Sensitivity Enhancement of Exciton Coupling by Fluorescence Detected Circular Dichroism (FDCD)

Exciton coupled circular dichroism,2,3 characterized by split Cotton effects, is a microscale chiroptical method that determines the absolute configurations or conformations of a variety of compounds. This method becomes particularly powerful when complex experimental curves agree with calculated couplets, e.g., vinblastine.4 A critical experimental finding was that the exciton split CD of molecules consisting of multiple interacting chromophores, identical5a or different,5b,c can be reproduced by pairwise summation of interacting chromophores, a principle which has been confirmed by theoretical calculations.6 Since the amplitude of the split CD (A value, ACD) is proportional to 2, chromophores with intense absorptions such as porphyrins with sharp Soret bands at ca. 415 nm ( 350 000) exhibit couplings at a distance of 40-50 A.7 Fluorescent chromophores further facilitate ng ∼ μg scale handling of sample and enhance their HPLC detection,8 e.g., 2-naphthoic acid, λmax 234 nm ( 57 500), and fluorescence emission λmax 360 nm upon irradiation at 234 nm. However, the sensitivity of conventional CD detection (obtained in transmission) remains limited because it depends on the intensity of dichroic absorption. In contrast, in fluorescence detected circular dichroism (FDCD), the sensitivity is greatly enhanced because it is based on direct mearurement of emitted radiation against a zero background. Moreover, in FDCD, only the CD-active and fluorescent transitions give rise to a signal,9,10 thus enabling the selective measurement of the CD of fluorophores in a multichromophoric molecule, e.g., proteins with single fluorescent tryptophans.11 FDCD using a modified phase-modulation spectrofluorometer12 has been used to determine the enantiomeric excess without physical separation of enantiomers.13 However, due to technical difficulties, high sensitivity has been achieved so far only when FDCD was combined with other techniques, such as laser-based detection for HPLC14 and on-column detection in capillary electrophoresis.15 Moreover, the FDCD of multichromophoric systems are not necessarily straightforward and have led to various observations, i.e., no exciton coupling due to stacking in the case of ethenoadenosines,16,17 weak coupling arising from nonradiative deactivation with poly(1-pyrene-alanine),18 and a complex CD pattern with polytryptophan.19 In the following we report extension of FDCD to exciton coupled systems consisting of identical chromophores with well-defined and intense electric transition moments, 1(S),2(S)-trans-cyclohexanediol bis(6-methoxy-2-naphthoate) (1), 1(R),2(R)-trans-cyclohexanediol bis(2naphthoate) (2), a steroidal 3â,6R-bis-(2-anthroate)8b (3), and ouabagenin 1,3,19-tris-(2-naphthoate) (4). This FDCD measurement results in 50-100-fold sensitivity enhancement over conventional CD.