Ultrafast chiroptical spectroscopy

Journal of Analytical Science & Technology (2011) 2 (Suppl A), A103-A107 Proceeding www.jastmag.org DOI 10.5355/JAST.2011.A103

Session Ⅲ – Bioanalysis Ⅱ

Open Access

Ultrafast chiroptical spectroscopy: Monitoring optical activity in quick time Hanju Rhee1,2*, Intae Eom1, Minhaeng Cho1,3* 1

2

Korea Basic Science Institute, Seoul 136-713, Republic of Korea Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 305-764, Republic of Korea 3 Department of Chemistry, Korea University, Seoul 136-701, Republic of Korea

*Corresponding author: Hanju Rhee, E-mail: [email protected] Minhaeng Cho, E-mail: [email protected] From 2011 International Symposium on Analytical Science and Technology (ISAST) Daejeon, Republic of Korea, 15-17 November, 2011

Abstract Optical activity spectroscopy provides rich structural information of biologically important molecules in condensed phases. However, a few intrinsic problems of conventional method based on electric field intensity measurement scheme prohibited its extension to time domain technique. We have recently developed new types of optical activity spectroscopic methods capable of measuring chiroptical signals with femtosecond pulses. It is believed that these novel approaches will be applied to a variety of ultrafast chiroptical studies.

Key words: optical activity; molecular chirality; femtosecond spectroscopy; heterodyned detection

Introduction Almost all biomolecules including proteins, peptides, and nucleic acids are chiral so that they exhibit a distinctive optical property known as optical activity (OA) [1], where OA originates from the differential interaction of chiral molecule with left- and righthanded radiations. Circular dichroism (CD) and optical

rotatory dispersion (ORD) are two important OA measurement techniques that have been extensively used to elucidate structural details of biologically important molecules in condensed phase [1-3]. Nevertheless, typical OA signal of which intensity is determined by rotational strength is extremely weak in comparison to absorption because the angular

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component of involved quantum transition between two vibrational or electronic states is about two orders of magnitude smaller than the linear component. Such weak signal problem has significantly limited further extensions of OA measurement technique into ultrafast time-resolved CD/ORD methods, even though such time-resolved OA measurement method will no doubt enable one to monitor structural changes of chiral molecules in unprecedentedly rapid timescale.[4-7] For example, it should be noted that biomolecules participating in certain chemical and biochemical reactions are dynamic in nature and such biological functions inevitably involve rapid changes of local conformations and structures of chemical groups on timescale ranging from femtosecond to nanosecond, e.g. protein folding/unfolding process, enzyme catalytic reaction, etc. [8-10] To unravel the underlying mechanisms of fundamental biological processes, ultrafast spectroscopic techniques have been widely used. Unlike other conventional tools like multidimensional NMR and X-ray crystallography, our OA spectroscopic method utilizing femtosecond laser pulse has intrinsically ultrafast time-resolvability achievable. In practice, however, due to intrinsic power and phase fluctuations of a train of mode-locked laser pulses, accurate and high precision measurement of such weak OA signal has been quite challenging, if a few conventional methods relying on differential measurement were of use. Recently, we have developed new chiroptical techniques based on free-induction-decay (FID) measurement much like pulsed NMR experiment [1113]. In contrast to the conventional differential method using both left- and right-circularly polarized lights and measuring the signal field intensities, we show that coherent OA-FID field created by a linearly-polarized (LP) femtosecond laser pulse can be detected. Since the signal field amplitude and phase are to be characterized to retrieve the OA properties from the measured field, we used heterodyne-detection methods. We found that the heterodyne-detection method is of use not only to perform phase-sensitive detection but also to amplify the weak OA-FID signal field. This approach has a few notable advantages over the conventional CD method: (1) background-free and non-differential measurement that dramatically enhances the measurement sensitivity, (2) simultaneous CD and ORD

measurement and (3) femtosecond time-resolution achievable. We believe that these novel aspects are unique in our approach and will allow us to make its extension to ultrafast OA application feasible. Here, we shall introduce two different types of heterodyne-detection methods used to measure the OAFID field and they will be referred to as active- and selfheterodyned detection techniques. Using these methods, we have successfully carried out vibrational and electronic CD/ORD measurements of small organic chiral molecules and DNA-dye aggregate system. Although we have not yet demonstrated any timeresolved CD/ORD measurements, the present method can be extended to such application in a straightforward manner by combining it with an appropriate pump-probe method such as temperature-jump (T-jump).

Experimental methods In conventional differential measurement method, both left- and right circularly-polarized (LCP and RCP) radiations are needed to obtain a CD spectrum that is defined as the difference between the resulting absorption spectra. In contrast, the present technique measures a time domain OA-FID signal coherently emitted by a chiroptical response of the sample. From linear response theory, we found that, when a linearly polarized incident femtosecond pulse passes through a chiral sample, the OA-FID ( E⊥ (ω ) ) field whose polarization direction is perpendicular to that of the incident LP field, is generated [11, 13]. Note that these two orthogonal polarization states of a given radiation are not coupled if the isotropic medium contains no chiral molecules. For a chiral molecule solution, we found that the perpendicular electric field spectrum is related to the parallel electric field spectrum as E⊥ (ω ) ∝ ∆χ (ω ) ⋅ E (ω ) .

(1)

Here, the proportionality function ∆χ (ω ) is the optical activity susceptibility that is a complex function. The imaginary and real parts of ∆χ (ω ) correspond to the CD (differential absorption) and ORD (differential retardation), respectively. In equation (1), E⊥ (ω ) and E (ω ) are the complex electric field spectra whose

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polarization directions are perpendicular and parallel to that of the incident linear polarization, respectively. Equation (1) suggests that the complex chiral susceptibility ∆χ (ω ) can be characterized by measuring both E (ω ) and E⊥ (ω ) , even though the whole chiral information is exclusively contained in the perpendicular field component, E⊥ (ω ) . These two FID fields, E (ω ) and E⊥ (ω ) , were obtained by using heterodyned detection methods that have often been used to characterize coherent optical fields. For instance, two-dimensional vibrational or electronic spectrum was obtained by allowing the thirdorder photon echo signal field to interfere with an independently injected local oscillator (LO) field. On the other hand, self-heterodyne-detected pump-probe spectroscopy is to measure the third-order pump-probe signal field by the probe pulse itself. Thereby, this type of detection method has known to be a selfheterodyning method. Similarly, in our cases the OAFID field was deliberately made to interfere with a LO field. Depending on whether the local oscillator (LO) that is heterodyned with the signal fields is controlled externally (active-heterodyned detection) or internally (self-heterodyned detection), the CD and ORD signals are obtained differently. In the active-heterodynedetection scheme (Fig. 1(a)), the LO (ELO) and FID signals (E⊥,||) are temporally separated and the time delay (τd) is controlled to be constant. As a result, the two fields interfere with each other in the frequency domain and the resulting heterodyned spectral interferogram, which is related to the signal and LO field spectra as S⊥ , (ω ) = 2 Re[ E⊥ , (ω ) ELO (ω )eiωtd ] . Here, these two spectral interferograms (S⊥,||) can be individually detected by aligning the analyzer (A) perpendicular or parallel to the first polarizer (P). A standard Fourier transform spectral interferometry (FTSI) procedure (FTSI[⋅⋅⋅]) [14], is then used to transform the spectral interferogram, S⊥ , (ω ) , which is a real function, into its complex electric field spectrum. As long as the LO and τd are fixed, the ratio of the FTSI transformed spectra gives information on the OA spectra as, FTSI [ S ⊥ (ω )] = δ (ω ) + iθ (ω ) ∝ ∆χ (ω ) , FTSI [ S (ω )]

(2)

where δ ( ω ) is the optical rotation and θ ( ω ) is the ellipticity. The imaginary and real parts of this ratio finally yield the CD (δ(ω)) and ORD (θ(ω)) spectra, respectively. In the self-heterodyned detection scheme (Fig. 1(b)), on the other hand, the incident field itself acts as both the excitation source and the LO [15]. Here, the polarization state of the incident light is either an elliptical polarization (for CD) or a rotated linear polarization (for ORD). In either case, the minor-axis field component is used as the LO. In this case, the logarithm of the ratio of the self-heterodyned spectra is given as  S (ω )  i ∆φ log  ⊥  ∝ Re {[δ (ω ) + iθ (ω ) ] e } ,  S (ω ) 

(3)

where ∆φ is the phase difference between ELO and E||. After the sample, ∆φ is fixed to π/2 (elliptical polarization) or 0 (rotated linear polarization) so that the CD and ORD are separately measured depending on the incident polarization state.

Fig. 1. Heterodyned OA-FID detection scheme. (a) Active- and (b) self-heterodyned detection methods. P: polarizer, S: sample, A: analyzer, PBS: polarizing beam splitter.

Results and discussions To demonstrate the experimental feasibility of our active-heterodyned detection method depicted in Fig. 1(a), we performed vibrational CD and ORD (VCD/VORD) measurements of a small organic chiral molecule, (1S)-β-pinene [16]. Figure 2(a) shows the heterodyned spectral interferograms measured for two different vibrational modes of (1S)-β-pinene: C-C (1000~1350 cm-1) and C-H (2850~3000 cm-1) stretching

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modes. The S⊥ (ω ) (red) and S (ω ) (blue) exhibit different spectral patterns (amplitude and phase). In particular, a fast oscillating feature of S⊥ (ω ) compared with that of S (ω ) indicates that the OA-FID carries more complicated positive/negative sign information in its phase and amplitude than the achiral FID. The VCD and VORD spectra obtained by using equation (2) are depicted in Fig. 2(b) and they are in good agreement with the results obtained by using a commercial VCD spectrometer. This proves that the present method properly works. In general, the VCD signal is exceedingly small in comparison to the background noise fluctuation so that typically it takes multiple hours to average out such fluctuating noise and to obtain a statistically meaningful VCD spectrum with the conventional VCD spectrometer. On the other hand, it is noteworthy that the dataacquisition time can be significantly reduced down to less than a minute by virtue of the dramatic enhanced sensitivity of the present active-heterodyne-detection method using femtosecond laser pulses.

Fig. 2. VCD and VORD spectra of (1S)-β-pinene measured by using the active-heterodyned detection method. (a) Experimentally measured heterodyned spectral interferograms, S ⊥ (ω ) (solid), S (ω ) (dashed). (b) Retrieved VCD (solid) and VORD (dashed) spectra from FTSI procedure.

Secondly, we have considered another interesting chiral system, cyanine dye aggregates bound to a double helical DNA. Cyanine dye molecule itself has no chiral center, but the DNA-templated cyanine dye complexes become chiral when bound to the minor groove of the helical DNA. As a result, the induced CD signal can be

finite in the visible frequency range. In Fig. 3, the electronic absorption and CD/ORD spectra of the DNAcyanine dye complexes, which were measured by using our self-heterodyne-detection method, are shown. A Davydov split CD pattern at around 600 nm is clearly observed and that indicates a head-to-tail excitonic coupling (J-type) between dye molecules assembled to DNA minor groove. The ORD spectrum shows a strong negative peak that is related to the split CD pattern through the Kramers-Kronig transformation.

Fig. 3. Electronic CD (middle) and ORD (bottom) spectra of DNA-cyanine dye aggregates measured by using the selfheterodyned detection technique. Top panel indicates UV visible absorption spectra of DNA-dye aggregates (solid) and dye monomer (dashed).

We have so far considered the steady-state vibrational and electronic CD/ORD experiments under equilibrium conditions. If the present OA-FID method is combined with a proper triggering method, one can observe the dynamical evolution of the system from a chiral perspective. As an example, let us consider a T-jump induced unfolding process of protein in water. Upon a T-jump excitation of OH or OD stretch overtones of normal or heavy water, the solvent bath temperature would be virtually instantaneously increased. Protein

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under thermally non-equilibrium state undergoes an unfolding process. A time-delayed probe pulse, which produces the OA-FID, is then used to monitor the subsequent structural relaxation in time. Currently, we are studying unfolding dynamics of myoglobin and DNA etc. with this T-jump-coupled vibrational or electronic OA measurement method.

Acknowledgement This work is supported by the KBSI grant (T31401).

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