Cyano-tryptophans as dual infrared and fluorescence spectroscopic labels to assess structural dynamics in proteins

Abstract

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.

4-Cyanoindole-2′-deoxyribonucleoside as a Dual Fluorescence and Infrared Probe of DNA Structure and Dynamics

“…To corroborate this notion, we examine the dependence of the C≡N stretching vibrational frequency (ν CN ) of 4CNI-NS on a solvent. Our results show that ν CN is sensitive to a solvent, similar to that observed for 4-cyanotryptohan (4CN-Trp) [13]. Therefore, this finding supports the idea that 4CNI-NS can be utilized to provide information about the local hydration and electrostatic environment of DNA in a site-specific manner.…”

“…More recently, Passow and Harki [10] have shown that 4CNI-NS can also be used as a universal DNA base. As demonstrated in previous studies [11,12,13], in comparison to indole and other indole derivatives, 4-cyanoindole (4CNI) (Figure 1) is significantly more fluorescent, has a long fluorescence lifetime, a red-shifted absorption spectrum, and an emission spectrum in the blue region of the visible spectrum. These qualities give 4CNI-NS a distinct advantage over 5NI-NS as it can serve as a fluorescence reporter.…”

Structures, dipole moments and excited state lifetime of isolated 4-cyanoindole in its ground and lowest electronically excited singlet states

“…A special class of indole derivatives, the cyanotryptophans are used as dual infrared and fluorescence spectroscopic labels to assess structural dynamics in proteins. 14 Markiewicz et al have shown that 5-cyanotryptophan fluorescence can be utilized as a sensitive probe of protein hydration. 15 The fluorescence lifetime of the different cyanoindoles in solution has been found to depend considerably on the solvent.…”

Synthesis and application of the blue fluorescent amino acid l-4-cyanotryptophan to assess peptide–membrane interactions

“…Indeed, several methods for syntheses of 4CN-Trp or its derivatives have been reported in the literature (Figure 1, Table S1, ESI †). 13,[17][18][19][20][21] For example, Bartoccini et al 20 described the synthesis of N-acetyl-4CN-Trp methyl ester from 4-pinacol boronated ester 1. However, the product was a racemic mixture and the yield was low.…”

“…Recently, another racemic synthesis of N-acetyl-4CN-Trp from 4-cyanoindole 2 was reported. 21 Since the experimental details were not described in that study, we attempted to reproduce its result by implementation of similar reaction conditions [22][23][24] known for tryptophan analogues but the desired product was obtained in very low yield in our hands. In the study of Hilaire et al, a short synthetic route to produce 4CN-Trp methyl ester from 4-bromo-L-tryptophan 3 in optically pure form was described.…”