Fluorescence-detected exciton-coupled circular dichroism: Scope and limitation in structural studies of organic molecules

Article abstract of DOI:10.1021/ja990936b

The potential of fluorescence-detected circular dichroism (FDCD) to extend the scope of the exciton chirality method is rooted in the increased sensitivity and selectivity of fluorescence as compared with absorbance measurements. In this paper, several pr

Full text of DOI:10.1021/ja990936b

VOLUME 121, NUMBER 38
SEPTEMBER 29, 1999
© Copyright 1999 by the
American Chemical Society
Fluorescence-Detected Exciton-Coupled Circular Dichroism: Scope
and Limitation in Structural Studies of Organic Molecules
Tatsuo Nehira, Craig A. Parish, Steffen Jockusch, Nicholas J. Turro, Koji Nakanishi,* and
Nina Berova*
Contribution from the Department of Chemistry, Columbia UniVersity, New York, New York 10027
ReceiVed March 22, 1999. ReVised Manuscript ReceiVed August 4, 1999
Abstract: The potential of fluorescence-detected circular dichroism (FDCD) to extend the scope of the exciton
chirality method is rooted in the increased sensitivity and selectivity of fluorescence as compared with absorbance
measurements. In this paper, several practical aspects of FDCD are addressed with model compounds possessing
a (1R,2R)-trans-1,2-cyclohexanediol skeleton and two chromophores attached to the diol through ester linkages.
We have probed the utility of fluorescent chromophores that have previously been shown to be excellent
chromophores for the conventional absorbance-based circular dichroism (CD) exciton chirality method. Certain
fluorophores, such as 2-naphthoate or 6-methoxy-2-naphthoate, provide FDCD spectra that are in good agreement
with the conventional CD. In other cases, a bischromophoric derivative results in an FDCD spectrum that
significantly deviates from the absorbance based data. Here it is shown that the extent of fluorescence
polarization, an estimate of sample anisotropy, is directly correlated with the ability to extract a meaningful
exciton-coupled FDCD spectrum. If fluorescence polarization is negligible, the solution is isotropic, and the
FDCD and conventional CD are in good agreement. Fluorescence lifetime measurements are used to address
the origin of solution anisotropy.
Introduction
The exciton chirality method1,2 requires no reference sample
since it nonempirically establishes absolute configurations or
conformations. This method is extremely versatile in that either
preexisting chromophores3,4 present in the molecule of interest
can be used or chromophores can be introduced by derivatization
of an appropriate functional group (e.g., alcohol or amine).5
Theoretical calculations of exciton-split CD spectra2 further
expand the scope of the exciton chirality method and allow for
rationalization of the origin of a bisignate curve. In addition, it
has been found that the exciton-split CD spectra of molecules
The exciton chirality method, based on exciton-coupled
circular dichroism,1,2 provides intense bisignate circular dichro-
ism (CD) spectra derived from the through-space coupling of
two or more chromophores in chiral substrates and is a valuable
tool for the determination of the absolute configuration or
conformation of a natural product in solution. The sign of the
split Cotton effect directly reflects the sense of chirality between
the electric transition moments of the interacting chromophores.
* To whom correspondence should be addressed: Koji Nakanishi,
Department of Chemistry, Columbia University, 3000 Broadway, New York,
NY 10027. E-mail: kn5@columbia.edu.
(1) Nakanishi, K.; Berova, N.; Woody, R. W. Circular Dichroism:
Principles and Applications; VCH: New York, 1994.
(2) Harada, N.; Nakanishi, K. Circular Dichroic Spectroscopy: Exciton
Coupling in Organic Stereochemistry; University Science Books: Mill
Valley, CA, 1983.
(3) Koreeda, M.; Weiss, G.; Nakanishi, K. J. Am. Chem. Soc. 1973, 95,
239-240.
(4) Parish, C. A.; Dong, J.-G.; Bornmann, W. G.; Chang, J.; Nakanishi,
K.; Berova, N. Tetrahedron 1998, 54, 15739-15758.
(5) Wiesler, W. T.; Berova, N.; Ojika, M.; Meyers, H. V.; Chang, M.;
Zhou, P.; Lo, L. C.; Niwa, M.; Takeda, R.; Nakanishi, K. HelV. Chim. Acta
1990, 73, 509-51.
10.1021/ja990936b CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/10/1999

8682 J. Am. Chem. Soc., Vol. 121, No. 38, 1999
Nehira et al.
containing several interacting chromophores, either identical6
or different,5,7 can be reproduced by summation of the pairwise
contributions of every possible bischromophoric interaction; this
pairwise additivity principle, which has been confirmed by
theoretical calculations,8 is critical for simplifying the interpreta-
tion of complex CD spectra.
of FDCD methods, including multidimensional FDCD18,19 and
lifetime-resolved FDCD,20-23 and several additional applica-
tions, such as FDCD-based detectors for capillary electrophore-
sis24 and HPLC,25 and the determination of enantiomeric
excess,26 have described the potential of this technique. In
FDCD, the excitation alternates between left- and right-circularly
polarized light, and the difference in the emission intensity is
measured. The resulting emission can be characterized in one
of two ways; either the emission is isotropic, or it is anisotropic,
and photoselection becomes relevant.27,28 In the case where
photoselection exists, the FDCD can be significantly distorted
so that the spectrum can no longer be directly related to
conventional CD. A number of modified instrumental setups29,30
have been used to attempt to eliminate the signal artifacts that
appear in FDCD spectra due to photoselection. However, the
development of FDCD as a general technique has been slowed
considerably since these spectropolarimeters were substantially
customized and were not commercially available. It is a
significant advantage that the FDCD setup used in all of the
studies described here and in our previous report11 relies on a
standard commercially available fluorescence attachment.
Fluorescence-detected circular dichroism (FDCD) spectra can
be obtained with enhanced sensitivity and greater selectivity
than that observed for conventional (absorbance) CD.9,10 While
CD measures the difference in absorption for left- and right-
circularly polarized light, FDCD detects the difference in
fluorescence intensity for left- and right-circularly polarized
excitations. Under standard conditions, when the emission of a
sample is directly proportional to absorbance, the same dichroic
information can be obtained from both processes. Since
fluorescence directly measures the amount of light emitted
against zero background while absorbance is determined from
an intensity difference of transmitted light, the fluorescence
signal typically can be observed at much lower concentrations
than absorbance. This enhanced sensitivity should allow for the
measurement of fluorescence-detected exciton split CD spectra
at sub-micromolar concentrations. Recently, we described FDCD
measurements that can be applied successfully to compounds
where the CD arises from exciton coupling of two or more
identical fluorophores. That study showed that excellent agree-
ment of FDCD with conventional CD and a remarkable 50-
100 fold enhancement in sensitivity could be attained under
favorable conditions, for example, when two strongly absorbing
and fluorescent chromophores couple through space.11 While
increased sensitivity is a significant asset of FDCD, it is the
selectivity of fluorescence detection that holds greater possibili-
ties in terms of practical applications of FDCD. The ability to
selectively analyze the chiral environment of fluorophores in
systems that also contain nonfluorescent chromophores would
clearly extend the overall scope of CD methods. This facet of
FDCD has been explored in only a few cases, such as proteins
that contain a single fluorescent tryptophan residue.12 While our
previous results confirm the CD/FDCD correlation for exciton
chirality, the role of selectivity in exciton-coupled FDCD is an
intriguing question; this can be examined directly by preparing
an exciton-coupled system containing one fluorophore and one
nonfluorescent chromophore. A more thorough analysis of
exciton-coupled FDCD was undertaken to clarify the practical
aspects and subtleties of this phenomenon.
In this paper, the main impetus of the FDCD analysis is the
identification of optimal fluorophores for fluorescence-detected
exciton chirality studies. The criterion for what constitutes a
reasonable fluorophore is the extent of agreement between the
spectra obtained from FDCD and conventional CD. The
fluorophores selected in this survey should have certain at-
tributes, many of which are analogous to those required in
exciton-coupled CD analysis. Namely, the fluorophore should
have (1) a large extinction coefficient, ꢀ, and a known direction
of the electric transition moment, µ; (2) high fluorescence
quantum yield; (3) chemical stability; (4) an appropriate
substituent, e.g., carboxyl, for derivatizing common functional
groups such as hydroxyls or amines; and (5) no or very limited
effects due to photoselection. With these qualities in mind, four
fluorophoric groups were selected for further analysis: 2-naph-
thoate 1,31,32 6-methoxy-2-naphthoate 2,11 2-anthroate 3,33 and
p-phenylbenzoate 4.34 In addition, the effect on FDCD of four
weakly fluorescent or nonfluorescent chromophores was ex-
amined: p-dimethylaminobenzoate 5,2 p-methoxycinnamate 6,35
(18) Thomas, M.; Patonay, G.; Warner, I. ReV. Sci. Instrum. 1986, 57,
1308-1313.
(19) Thomas, M. P.; Patonay, G.; Warner, I. M. Anal. Biochem. 1987,
164, 466-473.
(20) Geng, L.; McGown, L. B. Anal. Chem. 1992, 64, 68-74.
(21) Wu, K.; McGown, L. B. Appl. Spectrosc. 1991, 45, 1-3.
(22) Wu, K.; Geng, L.; Joseph, M. J.; McGown, L. B. Anal. Chem. 1993,
65, 2339-2345.
(23) Geng, L.; McGown, L. B. Appl. Spectrosc. 1994, 48, 167-175.
(24) Christensen, P. L.; Yeung, E. S. Anal. Chem. 1989, 61, 1344-1347.
(25) Synovec, R. E.; Yeung, E. S. J. Chromatogr. 1986, 368, 85-93.
(26) Geng, L.; McGown, L. B. Anal. Chem. 1994, 66, 3243-3246.
(27) Ehrenberg, B.; Steinberg, I. Z. J. Am. Chem. Soc. 1976, 98, 1293-
1295.
(28) Turner, D. H. Methods Enzymol. 1978, 49, 199-214.
(29) Lobenstine, E. W.; Turner, D. H. J. Am. Chem. Soc. 1980, 102,
7786-7787.
(30) Bicknese, S. F.; Maestre, M. F. ReV. Sci. Instrum. 1987, 58, 2060-
2063.
Prior to our contribution on fluorescence-detected exciton-
coupled systems,11 several studies on FDCD,12-17 extensions
(6) Liu, H. W.; Nakanishi, K. J. Am. Chem. Soc. 1982, 104, 1178-85.
(7) Wiesler, W. T.; Vazquez, J. T.; Nakanishi, K. J. Am. Chem. Soc.
1987, 109, 5586-92.
(8) Dong, J.-G.; Akritopoulou-Zanze, I.; Guo, J.; Berova, N.; Nakanishi,
K.; Harada, N. Enantiomer 1997, 2, 397-409.
(9) Turner, D. H.; Tinoco, I., Jr.; Maestre, M. J. Am. Chem. Soc. 1974,
96, 4340-4342.
(10) Tinoco, I., Jr.; Turner, D. H. J. Am. Chem. Soc. 1976, 98, 6453-
6456.
(11) Dong, J.-G.; Wada, A.; Takakuwa, T.; Nakanishi, K.; Berova, N.
J. Am. Chem. Soc. 1997, 119, 12024-12025.
(12) Lobenstine, E. W.; Schaefer, W. C.; Turner, D. H. J. Am. Chem.
Soc. 1981, 103, 4936-4940.
(13) Egusa, S.; Sisido, M.; Imanishi, Y. Macromolecules 1985, 18, 882-
889.
(31) Kawamura, A.; Berova, N.; Nakanishi, K.; Brunhilde, V.; Adam,
G. Tetrahedron 1997, 53, 11961-11970.
(32) Ikemoto, N.; Lo, L.-C.; Nakanishi, K. Angew. Chem. 1992, 31, 890-
891.
(14) Sisido, M. Makromol. Chem. Suppl. 1985, 14, 131-143.
(15) Muto, K.; Mochizuki, H.; Yoshida, R.; Ishii, T.; Handa, T. J. Am.
Chem. Soc. 1986, 108, 6416-6417.
(33) Akritopoulou-Zanze, I.; Nakanishi, K.; Stepowska, H.; Grzeszczyk,
B.; Zamojski, A.; Berova, N. Chirality 1997, 9, 699-712.
(34) Takeda, R.; Park, M. H.; Nakanishi, K. Isr. J. Chem. 1989, 29, 239-
42.
(35) Zhao, N.; Zhou, P.; Berova, N.; Nakanishi, K. Chirality 1995, 7,
636-51.
(16) Watanabe, K.; Muto, K.; Ishii, T. Biospectroscopy 1997, 3, 103-
111.
(17) Reigh, C.; Tinoco, I. J. Biopolymers 1980, 19, 833-848.

Fluorescence-Detected Exciton-Coupled CD
J. Am. Chem. Soc., Vol. 121, No. 38, 1999 8683
p-bromobenzoate 7,36 and p-methoxybenzoate 8.37 All of these
chromophores have been used extensively in conventional CD
studies. The FDCD and fluorescence properties of a wide range
of combinations of these fluorophores/chromophores conjugated
to (1R,2R)-trans-1,2-cyclohexanediol were investigated.
To determine the conditions under which the CD and FDCD
spectra were most closely correlated, the effects of fluorescence
polarization and fluorescence lifetime were addressed. Fluo-
rescence polarization (PF),28,38 the ratio of the difference between
the intensity of the vertically and horizontally polarized emission
components (FV - FH) to the sum of these two values (FV +
FH) originating from a vertically plane-polarized excitation, can
be used as a relative measure of sample anisotropy. The current
studies have clarified that with systems in which the exciton-
coupled FDCD spectrum deviated significantly from conven-
tional CD, fluorescence polarization was relatively large; in
cases where PF was negligible, photoselection effects were not
observed, and good agreement was observed between FDCD
and CD.
and by single-photon counting (pulse method) using a FL900CDT
spectrometer (Edinburgh Analytical Instruments). Since lifetime de-
terminations required adequate emission for measurement accuracy,
only compounds showing reasonable emission intensities were analyzed.
Rotational Correlation Time. Brownian movement in solution is
approximated by the rotational correlation time (φ, in ns),38 which can
be calculated from the following equation: φ ) (ηV)/(RT), where η is
the viscosity of the solvent (cP), V is the volume of the rotating unit
(cm3/mol) derived from the molecular weight and the density of the
molecule (the density of each compound was assumed to be 1.3 g/cm3),
T is temperature (K), and R ) 8.314 × 107 erg‚mol-1‚K-1
.
FDCD Measurements. To avoid intermolecular fluorescence quench-
ing,38 solutions for FDCD measurement had maximal absorbance below
0.2. In all cases, the same solution was used for both CD and FDCD
measurements, ensuring that any observed difference between CD and
FDCD spectra was not derived from sample preparation. The cell for
CD, FDCD, and fluorescence measurements was a 1 cm square
fluorescence quartz cell.
A JASCO-720 spectropolarimeter was fitted with a JASCO prototype
FDCD attachment (containing a Hamamatsu phonics R376 photomul-
tiplier tube), with the fluorescence detector placed at 90° to the
excitation beam.28 The transmitted light was trapped by a Rayleigh
horn beam trap placed between the sample and the CD detector. The
bandwidth was either 2.0 nm (highly fluorescent samples) or 5.0 nm
(weakly fluorescent samples), and the voltage (HT) applied to the
detector was always kept between 400 and 800 V, corresponding to a
maximal DC output signal voltage from the detector of 1.0 V.
For FDCD measurements, the emission is observed over a range of
irradiation wavelengths. The emitted light that is detected can be
controlled by varying the cutoff of a long-pass filter placed between
the sample and the fluorescence detector. Long-pass filters used were
JASCO UV34 (340 nm), L38 (380 nm), and L42 (420 nm), CVI WG-
320 (320 nm), and Coherent-Ealing WG-360 (360 nm). The appropriate
filter was chosen in order to avoid scattered light from the excitation
wavelengths which could contaminate the observed emission and to
maximize the emitted light signal. In cases where scatter of the
excitation wavelengths overlapped with the emission range, it was of
primary concern to adjust the filter to a longer wavelength to eliminate
the scattered excitation light. Analysis of the emission spectrum of each
compound was used to select the filter cutoff prior to FDCD
measurement. In experiments with a linear polarizer (plastic sheet
polarizer), the polarizer was placed between the fluorescence detector
and the long-pass filter. Rotation of the linear polarizer allowed for
FDCD measurements detecting different orientations of light.17,41
FDCD raw data represent an excitation spectrum that corresponds
to the difference in emission (FL - FR) and the total emission (FL +
FR) resulting from differential absorption of left- and right-circularly
polarized light. These data were converted into a conventional CD
spectrum by the following equation: ∆ꢀ ) ꢀL - ꢀR ) {2(1 - 10-A)-
S}/{cd10-A ln10}, where A is the absorbance, c is the concentration
(M), d is the cell length (cm), S ) k (FL - FR)/(FL + FR), and k is a
derived instrumental constant (-3.49 × 10-5).9,11 The negative value
of k inverts S so that FDCD and CD give Cotton effects with the same
sign. (Note: Although the data derived from absorption and fluores-
cence measurements are the same, conventional CD is based on the
detection of transmitted light, an indirect estimation of differential
absorption. Since the spectropolarimeter takes into account the indirect
nature of absorbance measurements, the fluorescence signal, which is
measured directly by the detector, initially appears opposite in sign.)
Sample Preparation. Homochromophoric and Heterochro-
mophoric Systems. All bis-ester model compounds were prepared from
(1R,2R)-trans-1,2-cyclohexanediol and appropriate carboxylic acid(s)
by either general procedure A or B, unless otherwise indicated. Relevant
spectroscopic data for each compound is presented in Tables 1-5. Full
characterization of compounds 1-4 and 9-30 is provided in the
Supporting Information. UV-vis absorbance data for weakly or
nonfluorescent chromophores 5-8 have been previously reported2,35
and fluorescence measurements for weakly fluorescent chromophores
Experimental Section
Abbreviations. The following abbreviations were used: CD, circular
dichroism; FDCD, fluorescence-detected circular dichroism; DBU, 1,8-
diazabicyclo[5.4.0]undec-7-ene; EDC, 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride; DMAP, 4-(dimethylamino)pyridine;
ΦF, fluorescence quantum yield; PF, fluorescence polarization; τF,
fluorescence lifetime; φ, rotational correlation time; λex, excitation
wavelength; λem, emission wavelength; λmax, UV maximum wavelength;
λ
ext, CD extremum wavelength; ꢀ, molar extinction coefficient; ∆ꢀ,
molar circular dichroism; FL and FR, fluorescence intensity for left-
and right-circularly polarized excitation, respectively.
General. All UV/vis and CD spectra were recorded in acetonitrile
(Aldrich, spectrophotometric grade), unless otherwise noted, on a
Perkin-Elmer Lambda 40 UV/vis spectrometer and JASCO J-720
spectropolarimeter, respectively. In all cases, solution concentration was
calculated based on sample weight. 1H NMR spectra were recorded on
a Bruker 300 or 400 MHz spectrometer. Chemical ionization (CI) was
measured on NERMAG R1010 mass spectrometer with NH3 as the
ionization gas. Model compounds were prepared with appropriate
materials purchased from Aldrich and TCI America (2-anthroic acid).
Dichloromethane was distilled from calcium hydride under nitrogen
and anhydrous acetonitrile used for syntheses was purchased from
Aldrich. Analytical and preparative TLC were run on Analtech
precoated silica gel plates (20 × 20 cm), 250 and 500 µm, respectively.
Flash column chromatography was performed with Selecto Scientific
silica gel, 32-63 mesh.
Fluorescence Properties. Fluorescence excitation and emission
spectra were measured in acetonitrile on a SPEX FluoroMax-2
spectrometer. The fluorescence quantum yields for all model compounds
in this report were determined by a relative method.39 The reference
solution for determination of quantum yield was 9,10-diphenylan-
thracene in acetonitrile, the quantum yield (0.90) of which was
determined based on the previously reported quantum yield in ethanol
(0.95) by employing the refractive index squared correction.39 Fluo-
rescence polarization experiments38 were performed on the SPEX
FluoroMax-2 spectrometer with Glan Thompson polarizers. The
absorption maxima and other specific wavelengths of interest, if
necessary, were used as excitation wavelengths, and vertical and
horizontal components in emission were observed at emission maxima.
Fluorescence lifetimes38,40 were determined by the phase and
modulation method using a SPEX Fluorolog 2T2 double spectrometer
(36) Lin, Y.-Y.; Risk, M.; Ray, S. M.; Van Engen, D.; Clardy, J.; Golik,
J.; James, J. C.; Nakanishi, K. J. Am. Chem. Soc. 1986, 103, 6773-6775.
(37) Shapiro, S. Enantiomer 1996, 1, 151-166.
(38) Lakowicz, J. R. In Principles of Fluorescence Spectroscopy;
Plenum: New York, 1984; pp 111-153.
(39) Morris, J. V.; Mahaney, M. A.; Huber, J. R. J. Phys. Chem. 1976,
80, 969-974.
(40) Slavik, J. In Fluorescent probes in cellular and molecular biology;
CRC Press: Boca Raton, FL, 1994; pp 241-268.
(41) Lobenstine, E. W.; Turner, D. H. J. Am. Chem. Soc. 1979, 101,
2205-2207.

8684 J. Am. Chem. Soc., Vol. 121, No. 38, 1999
Nehira et al.
Table 1. UV and Fluorescence Properties of Fluorophoresa,b
a ΦF ) fluorescence quantum yield, PF ) fluorescence polarization, τF ) fluorescence lifetime, φ ) rotational correlation time. b All data were
obtained in acetonitrile. c (1R,2R)-trans-1,2-Cyclohexanediol monoester. d Methyl ester.
5 and 6 are presented in Table 2. The terms “homochromophoric” and
“heterochromophoric” systems are used to describe bis-ester derivatives
containing two identical and two different chromophores, respectively.
General Procedure A. Monoester. To a solution of (1R,2R)-trans-
1,2-cyclohexanediol (6.1 mg, 0.053 mmol) and p-phenylbenzoic acid
(11.1 mg, 0.056 mmol) in dichloromethane (1.5 mL) were added DMAP
(7.6 mg, 0.062 mmol) and EDC (16.7 mg, 0.0871 mmol) under nitrogen.
The reaction mixture was stirred at room-temperature overnight, and
the solvent was removed under reduced pressure. The solid residue
was purified by flash column chromatography (silica gel, hexane/EtOAc
) 5/1) to give the (1R,2R)-trans-1,2-cyclohexanediol 1-(p-phenylben-
zoate) (6.5 mg, 42%)
Bis-ester. To a solution of (1R,2R)-trans-1,2-cyclohexanediol 1-(p-
phenylbenzoate) (6.5 mg, 0.022 mmol) and p-bromobenzoic acid (15.2
mg, 0.0756 mmol) in dichloromethane (1.5 mL) were added EDC (17.0
mg, 0.0887 mmol) and DMAP (13.2 mg, 0.108 mmol) under nitrogen.
The reaction mixture was stirred overnight, and the solvent was removed
under reduced pressure. The solid residue was purified by flash column
chromatography (silica gel, hexane/EtOAc ) 10/1) to afford (1R,2R)-
trans-1,2-cyclohexanediol 1-(p-bromobenzoate)-2-(p-phenylbenzoate)
(10.5 mg, 99%).
overnight. To the reaction mixture, additional 2-anthroic acid (10.2 mg,
0.046 mmol), EDC (9.0 mg, 0.052 mmol), and DMAP (10.0 mg, 0.074
mmol) were added and stirred for 8 h. The solvent was removed under
reduced pressure, and the solid residue was purified by flash column
chromatography (silica gel, hexane/EtOAc ) 8/1) to give (1R,2R)-trans-
1,2-cyclohexanediol 1-(2-anthroate)-2-(6-methoxy-2-naphthoate) (9.7
mg, 99%).
Results and Discussion
Isolated Chromophores. The scope of exciton-coupled
FDCD was probed with derivatives of (1R,2R)-trans-1,2-
cyclohexanediol, a simple and conformationally rigid skeleton
with two hydroxyl groups suitable for fluorophoric derivatiza-
tion. The strategy for determining favorable fluorophores for
FDCD studies involved the systematic preparation and analysis
of an extensive series of chromophoric derivatives. This report
focuses on four chromophores that were expected to have
advantageous fluorescence properties (Table 1) and four other
chromophores previously reported as being suited for conven-
tional exciton-coupled CD studies (Table 2).
General Procedure B. 6-Methoxy-2-naphthoylimidazole. A mix-
ture of 6-methoxy-2-naphthoic acid (65.1 mg, 0.322 mmol) and 1,1′-
carbonyldiimidazole (84.1 mg, 0.519 mmol) in acetonitrile (4.0 mL)
was stirred overnight under a nitrogen atmosphere. After the solvent
was removed under reduced pressure the resulting solid residue was
purified on flash column chromatography (silica gel, hexane/EtOAc
) 3/1) to give 6-methoxy-2-naphthoylimidazole (67.4 mg, 83%).
Monoester. To a solution of (1R,2R)-trans-1,2-cyclohexanediol (27.9
mg, 0.240 mmol) and 6-methoxy-2-naphthoylimidazole (67.4 mg, 0.267
mmol) in acetonitrile (2.5 mL), was added DBU (eight drops) under a
nitrogen atmosphere. The solution was stirred at room temperature for
1 h, and the solvent was removed under reduced pressure. The solid
residue was purified by flash column chromatography (silica gel,
hexane/EtOAc ) 5/1 and then 3/1) to give (1R,2R)-trans-1,2-cyclo-
hexanediol 1,2-bis-(6-methoxy-2-naphthoate) (14.1 mg, 12%) and
(1R,2R)-trans-1,2-cyclohexanediol 1-(6-methoxy-2-naphthoate) (47.8
mg, 66%).
The first four groups have been described as fluorophores
with high fluorescence quantum yields and well-defined orienta-
tions of the strong electric transition moments that produce
exciton couplets: 2-naphthoate 1,32 6-methoxy-2-naphthoate 2,11
2-anthroate 3,33 and p-phenylbenzoate 4.34 For the purpose of
FDCD analysis, quantum yield (ΦF), fluorescence polarization
(PF), and fluorescence lifetime (τF) were measured in acetonitrile
(Table 1). All four fluorophores (1-4) have relatively high
quantum yields, those of 2-4 exceeding 0.6, while that of
2-naphthoate 1 is ∼0.3. 2-Naphthoate, 6-methoxy-2-naphthoate,
and 2-anthroate 1-3 exhibit relatively small fluorescence
polarization (PF < 0.0035) and long fluorescence lifetimes (τF
g 5.0 ns). Thus, the extent of fluorescence polarization can be
correlated with the value of the fluorescence lifetime. It is well
accepted that a high level of fluorescence polarization is
observed when a molecule is unable to undergo sufficient
Brownian motion to randomize molecular orientations before
emission. In this case, when the probability of excitation depends
on the sense of polarization of the incident light, the measured
difference in fluorescence obtained by FDCD is no longer
Bis-ester. To a solution of (1R,2R)-trans-1,2-cyclohexanediol 1-(6-
methoxy-2-naphthoate) (5.8 mg, 0.019 mmol) and 2-anthroic acid (8.8
mg, 0.039 mmol) in dichloromethane (1.0 mL), were added under a
nitrogen atmosphere EDC (9.6 mg, 0.050 mmol) and DMAP (5.3 mg,
0.043 mmol), and the mixture was stirred at room-temperature

Fluorescence-Detected Exciton-Coupled CD
J. Am. Chem. Soc., Vol. 121, No. 38, 1999 8685
Table 2. UV and Fluorescence Properties of Weakly or
and CD seen in bis-2-naphthoate 9 and bis-6-methoxy-2-
naphthoate 10,11 and clearly demonstrated the enhanced sensi-
tivity of FDCD. In the present study, 9 and 10 were used as
standards to compare the extent of agreement between FDCD
and CD for all new derivatives. In addition, a more detailed
analysis of the fluorescence polarization and lifetime of 9 and
10 was performed in order to determine the extent of solution
anisotropy for these systems (Table 3).
Nonfluorescent Chromophoresa,b
It was expected that the fluorescence properties of bis-2-
naphthoate 9 would be similar to those of the isolated chro-
mophore in 1. Despite the fact that the two fluorophores in 9
are identical to that in 1, the bis-ester showed a slight broadening
of its emission spectrum from 400 to 500 nm as compared to
the monochromophoric ester (Figure 1). The fluorescence
lifetime and polarization of 1 and 9 were essentially identical
(Tables 1 and 3).
Based on the emission spectrum of 9 (Figure 1B), a 320 nm
long-pass filter was chosen for FDCD measurement since most
emission occurs above 340 nm and excitation wavelengths
between 200 and 300 nm were sufficient for observation of the
exciton couplet. The FDCD raw data (FL - FR; FL + FR) for 9
in acetonitrile are shown in Figure 2A. Using the conversion
equation (see Experimental Section), these data were translated
into an FDCD spectrum (∆ꢀ/λ). A comparison of the converted
FDCD and the spectrum obtained from conventional CD clearly
showed excellent agreement (Figure 2B). In this case, the ratio
of the lifetime (τF ) 8.0 ns) to the rotational correlation time
(φ ) 0.045 ns) indicated negligible photoselection effects.
Indeed, this was confirmed by the low fluorescence polarization
value (PF ) 0.004). In addition, a very close agreement between
FDCD and CD was seen in the case of bis-6-methoxy-2-
naphthoate 10 (Figure 3A). As with 9, the fluorescence polari-
zation, fluorescence lifetime and the rotational correlation time
of 10 (Table 3) indicated that this solution could also be con-
sidered isotropic. The FDCD analyses of 9 and 10 thus demon-
strate the utility of 2-naphthoate and 6-methoxy-2-naphthoate
chromophores for fluorescence-based CD measurements.
a ΦF ) fluorescence quantum yield, PF ) fluorescence polarization.
b All data for methyl esters in acetonitrile except where indicated. c In
ethanol, ref 2. d Reference 35. e Data for compound (1R,2R)-trans-1,2-
cyclohexanediol monoester. f Not applicable due to weak fluorescence.
necessarily related to the difference in absorbed light measured
by CD.27 This type of anisotropy is generally observed either
when a molecule is very large and/or rigid (e.g., biomacromol-
ecules), or when the solvent is viscous and rotation is hindered.
To understand the source of any fluorescence anisotropy
observed in these experiments, rotational correlation times (φ)
were compared with fluorescence lifetimes (Table 1).38 All four
fluorophores have calculated rotational correlation times (φ)
∼0.03 ns, and, thus, are able to undergo >150 rotations since
their emission lifetimes range between 5.0 and 11.0 ns. This
randomization of the excited state leads to very small observed
values of fluorescence polarization, indicating that samples
containing only these fluorophores should be essentially iso-
tropic. In contrast, the fluorescence lifetime (τF ) 0.8 ns) of
p-phenylbenzoate 4 is only 25 times longer than its rotational
correlation time (φ ) 0.031 ns), indicating that the emission of
this fluorophore is more inclined to photoselection effects. The
higher anisotropy of the emission of 4 is confirmed by the
increased fluorescence polarization (PF ) 0.034).
The cyclohexanediol scaffold was derivatized not only with
strong fluorophores, but also with moderately fluorescent or
nonfluorescent chromophores. Chromophores previously de-
scribed as being suited for conventional exciton-coupled CD
studies, namely p-dimethylaminobenzoate 5,2 p-methoxycin-
namate 6,35 p-bromobenzoate 7,36 and p-methoxybenzoate 8,37
were employed for these studies (Table 2). The p-dimethylami-
nobenzoate chromophore, 5, has a weak fluorescence signal
(ΦF ) 0.018) and a relatively large fluorescence polarization
(PF ) 0.02). A second weak-fluorophore, p-methoxycinnamate
6, has even lower quantum yield (ΦF ) 0.0005) and, moreover,
larger fluorescence polarization (PF ) 0.4) than 5. Two
chromophores, p-bromobenzoate 7 and p-methoxybenzoate
8, did not exhibit significant emission and were considered
nonfluorescent.
While 2-anthroate 3 was previously described as a fluorophore
for FDCD,11 attempts to prepare bis-2-anthroate 11 were
complicated by the light sensitivity of this compound. Although
small amounts of 11 could be purified, light exposure led to
significant decomposition as monitored by silica thin-layer
chromatography; therefore, further studies with 11 were not
pursued. However, in certain cases, compounds containing only
a single 2-anthroate fluorophore were stable under conditions
employed for routine FDCD measurements (see below); the
factors affecting the stability of compounds containing 2-an-
throate have not been established.
Although the fluorescence quantum yield of p-phenylbenzoate
4 is 0.66, its relatively large PF (Table 1) is a significant
drawback for FDCD. As expected, bis-ester 12 showed a
relatively large fluorescence polarization (Table 3), and the
agreement between FDCD and CD (Figure 3B) was less
satisfactory than that observed for 9 and 10. The FDCD
spectrum, when compared with the CD spectrum, exhibited a
difference in intensity and position of corresponding Cotton
effects. Thus, this is a case where photoselection effects cannot
be neglected.
Bis-derivatives containing one of the two weak-fluorophores,
p-dimethylaminobenzoate 5 and p-methoxycinnamate 6, were
also prepared. The CD and FDCD of bis-p-dimethylaminoben-
zoate 13 were very similar in both position and intensity (Table
3), but the low quantum yield (0.021) and moderate fluorescence
polarization (PF ) 0.01) make p-dimethylaminobenzoates
Bischromophoric derivatives. All model compounds de-
scribed here possess a (1R,2R)-trans-1,2-cyclohexanediol skel-
eton and two chromophores attached to the diol through ester
linkages. Our initial report of fluorescence-detected exciton-
coupled CD described the excellent agreement between FDCD

8686 J. Am. Chem. Soc., Vol. 121, No. 38, 1999
Nehira et al.
Table 3. Bis-homochromophoric Esters of (1R,2R)-trans-1,2-Cyclohexanediola,b
a ΦF ) fluorescence quantum yield, PF ) fluorescence polarization, τF ) fluorescence lifetime, φ ) rotational correlation time, λext ) extremum
wavelength. b All data were obtained with acetonitrile. c Due to photochemical instability, the data for 11 were inconsistent. d Not determined.
Figure 1. Excitation and emission spectra of the naphthoate chromophore. (A) Excitation (solid line) and emission (dashed line) spectra of (1R,2R)-
trans-1,2-cyclohexanediol mono-naphthoate (1). (B) Excitation (solid line) and emission (dashed line) spectra of (1R,2R)-trans-1,2-cyclohexanediol
bis-naphthoate (9).
generally unsuitable for FDCD. In addition, this chromophore
is believed to give rise to a twisted intramolecular charge-
transfer (TICT) excited state,42 which may lead to environment-
sensitive fluorescence that distorts FDCD data. In certain cases,
it may be that the TICT state leads to high levels of emission
anisotropy. Therefore, this chromophore cannot be used for
exciton-coupled FDCD studies.
Bis-p-methoxycinnamate 14 (Table 3) has an extremely large
fluorescence polarization of 0.4 and the FDCD was greatly
perturbed by photoselection effects.27,30,43 Comparison of its CD
spectrum with that obtained by conventional CD revealed no
similarity. Although p-methoxycinnamate is a valued red-shifted
chromophore for absorbance-based exciton chirality studies,
similar to p-dimethylaminobenzoate, it is not useful for FDCD
studies.
The above studies on bis-homochromophoric cyclohexanediol
derivatives indicated that fluorophores with high quantum yield
and small fluorescence polarization gave rise to similar FDCD
and CD spectra. When fluorescence polarization was large,
regardless of quantum yield, FDCD spectra were no longer
correlated with conventional CD.
While it was demonstrated that certain pairs of identical
fluorophores lead to well-defined exciton-coupled FDCD, the
effect of having nonidentical fluorophores in the fluorescence-
detected exciton-coupled system has not been addressed. It was
of interest to determine the type of FDCD that would result
from the coupling of a single fluorophore with a nonfluorescent
chromophore. The selectivity of fluorescence detection may lead
to a CD spectrum where only the Cotton effect of the
fluorophoric group is observed. Alternatively, it was possible
that the dipole-dipole exciton coupling interaction would allow
for the manifestation of the entire bisignate curve. Similar to
the previous cases, heterochromophoric systems were analyzed
(42) Grabowski, Z. R.; Dobkowski, J. Pure Appl. Chem. 1983, 55, 245-
252.
(43) Tinoco, I.; Ehrenberg, B.; Steinberg, I. Z. J. Am. Chem. Soc. 1977,
66, 916-920.

Fluorescence-Detected Exciton-Coupled CD
J. Am. Chem. Soc., Vol. 121, No. 38, 1999 8687
Figure 2. FDCD spectrum of (1R,2R)-trans-1,2-cyclohexanediol 1,2-bis-(2-naphthoate) (9). (A) Raw data obtained from spectropolarimeter. (B)
Processed FDCD spectrum (dashed line), as described in the Experimental Section. The FDCD spectrum is compared with the conventional CD
spectrum (solid line). All spectra were obtained in acetonitrile (1.93 µM).
Figure 3. FDCD spectra of homochromophoric-bis-esters of (1R,2R)-trans-1,2-cyclohexanediol. FDCD (dashed lines), CD (solid lines). (A) Bis-
6-methoxy-2-naphthoate (10) in acetonitrile (2.24 µM), (B) bis-p-phenylbenzoate (12) in acetonitrile (5.48 µM).
by comparing the converted FDCD spectra with conventional
CD and searching for further correlation between fluorescence
polarization and lifetime measurements.
of 15 indicated that it has very small levels of fluorescence
polarization (Table 4, PF < 0.001).
Compounds 16, 17, and 18, containing different combinations
of the fluorescent 2-naphthoate 1, 6-methoxy-2-naphthoate 2
and 2-anthroate 3, and the nonfluorescent p-methoxybenzoate
8, also showed satisfactory agreements between their FDCD
and CD curves (Figure 6). In all three cases, their polarizations
were relatively small (Table 4, PF < 0.009) and their fluores-
cence lifetimes were relatively long (Table 4, τF > 5.0 ns). The
relationship between the fluorescence lifetime, the rotational
correlation time of a molecule, and fluorescence polarization is
also evident by 16-18; e.g., in the case of hetero-bis-ester 16,
where τF ) 11 ns and φ ) 0.053 ns, more than 200 rotations
can occur during its emission lifetime. This situation led to low
levels of fluorescence polarization (PF ) 0.002), which, in turn,
gave rise to the reasonable agreement between FDCD and CD.
The long τF and small PF relation was also seen in cases 17
and 18, which showed satisfactory agreement between CD and
FDCD.
In certain cases, bis-derivatives exhibited high ΦF, small PF,
and good agreement between FDCD and CD (Table 4). The
heterochromophoric system 15, containing the fluorescent
2-anthroate 3 and nonfluorescent p-methoxybenzoate 8, provided
FDCD spectra that were in excellent agreement with CD. Thus,
when these two chromophores couple, although only one is a
fluorophore (2-anthroate, ΦF ) 0.62), the resulting exciton
couplet can be surprisingly well represented by FDCD. Excita-
tion and emission spectra of 2-anthroate 3 as a single fluorophore
(Figure 4A) and bis-ester 15 (Figure 4B) were very similar,
confirming that the nonfluorescent p-methoxybenzoate is silent
in terms of fluorescence. The emission spectrum of the hetero-
bis-ester 15 (Figure 4B) indicated that most of the emission is
at wavelengths greater than 400 nm. The exciton couplet is
expected around the absorbance maxima of the two chro-
mophores, between 250 and 260 nm. With these data in mind,
circularly polarized excitation wavelengths from 200 to 320 nm
were scanned and a cutoff filter at 380 nm was employed. The
resulting raw data (Figure 5A) were converted into an FDCD
spectrum which closely resembled the absorbance-based CD
(Figure 5B). It is clear that there is good agreement between
the two curves, and the analysis of the fluorescence properties
It is remarkable that the presence of only one fluorophore in
esters 15 and 18 still led to exciton coupled FDCD. Since in
this case the fluorescent and nonfluorescent chromophores
interact through space as a coupled oscillator forming two new
split energy levels, the light absorbed by either chromophore
can be emitted by the fluorophore. The interaction between the

8688 J. Am. Chem. Soc., Vol. 121, No. 38, 1999
Nehira et al.
Table 4. Bis-heterochromophoric Esters of (1R,2R)-trans-1,2-Cyclohexanediol Showing Good Agreements between FDCD and CDa,b
a ΦF ) fluorescence quantum yield, PF ) fluorescence polarization, τF ) fluorescence lifetime, φ ) rotational correlation time, λext ) extremum
wavelength. b All data were obtained in acetonitrile. c Determined by phase and modulation method. d Determined by pulse method.
Figure 4. Excitation and emission spectra of 2-anthroate and its derivative. (A) Excitation (solid line) and emission (dashed line) spectra of
2-anthroate (3). (B) Excitation (solid line) and emission (dashed line) spectra of (1R,2R)-trans-1,2-cyclohexanediol 1-(2-anthroate)-2-(p-
methoxybenzoate) (15).
fluorophore and non- (or weak-) fluorophore in exciton-coupled
systems leads to bisignate curves in FDCD where the Cotton
effect derived from the nonfluorophore is apparent and both
CD and FDCD couplets are almost identical. Therefore, the
selectivity of FDCD does not extend to systems in which two
chromophores are involved in through-space dipole-dipole
interactions. Presumably, an additional nonfluorescent chro-
mophore that is isolated from the exciton-coupled system would
be silent in FDCD measurements. This case will be submitted
for further studies to probe the selectivity of FDCD analysis.
Additional FDCD studies of nonfluorescent chromophores as
parts of exciton-coupled systems may allow for a better
understanding of the fundamental nature of the exciton coupling
phenomenon.
Exciton-coupled CD2 and fluorescence energy transfer44 are
both derived from dipole-dipole chromophoric interactions. The
heterochromophoric bis-ester 16, containing 2-anthroate and
6-methoxy-2-naphthoate, was used to investigate the effect of
energy transfer on FDCD.45,46 As shown in Figure 7A, there is
an overlap between the emission of 6-methoxy-2-naphthoate 2
(donor) and the absorption of 2-anthroate 3 (acceptor).45 In fact,
the emission spectrum of 16 (solid line, Figure 7B) did not show
the emission corresponding to that of the donor 6-methoxy-2-
naphthoate; this peak was observed in the mixture of the two
(44) Lakowicz, J. R. In Principles of Fluorescence Spectroscopy;
Plenum: New York, 1984; pp 303-339.
(45) Schnepp, O.; Levy, M. J. Am. Chem. Soc. 1962, 84, 172-177.
(46) Latt, S. A.; Cheung, H. T.; Blout, E. R. J. Am. Chem. Soc. 1965,
87, 995-1003.

Fluorescence-Detected Exciton-Coupled CD
J. Am. Chem. Soc., Vol. 121, No. 38, 1999 8689
Figure 5. FDCD spectrum of (1R,2R)-trans-1,2-cyclohexanediol 2-anthroate, p-methoxybenzoate (15). (A) Raw data obtained from spectropolarimeter.
(B) Processed FDCD spectrum (dashed line), as described in the Experimental Section. The FDCD spectrum is compared with the conventional CD
spectrum (solid line). All spectra were obtained in acetonitrile (1.26 µM).
Figure 6. FDCD spectra of hetero-bis-esters of (1R,2R)-trans-1,2-cyclohexanediol. FDCD (dashed lines), CD (solid lines) (A) 6-Methoxy-2-
naphthoate, 2-anthroate (16) in acetonitrile (1.49 µM), (B) 6-Methoxy-2-naphthoate, 2-naphthoate (17) in acetonitrile (3.60 µM), (C) 6-Methoxy-
2-naphthoate, p-methoxybenzoate (18) in acetonitrile (4.41 µM).
Figure 7. Fluorescence properties of (1R,2R)-trans-1,2-cyclohexanediol 2-anthroate, 6-methoxy-2-naphthoate (16). (A) Overlap of 6-methoxy-2-
naphthoate emission (2) (dashed line) and 2-anthroate absorbance (3) (solid line). (B) Emission spectra of 16 (solid line) and an equimolar mixture
of 3 and 2 (dashed line).
monochromophoric compounds 2 and 3 (Figure 7B). Despite
this evidence of energy transfer, 16 showed good agreement
between FDCD and CD.
In the cases of compounds 19-21 (Table 4), good agreements
between CD and FDCD were observed. Although there was a
slight increase in the fluorescence polarization (PF ≈ 0.02) as
compared with derivatives 9 and 10, the ability of FDCD to
provide the same dichroic information that is obtained from
conventional CD was not compromised. In exciton-coupled
systems where higher values of PF are observed, an independent
method for confirming the presence of fluorescence anisotropy
may be required. One simple method for checking this, which
has previously been described and does not require instrumental
modification,17,41 is the placement of a linear polarizer between
the sample and the fluorescence detector. The linear polarizer
will allow only certain orientations of light to reach the detector.
The FDCD and CD spectra obtained at different orientations
of the polarizer should be compared. The angle of the linear

8690 J. Am. Chem. Soc., Vol. 121, No. 38, 1999
Nehira et al.
Table 5. Bis-heterochromophoric Esters of (1R,2R)-trans-1,2-Cyclohexanediol leading to poor agreement between FDCD and CDa,b
a ΦF ) fluorescence quantum yield, PF ) fluorescence polarization, τF ) fluorescence lifetime, φ ) rotational correlation time, λext ) extremum
wavelength. b All data were obtained in acetonitrile. c Determined by phase and modulation method. d Determined by pulse method. e Not determined.
f In cyclohexane. g Very weak FDCD signal.
polarizer will have no effect on FDCD when the solution is
isotropic;17 however, in the case where photoselection is
relevant, rotation of the linear polarizer should result in
nonidentical FDCD spectra. In the case of 21, the use of a linear
polarizer had no effect on the measured FDCD (data not
shown).
in this study. No agreement is seen between FDCD and CD for
23 (Figure 8B). This compound can only rotate approximately
10 times during its emission lifetime and clearly photoselection
effects play an important role in the FDCD spectrum of this
compound. Since it is unlikely that this situation will result in
a randomized excited state, fluorescence polarization is high
(PF ) 0.04). In contrast to the result observed with 21, the
presence of photoselection artifacts for the analysis of 23 was
further confirmed since the use of a linear polarizer lead to
altered FDCD spectra (data not shown). The use of modified
spectropolarimeters29,30 may alleviate these effects, but this type
of analysis requires substantial instrumental modification which
was beyond the scope of the current studies.
Compounds containing p-dimethylaminobenzoate (24-27)
and p-bromobenzoate chromophores (27 and 28) were weakly
fluorescent and were sensitive to photoselection artifacts.
Compound 28 gave results similar to 22. The FDCD of 28,
possessing weak fluorescence (ΦF ) 0.053) and significant
fluorescence polarization (PF ) 0.02), showed a distorted and
weak spectrum as compared with conventional CD analysis.
Compounds containing p-dimethylaminobenzoate and p-bro-
mobenzoate, in general, showed significant photoselection
effects and the agreement between FDCD and CD was not
satisfactory. Heterochromophoric bis-esters 29 and 30, contain-
Compounds that displayed larger fluorescence polarization,
regardless of quantum yield, gave less satisfactory agreement
between FDCD and CD (Table 5). The fluorescence polarization
of 22, containing 2-naphthoate 1 and p-bromobenzoate 7, varied
237 nm
significantly depending on the excitation wavelength (PF
) 0.02; PF
278 nm
) 0.006). Its fluorescence lifetime was
relatively short (τF ) 2.5 ns) compared to systems that showed
good CD/FDCD agreement. The CD and FDCD were similar
in the area where PF is small (>260 nm), but were different
where PF is larger (230-250 nm) (Figure 8A). Although the
overall bisignate shape of the FDCD curve was maintained, the
position and intensity of the peaks differed significantly from
those of the CD spectrum.
Compound 23, with p-phenylbenzoate 4 and p-bromobenzoate
7, has a short fluorescence lifetime of 0.6 ns and rotational
correlation time of 0.051 ns (Table 5). The fluorescence
polarization of 0.04 is larger than that for 22 (PF ) 0.02), while
the fluorescence lifetime is shorter than any other compound

Fluorescence-Detected Exciton-Coupled CD
J. Am. Chem. Soc., Vol. 121, No. 38, 1999 8691
Figure 8. FDCD spectra of hetero-bis-esters of (1R,2R)-trans-1,2-cyclohexanediol. FDCD (dashed lines), CD (solid lines) (A) 2-Naphthoate,
p-bromobenzoate (22) in acetonitrile (4.81 µM), (B) p-Phenylbenzoate, p-bromobenzoate (23) in acetonitrile (6.34 µM).
ing p-methoxycinnamate, displayed the largest photoselection
artifacts in FDCD. Despite the utility of this chromophore in
absorbance-based CD studies, the p-methoxycinnamate chro-
mophore is thus unsuited for FDCD studies.
polarization of fluorescence. Only isotropic samples, i.e.,
samples with fluorescence polarization below 0.01 yield exciton
coupled FDCD curves corresponding closely to those of
absorbance-based CD. A short fluorescence lifetime leads to
an increase in polarization since the molecule does not rotate
sufficiently to fully randomize molecular orientation before light
emission. Clear exciton-coupled FDCD spectra may be observed
even in the case where one of the chromophores is nonfluo-
rescent.
In summary, the value of fluorescence polarization (PF) is
crucial for predicting the agreement between circular dichroic
spectra obtained by absorbance and fluorescence. Although
additional studies are necessary, the present results suggest that
when the fluorescence polarization is smaller than 0.01, a good
agreement between the shape of the exciton-coupled CD and
FDCD curves can be expected. To our knowledge, this
represents the first extensive quantitative analysis of the
relationship between FDCD and fluorescence polarization. This
trend is valid among all model compounds described in this
report. Namely, measurements of fluorescence polarization can
be used to check whether the sample is suitable for exciton-
coupled FDCD studies; in addition, the fluorescence quantum
yield ΦF of the compound should be larger than 0.2 in order to
achieve a significant sensitivity enhancement. Further investiga-
tions are ongoing to clarify the factors involved in general
FDCD/CD correlations as well as in exciton-coupled FDCD.
Acknowledgment. This work was supported by Grant GM-
34509 from the National Institutes of Health (K.N./N.B.), a grant
from the Uehara Memorial Foundation (T.N.) and a National
Institutes of Health National Research Service Award (C.A.P.).
We gratefully acknowledge JASCO for making available to us
the fluorescence detector used in these studies. We thank Dr.
Akio Wada (JASCO), Dr. Takashi Takakuwa (JASCO), and
Dr. Jian-Guo Dong (Columbia University) for helpful discus-
sions. We acknowledge Ms. Jaroslava Paruchova for the
preparation of some of the described compounds.
Supporting Information Available: Detailed characteriza-
tion of compounds 1-4 and 9-30, including H NMR, mass
spectrometry, UV-vis absorbance, CD, fluorescence emission
and excitation, fluorescence quantum yield and polarization, and
FDCD data (PDF). This material is available free of charge via
the Internet at http://pubs.acs.org.
Conclusion
1
FDCD, which in general allows measurements with much
higher sensitivity than conventional CD, can be used for the
determination of absolute configuration of small molecules by
the exciton chirality method. Optimal fluorophores for the
fluorescence-detected exciton chirality method have strong
absorbance, high fluorescence quantum yield, and negligible
JA990936B