6. Brini, M., Marsault, R., Bastianutto, C., Pozzan, T., and Rizzuto, R. (1994) Cell Calcium 16, 259–268. 7. Kishi, Y., Tanino, H., and Goto, T. (1972) Tetrahedron Lett. 27, 2747–2748. 8. Inoue, S., Sugiura, S., Kakoi, H., Hasizume, K., Goto, T., and Iio, H. (1975) Chem. Lett., 141–144. 9. Shimomura, O., Musicki, B., and Kishi, Y. (1989) Biochem. J. 261, 913–920. 10. Freifelder, M., and Hasbrouck, R. (1960) J. Am. Chem. Soc. 82, 696–698. 11. Cook, A. H., Heilbron, I., and Levy, A. L. (1948) J. Chem. Soc., 201–206. 12. Percec, V., and Tomazos, D. (1989) J. Polym. Sci. Part A: Polym. Chem. 27, 999–1015. 13. Alvi, K. A., Crews, P., and Longhhead, D. (1991) J. Nat. Prod. 54, 1509–1515. 14. Bidd, I., and Whiting, M. C. (1984) Tetrahedron Lett. 25, 5949– 5950.
A Cytotoxicity Assay with Bindschedler’s Green Leuco Base Shiro Yamashoji Kobe Gakuin Women’s Junior College, 27-1 Hayashiyamacho, Nagata-ku, Kobe, 653 Japan Received July 1, 1996
Bioassays using cell cultures are expected to be useful alternatives to animal testing, which is expensive ANALYTICAL BIOCHEMISTRY
240, 310–312 (1996) ARTICLE NO. 0366
0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
08-22-96 16:10:05
abnt
AP: Anal Bio
NOTES & TIPS
cantly altered after 30 min but was significantly decreased after 60 and 120 min when compared to the 5min value. The intensity of the signal generated from the bands developed with SuperSignal ULTRA was not significantly altered throughout the 120-min protocol. Similar results were obtained for each substrate with the 10- and 5-mg bands. Summary
FIG. 2. (Top) Representative Western blot for b-actin developed 5 min after exposure to SuperSignal and SuperSignal ULTRA. Membranes were incubated with 1:100,000 dilutions of primary and secondary antibody for 30 min each. (Bottom) Comparison of signal intensity generated by SuperSignal and SuperSignal ULTRA horseradish peroxidase substrates for b-actin band in 20 mg of total rat liver protein over a 2-h period following development with the HRP substrate. * indicates significant difference (P õ 0.05) from 5-min value. † indicates significant difference (P õ 0.05) from same time point in the other group.
tions of primary and secondary antibody and exposed to SuperSignal ULTRA and SuperSignal is shown in Fig. 2 (top). Each amount of protein loaded exhibited a significantly greater intensity when developed with SuperSignal ULTRA than with SuperSignal. Five minutes after development with the HRP substrates, the densitometric values for the bands on the SuperSignal side of the membranes (N Å 7) averaged 119 { 60, 40 { 19, and 4 { 2 densitometric units while the bands on the SuperSignal ULTRA side averaged 1176 { 183, 603 { 99, and 86 { 19 units for the 20-, 10-, and 5-mg bands, respectively. The relationship between signal intensity and time after development for the 20-mg band developed with SuperSignal and SuperSignal ULTRA substrates is shown in Fig. 2 (bottom). The signal intensity generated with SuperSignal was approximately 10% of the signal generated by the corresponding bands incubated with SuperSignal ULTRA for the initial 30 min following development, and the absolute signal intensity of the two groups was statistically different throughout the 2-h protocol. Analysis of the signal intensity with time indicated that the absolute level of signal from the bands incubated with SuperSignal was not signifi-
The present data demonstrate that with equivalent amounts of protein, the chemiluminescent signal generated with SuperSignal ULTRA is greater than that generated with SuperSignal HRP substrate. In turn, the signal intensity generated by SuperSignal is greater than that by ECL. An important observation is that the intense amount of light development with SuperSignal ULTRA, and even SuperSignal, necessitates optimization of assay conditions to eliminate undesirable cross-reactivity and/or overexposure of film. In conclusion, relative to ECL, the use of more potent HRP substrates should allow the use of more dilute antibody solutions and could permit the detection of extremely low levels of protein in blotting protocols. REFERENCES 1. Kricka, L. J. (1991) Clin. Chem. 37, 1472–1481. 2. Whitehead, T. P., Kricka, L. J., Carter, T. J. N., and Thorpe, G. H. G. (1979) Clin. Chem. 25, 1531–1546.
Synthesis of Coelenterazine Guadalupe Gonzalez-Trueba,* Cristina Paradisi,*,1 and Mario Zoratti† *Dipartimento di Chimica Organica, Centro Meccanismi Reazioni Organiche del CNR, Via Marzolo 1, 35131 Padova, Italy; and †Dipartimento Scienze Biomediche, Centro Biomembrane del CNR, Via Trieste 75, 35131 Padova, Italy Received June 3, 1996
Coelenterazine, 2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)-8-benzyl-3,7-dihydroimidazo [1,2-a]pyrazin-3one, is the prosthetic group of aequorin, a coelenterate protein. The aequorin–coelenterazine complex emits light upon binding Ca2/ and has long been used as a Ca2/ indicator in biological systems (1). The method has taken added significance with the development of 1
To whom correspondence should be addressed. Fax: /39 49 8275239. ANALYTICAL BIOCHEMISTRY
240, 308–310 (1996) ARTICLE NO. 0365
0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
08-22-96 16:10:05
abnt
AP: Anal Bio
309
NOTES & TIPS
SCHEME 1
SCHEME 2
SCHEME 3
AID
AB $NAT
/
6m1d$$$nat
08-22-96 16:10:05
abnt
AP: Anal Bio
310
NOTES & TIPS
recombinant aequorins targeted to various subcellular compartments (2–6). Coelenterazine must in all cases be added to the system for the measurements to be possible. While coelenterazine is commercially available, its steep price induced us to attempt to synthesize it following published protocols (Scheme 1) (7–9). We encountered difficulties which were circumvented by introducing some modifications in the procedures, which we report here in the hope of facilitating the task of others wishing to do the same. The major problem we met concerned the synthesis of 1, one of the starting materials of Scheme 1, by the procedure of Freifelder and Hasbrouck (10) (Scheme 2). In our hands, several attempts to use this procedure, which was referred to in the already mentioned reports on the synthesis of coelenterazine (7, 9), yielded a gummy untreatable material. A successful alternative route to 1 is summarized in Scheme 3. The procedure used, which reproducibly gave pure 1 in good yields, is as follows. A 90% aqueous solution of glycinonitrile (32 mmol), freshly prepared from the commercially available hydrogen sulfate (11), was added dropwise to a stirred solution of acetophenone (26 mmol in 25 ml of toluene) in the presence of p-toluensulfonic acid (0.26 mmol) at 1007C. The mixture was kept at reflux for 6 h while the water was removed with a Dean-Stark apparatus. Dried powdered NaOH (26.3 mmol) and benzyl chloride (26 mmol) were then added at room temperature and the mixture was kept under stirring overnight. After extraction with water the organic layer was treated with 2 eq of aqueous HCl and the product (white solid) isolated from the aqueous layer by solvent removal under reduced pressure. The free amine [1H NMR (CDCl3 , TMS) d (ppm): 2.10 (broad s, NH2), 3.04 (d, J Å 5.0 Hz, CH2), 3.96 (t, J Å 5.0 Hz, CH), 7.26–7.37 (m, 5H)] was obtained as an oil by the method of Cook et al. (11). We found it convenient to use the free amine in step (a) of Scheme 1 with chloroform instead of pyridine as solvent (the yield of 2 was 68% after purification by flash chromatography, eluant CH2Cl2/AcOEt 9:1 v/v, then MeOH). Other suggestions concern the intermediates, both quite unstable, involved in steps (d) and (e). The acid chloride 3 is generally prepared in situ due to its high reactivity. We found that, once purified, it could be stored under inert atmosphere and refrigeration. It was prepared by stirring a mixture of 4-acetoxyphenylacetic acid (12) with SOCl2 (50% molar excess) and a drop of DMF (12) overnight at room temperature. The product was distilled under reduced pressure (it is essential that all unreacted SOCl2 be removed prior to heating the crude) to yield a white crystalline solid. 1H NMR (CDCl3 , TMS) d (ppm): 2.30 (s, CH3), 4.13 (s, CH2), 7.10 (dd, Jortho Å 6.5 Hz, Jmeta Å 2.1 Hz, 2H), 7.28 (dd, 2H). The bromide 4, prepared according to published procedures (13), must also be stored under inert atmosphere.
An alternative way to carry out step (d) involved conversion of 3 to 1-[4-(acetoxy)phenyl]-3-chloro-2-propanone by the addition of diazomethane to an ether solution of 3 [rather than the inverse addition to arrive at the diazoketone (8)] followed by halogen exchange with nBu4Br in 1-bromopropane (14). Finally, we found it convenient to purify coelenterazine by flash chromatography under nitrogen using CH2Cl2/MeOH 9:1 v/v as eluant. By improving the procedures as described, the synthesis of milligram quantities of coelenterazine could reproducibly be accomplished in a few weeks. REFERENCES 1. Blinks, J. R., Mattingly, P. H., Jewell, B. R., van Leeuwen, M., Harrer, G. C., and Allen, D. G. (1978) Methods Enzymol. 57, 292–328. 2. Knight, M. R., Campbell, A. K., Smith, S. M., and Trewavas, A. J. (1991) Nature 352, 524–526. 3. Brini, M., Marsault, R., Bastianutto, C., Alvarez, J., Pozzan, T., and Rizzuto, R. (1995) J. Biol. Chem. 270, 9896–9903. 4. Rizzuto, R., Simpson, A. W. M., Brini, M., and Pozzan, T. (1992) Nature 358, 325–327. 5. Montero, M., Brini, M., Marsault, R., Alvarez, J., Sitia, R., Pozzan, T., and Rizzuto, R. (1995) EMBO J. 14, 5467–5475. 6. Brini, M., Marsault, R., Bastianutto, C., Pozzan, T., and Rizzuto, R. (1994) Cell Calcium 16, 259–268. 7. Kishi, Y., Tanino, H., and Goto, T. (1972) Tetrahedron Lett. 27, 2747–2748. 8. Inoue, S., Sugiura, S., Kakoi, H., Hasizume, K., Goto, T., and Iio, H. (1975) Chem. Lett., 141–144. 9. Shimomura, O., Musicki, B., and Kishi, Y. (1989) Biochem. J. 261, 913–920. 10. Freifelder, M., and Hasbrouck, R. (1960) J. Am. Chem. Soc. 82, 696–698. 11. Cook, A. H., Heilbron, I., and Levy, A. L. (1948) J. Chem. Soc., 201–206. 12. Percec, V., and Tomazos, D. (1989) J. Polym. Sci. Part A: Polym. Chem. 27, 999–1015. 13. Alvi, K. A., Crews, P., and Longhhead, D. (1991) J. Nat. Prod. 54, 1509–1515. 14. Bidd, I., and Whiting, M. C. (1984) Tetrahedron Lett. 25, 5949– 5950.
A Cytotoxicity Assay with Bindschedler’s Green Leuco Base Shiro Yamashoji Kobe Gakuin Women’s Junior College, 27-1 Hayashiyamacho, Nagata-ku, Kobe, 653 Japan Received July 1, 1996
Bioassays using cell cultures are expected to be useful alternatives to animal testing, which is expensive ANALYTICAL BIOCHEMISTRY
240, 310–312 (1996) ARTICLE NO. 0366
0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
08-22-96 16:10:05
abnt
AP: Anal Bio