FIuorescence of Substituted 7-H ydroxycoumarins William R. Sherman and Eli Robins Department of Psychiatry, Washington University School of Medicine, St. Louis, Mo. 63110
THE ABSORPTION SPECTROPHOTOMETRIC measurement of products of the reaction of enzymes with natural and synthetic substrates is well known. Utilization of such substrates has made possible the study of enzymes in amounts too small to be measured by older methods. An extension of this technique is the measurement of fluorescent products of enzyme action on a synthetic fluorogenic substrate. In analogy with absorptiometry, the fluorometric method depends on a decrease in the fluorescence intensity when the fluorescent molecule is joined to some other system, the enzyme determinant, Two examples of this technique are the measurement of @-glucosidase by the fluorescence of 4methyl-7-hydroxycoumarin released by the action of the enzyme on 7-(P-~-g~ucopyranosy~oxy)-4-methy~coumarin (1) and the measurement of arylsulfatase by its action on the sulfate ester of 4-methyl-7-hydroxycoumarin (2). This study was begun in an effort to increase the sensitivity of these methods by increasing the fluorescence intensity of the fluorescent moiety of the enzyme substrate. In a recent effort of a similar nature (3), fluorescein digalactoside was prepared and used to measure the activity of single enzyme molecules. The complicated kinetics to be expected from a single fluorescent molecule bearing two sites for enzyme action led us to investigate the effect of substituent variations on the fluorescence of 7-hydroxycoumarins. Previous studies on the fluorescence intensities of variously substituted 7-hydroxycoumarins can only be evaluated in a qualitative way in that they have either used sunlight to excite fluorescence and were visually evaluated (4) or they neglected to consider variations in excitation and fluorescence Xmax (5). The suggestion that carboxy-, acetyl-, cyano-, and phenylsubstituents in the 3-position of 7-hydroxycoumarins enhance fluorescence ( 4 ) relative to 4-H or 4-methyl-7-hydroxycoumarin formed the basis for our study. In the course of this work we have found what appears to be a correlation of fluorescence intensity and the Hammett substituent constant.
EXPERIMENTAL 7-Hydroxycoumarins. 3-Carboxamido-7-hydroxycoumarin was prepared by the following procedure. 7-Hydroxy coumarin-3-carbonylchloride was prepared by heating 10 grams (0.048 mole) of 3-carboxy-7-hydroxycoumarin (6) under reflux with a mixture of thionyl chloride (20 ml, 0.28 mole) and 1P-dioxane for 1 hr. The solution was cooled to room temperature and the acid chloride precipitated with (1) D. Robinson, Biochem J., 63, 39 (1956). (2) W. R. Sherman and E. F. Stanfield, ibid., 102, 905 (1967). (3) B. Rotman, J. A. Zderic, and M. Edelstein, Proc. Nut. Acad. Sci., 50, 1 (1963).
(4) V. Balaiah, T. R. Seshadri, and V. Venkateswarlu, Proc. Znd. Acud, Sci., 16A, 68 (1942). ( 5 ) R. H. Goodwin and F. Kavanagh, Arch. Biochem., 27, 152 ( 1950). (6) H. v.
Pechmann and E Graezer, Chem. Ber., 34, 378 (1901).
0
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light petroleum ether. If the system sepzrated into two liquid phases, a small amount of benzene was added to maintain phase homogeneity. In this way 9.6 grams (88%) of crude product was obtained. This may be crystallized from 1,4dioxane as follows; 0.5 gram is taken up in 4 ml of dioxane at room temperature (heating causes polymerization), treated with charcoal and precipitated as short yellow needles by the addition of light petroleum ether. The product does not melt to 300" C, and in acetonitrile solution displays a broad hydroxyl absorption at 3250 cm-1 and a strong, sharp carbonyl band at 1790 cm-l. Anal. Calcd for CloHsC104: C, 53.40; H, 2.24. Found C, 53.44; H, 3.48. 3-Carboxamido-7-hydroxycoumarin was prepared from the crude, unrecrystallized, acid chloride (2.8 grams, 0.124 mole) by solution in anhydrous 1,4-dioxane (100 ml) and treatment with anhydrous ammonia for 10 min. The precipitate which formed was collected and washed on the filter with 1 M acetic acid. The product was dissolved in 20 ml of N,N-dimethylformamide at room temperature and gradually diluted with an equal volume of water while bringing it to a boil. After cooling the yield of light yellow needles was 1.5 gram (59%) mp 303" C (dec.). Anal. Calcd for C10H704N: C, 58.54; H 3.44; N, 6.83. Found C , 58.54; H, 3.32; N, 6.87. VOL. 40, NO. 4, APRIL 1968
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Table I. Fluorescence of Substituted FHydroxycoumarins
Ho
7-Hydroxycoumarin 3-Benzoyl4PhenylUnsubstituted CMethyl3-Carboxy3-Carboxamido3-Phenyl3-Acetyl3-Carbethoxy3-Cyano-
Excitation Reference
, , , A,
(7) (8)
(6) (9) (4)
(6)
(4
mfi 415 365 376 367 396 398 420 419 398 408
axo
First order Raman scatter of excitation X A,,
mp
476 414 422 416 453 456 48 1 480 456 469
Unsubstituted 7-hydroxycoumarin was obtained from the Aldrich Chemical Co., Milwaukee, Wis., 4-methyl-7-hydroxycoumarin from the Eastman Organic Chemicals Co., Rochester, N. Y . All other 7-hydroxycoumarins were prepared by the literature procedures cited in Table I. Fluorescence Measurements. All fluorescence data were obtained on a n Aminco-Bowman fluorescence spectrometer (American Instrument Co., Silver Spring, Md.). The instrument was equipped with a n Osram XB165 xenon arc, a 1P28 photomultiplier tube and measurements were made using slit arrangement 4. All excitation and fluorescence maxima are corrected to mercury lines. The fluorescence intensity values in Table I are relative to 4-methyl-7-hydroxycoumarin on a n equimolar basis, under optimal conditions of pH and wavelength, but are not corrected in this Table for variations in source intensity, grating transmission, or photomultiplier sensitivity; the results in Figure 1 are corrected approximately in this regard using the data of White, Ho, and Weimer (10). Fluorescence was measured at 10-7M except for 3-benzoyl- and 4-phenyl- which were measured a t IO-jM. The absorption maxima and molar absorptivity were measured on a Zeiss PMQIl (Carl Zeiss Inc., N. Y . ) . All samples were examined at the pH of maximum fluorescence intensity: 8.5 for all except unsubstituted 7-hydroxycoumarin, 4-methyl- and 4-phenyl-7-hydroxycoumarin which were measured a t pH 10. DISCUSSION
Fluorescence of 7-Hydroxycoumarins. An examination of the order of increasing fluorescence intensity in Table I suggests that there might be a correlation between the Hammett u constants of substituents in the 3-position and fluorescence. When the fluorescence intensity, corrected by the data of White, Ho, and Weimer (IO),is plotted against Hammett u values for different types of substituent interactions, the data correlate best with the Hammett u values obtained for meta substituted benzoic acids (11) (Figure 1). It should be (7) R. J. Pandya and K. C. Pandya, Agra Uniu. J. Res., 4, 345 (1955). (8) H. v, Pechrnann, Ber., 16,2119 (1883). (9) W. Baker, J. Chem. SOC.,1927,2898. (10) C. E. White, M. Ho, and E. Q. Weimer, ANAL.CHEM., 32, 438 (1960). (11) From the compilation of H. H. Jaffe, Chem. Reus., 53, 191 (1953).
804
ANALYTICAL CHEMISTRY
b
Fluorescence max, mp 468 515 454 449 450 445 456 458 445 450
Fluorescence intensity relative to Crnethyl7- hydroxycoumarin 6 . 4 x 10-4 1 . 2 x 10-2 0.96 1.0 1.9 2.5 2.7 3.1 3.6 3.6
Absorption Xmax
ml.c
412 372 365 359 385 400 412 413 402 407
€
43,000 17,700 18,500 17,000 36,700 39,300 37,700 43,500 38,700 41.600
pointed out that the original Hammett u meta value for phenyl which we use in Table I has been criticized (12) and that the currently used value of f0.06 is close to the Taft UI (13) given in Figure 1. The closeness of fit encourages us to use the original value. The basis for the apparent correlation of fluorescence intensity with the meta constant is not known to us. The structure of the 3-substituted coumarins suggests that they are simply vinylogs of a para-substituted phenol, however thepara substituent values are a poor predictor of fluorescence. It is understandable that the resonance-stabilizing quinonoid structures which may arise from the 7-phenolate group would increase the ultraviolet absorption coefficient of the 3-substituted coumarins. However, it is clear that the fluorescence intensity does not depend only on the development of such a conjugated system as all of the E values of the 3-substituted coumarins are similar. Further, the Taft u resonance values U~ and uR- (13) d o not correlate well with fluorescence. Similarly, the more purely inductive electron withdrawing measures of uI (14) d o not fit the fluorescence well. It thus seems that the fluorescence enhancement which we observe is not the result of simple resonance or inductive electron withdrawal from the 3-position of the 7-hydroxycoumarins. Zimmerman (15) has described the redistribution of the electron density of a substituted benzene from the ground state to the first excited state. In the case of a n electron withdrawing group, which in the ground state reduces electron density at the ortho and para positions, the excited state has electron density reduced at the ortho and meta positions. With such a marked reorganization of the electronic structure in the excited state, it is surprising to find any correlation with the Hammett substituent constants which are obtained a t ground state conditions. The fact that the correlation (if not simply fortuitous) is with the u meta values may indicate that the changes in ground state electron density at the meta position of benzoic acid which result from variation of sub(12) N. N. Lichtin and H. P. Leftin, J . Am. Chem. Soc., 74, 4207 (1952). (13) R. W. Taft, Jr., ibid., 79, 1045 (1957). (14) M. Charton, J . Org. Chem., 29, 1222(1964). (15) H. E. Zimmerman and V. R. Sandel, J. Am. Chem. SOC.,85, 915 (1963).
stituents are paralleled by the substituent effects on the meta position (in the case of the coumarins, the 6 and 8 positions) in the excited state. Why this might bring about fluorescence enhancement of the 7-hydroxycoumarins is not known. The low fluorescence intensity of 3-benzoyl-7-hydroxycoumarin has been observed previously (16). The u meta value for benzoyl has not been reported; however, u pura is +0.46-i.e. similar to acetyl and therefore not unusual in character. The other spectral properties of the 3-benzoylcompound are so similar to the remaining 3-substituted-7hydroxycoumarins that it seems this coumarin must undergo quenching. A comparison of the fluorescence intensities of the 4-substituted with the 3-substituted-7-hydroxycoumarins is of interest. The decrease in molar absorptivity resulting from moving the substituent to a position where no direct resonance interaction can occur is a possible explanation for the decreased fluorescence of 4-H- and 4-methyl-7-hydroxycoumarin when compared with the 3-substituted-7-hydroxycoumarins. The further decrease of fluorescence which occurs in the 4-phenyl case is also reported to occur with 4carboxy-7-hydroxycoumarin ( 4 ) and perhaps is the result of reduced electron density at the 4-position. (16) S. Rangaswami, T. Seshadri, and V. Venkateswarlu, Proc. Ind. Acad. Sci.. A13, 316 (1941).
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- -
Analytical Applications of 3 Substituted 7 Hydroxycoumarins. Raman and Rayleigh-Tyndall light scattering from the solvent in which the fluorescent molecule is dissolved are sensitivity-limiting factors and, depending on the nearness of the scatter maximum to the fluorescence maximum, the interference will be greater or lesser. All of the 3-substituted7-hydroxycoumarins have their fluorescence maxima between the first order Rayleigh-Tyndall scatter and the Raman scatter (see Table I). Of these compounds the 3-phenyl-, 3-acetyl-, and 3-cyano-derivatives have their fluorescence maxima near the scatter minimum which occurs in passing from the Rayleigh-Tyndall wavelength to the Raman wavelength. The amount of scattered light at this point is about four times the amount which occurs at the wavelength of the fluorescence maximum of 4-methyl-7-hydroxycoumarin. It is therefore unlikely that the increased fluorescence intensity of the 3-substituted-7-hydroxycoumarins,would result in any increase in the sensitivity of measurement of these compounds over that which can be obtained with 4-methyl-7-hydroxycoumarin. RECEIVED for review December 22, 1967. Accepted February 2, 1968. This work was supported by Public Health Service Research Career Program Award GM-21,863 to W.R.S. and by the following grants; NB-05159, MH04591 and a grant from the National Multiple Sclerosis Society.
A New Mixed Carrier for the Spectrographic Determination of Impurities in High Purity Uranium Oxides G . T. Day, P. A. Serin, and Klaas Heykoop' Analytical Laboratories, Eldorado Mining and Refining Ltd., Port Hope, Ontario, Canada
T o OVERCOME the very complex spectrum of uranium and its associated heavy background, Scribner and Mullin (I) used the method of fractional distillation of impurities by the addition of a spectrometric carrier. Since the introduction of this procedure, various carriers have been proposed as alternatives and additions to the 2% G a 2 0 3carrier originally used, many giving improvements for certain elements. The Ga2O3carrier has been replaced in several laboratories by AgCl which gives improved precision and sensitivity for many elements. The increased sensitivities are due to the formation of volatile chlorides by reaction of impurities with the thermally dissociated AgCI. Fluoride carriers have also been investigated. N a F was used by Belgisanin (2) to increase the detectability of boron. More recently, King and Neff (3) have used a combined AgCl1 Present address, Ontario Provincial Police, Peterborough Detachment.
(1) B. F. Scribner and H. R. Mullin, J. Res. Natl. Bur. Std., 37, 379, 1946.
( 2 ) N. Belgisanin, Rec. Trav. Inst. Recherches Structure Matiere (Belgrade),2, 27, 1953. (3) H. G. King and C . M. Neff, Appl. Spectry., 17,51,1963.
AgF carrier which showed significant increases in precision and sensitivity for most refractory elements over AgCl alone. Other mixed fluoride-chloride combinations have been used with success, notably AgCl-LiF (4) and AgC1-SrF2 (5). This type of carrier could not be used in our laboratory because silver is a specification element in our product. A carrier showing similar properties but not containing silver was therefore sought. EXPERIMENTAL
Prior to the investigation of a mixed carrier, all samples in this laboratory were analyzed by the 2% G a 2 0 3carrier method ( I ) . A fluoride carrier was sought mainly to increase the sensitivity of boron and improve the precision of the refractory elements. Strontium fluoride was chosen from the fluorides studied as a suitable compound and G a 2 0 3 was added to produce a smoother burn. Best results were obtained with a mixture of one part SrF2 and two parts of G a 2 0 3 added to U30s to give a 4 x mixture. It is interesting to note that the fluorine content in a 100-mg charge is 0.40 mg, which is almost (4) C. E. Pepper, A. J. Pardi, and M. G. Atwell, Ibid., p 114. (5) R. S. Vogel, U.S. At. Energy Comm. Rept. MCW 1471, Feb. 1962. VOL 40, NO. 4, APRIL 1968
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