The steady state and time-resolved fluorescence and infrared (IR) properties of 4- and 5-cyanotryptophan (CNTrp) are investigated and compared, and the tryptophan (Trp) analogs are found to be very attractive to study structural and dynamic properties of proteins. The position of the nitrile substitution as well as the solvent environment influences the spectroscopic properties (solvatochromism). Similar to native Trp, electronic (nanosecond) lifetime and emission spectra are modulated by the environment, making CNTrps attractive fluorescent probes to study the structural dynamics of proteins in complex media. The nitrile absorption in the IR region can provide local structural information as it responds sensitively to changes in electrostatics and hydrogen bond (HB) interactions. Importantly, we find that 4CNTrp exhibits a single absorption in the nitrile stretch region, while the model compound 4CN-indole (4CNI) shows two. Even though the spectrum of the model compound is perturbed by a Fermi resonance, we find that 4CNTrp itself is a useful IR label. Moreover, if the nitrile group is substituted at the 5 position, the Trp analog predominantly reports on its HB status. Because the current literature on similar compounds is too limited for a detailed solvatochromic analysis, we extend the available data significantly. Only now are microscopic details such as the mentioned sensitivity to electrostatics coming to light. The vibrational lifetime of the CN moiety (acting on a picosecond time scale in contrast to the nanosecond time scale for fluorescent emission) allows for its application in 2D-IR spectroscopy in the low picosecond range. Taken together, the benefits of CNTrps are that they absorb and emit separately from the naturally occurring Trp and that in these dual fluorescence/vibrational labels, observables of IR and fluorescence spectroscopy are modulated differently by their surroundings. Because IR absorption and fluorescence operate on different time and length scales, they thus provide complementary structural information.

In conventional two-dimensional infrared (2D-IR) spectroscopy, the inherently short vibrational lifetimes limit the time window to observe molecular dynamics typically to tens of picoseconds. The rather complex dynamics of organized molecular systems (e.g., glass formers, polymers, membranes, proteins), however, span a wide range of timescales from femto-to microseconds and beyond. Vibrationally Promoted Electronic Resonance (VIPER) 2D-IR negates the limitations of 2D-IR spectroscopy, for its signal decays with the electronic lifetime. Here, we present 2-Isopropylthioxanthone as the first VIPER 2D-IR probe to exploit intersystem crossing, thereby covering even the microsecond timescale. We achieved the required signal-tonoise ratio and resolution by introducing the Fouriertransform approach to the VIPER 2D-IR pulse sequence. Now, we are in a position to monitor dynamics via spectral diffusion several orders of magnitude beyond the vibrational lifetime of 2D-IR labels.

In conventional two‐dimensional infrared (2D‐IR) spectroscopy, the inherently short vibrational lifetimes limit the time window to observe molecular dynamics typically to tens of picoseconds. The rather complex dynamics of organized molecular systems (e.g., glass formers, polymers, membranes, proteins), however, span a wide range of timescales from femto‐ to microseconds and beyond. Vibrationally Promoted Electronic Resonance (VIPER) 2D‐IR negates the limitations of 2D‐IR spectroscopy, for its signal decays with the electronic lifetime. Here, we present 2‐Isopropylthioxanthone as the first VIPER 2D‐IR probe to exploit intersystem crossing, thereby covering even the microsecond timescale. We achieved the required signal‐to‐noise ratio and resolution by introducing the Fourier‐transform approach to the VIPER 2D‐IR pulse sequence. Now, we are in a position to monitor dynamics via spectral diffusion several orders of magnitude beyond the vibrational lifetime of 2D‐IR labels.

Following up on our previous work on vibrationally resolved electronic absorption spectra including the effect of vibrational pre-excitation [J. von Cosel et al., J. Chem. Phys. 147, 164116 (2017)], we present a combined theoretical and experimental study of two-photon induced vibronic transitions in polyatomic molecules that are probed in the Vibrationally Promoted Electronic Resonance experiment using two-photon excitation (2P-VIPER). In order to compute vibronic spectra, we employ time-independent and time-dependent methods based on the evaluation of Franck-Condon overlap integrals and Fourier transformation of time-domain correlation functions, respectively. The time-independent approach uses a generalized version of the FCclasses method, while the time-dependent approach relies on the analytical evaluation of Gaussian moments within the harmonic approximation including Duschinsky rotation effects. For the Coumarin 6 dye, two-dimensional 2P-VIPER experiments involving excitation to the lowest-lying singlet excited state (S1) are presented and compared with corresponding one-photon (1P)-VIPER spectra. In both cases, coumarin ring modes and a CO stretch mode show VIPER activity, albeit with different relative intensities. Selective pre-excitation of these modes leads to a pronounced redshift of the low-frequency edge of the electronic absorption spectrum, which is a prerequisite for the VIPER experiment. Theoretical analysis underscores the role of interference between Franck-Condon and Herzberg-Teller effects in the two-photon experiment, which is at the root of the observed intensity distribution.