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

Abstract

Facile chemical synthesis of l-4CN-Trp and incorporation into a pHLIP peptide enabled FRET study on peptide–membrane interactions.

PET and FRET utility of an amino acid pair: tryptophan and 4-cyanotryptophan

“…The following considerations motivate this work: (1) spectroscopic techniques based on FRET and PET are widely used to assess protein conformations, conformational dynamics, and interactions; (2) evidence from several recent studies 10,11 indicate that using dye-based fluorophores in FRET and PET applications can yield skewed results, due to specific or preferred probe-probe interactions. Therefore, development of amino acid-based FRET and PET pairs that are intrinsically less-perturbative and hence can minimize such pitfalls is needed; (3) the absorption spectrum of 4CN-Trp is significantly red-shifted from that of Trp, allowing selective excitation of its fluorescence (e.g., using λ ex = 330−360 nm) or that of Trp (e.g., using λ ex = 270 nm) in PET or FRET applications; (4) 4CN-Trp is only one atom larger than Trp, making it less perturbative to proteins than fluorescent dyes; (5) 4CN-Trp can now be conveniently synthesized via chemical 28 and bilogical 29 means; and (6) it is possible to incorporate 4CN-Trp into proteins genetically via amber codon suppression or chemically using a post-translational modification method. 30,31 Given the unique photophysical property 13,16,32 of 4CN-Trp and the less-perturbating nature of this unnatural amino acid and Trp, we believe that this pair will find valuable applications in biochemistry and biophysics, especially in cases where using dye-based PET or FRET reporters is deemed inappropriate.…”

4‐Aminophthalimide Amino Acids as Small and Environment‐Sensitive Fluorescent Probes for Transmembrane Peptides

“…Unlike tryptophan, which needs to be excited in the UV‐B range and shows only weak fluorescence, however, 1 can be excited at the edge of the visible light range (outside the biopolymer absorbance range) and shows bright fluorescence through a large apparent Stokes shift. Also, the more recently published 4‐cyanotryptophan meets these criteria only as a FRET donor Compound 2 is based on preparation through Cu I ‐catalyzed alkyne–azide cycloaddition.…”

Fluorescent amino acids as versatile building blocks for chemical biology

“…A key step in the synthesis of 4-cyanotryptophan is the palladium (Pd)-catalysed incorporation of the cyano group at position 4 of L-tryptophan. Remarkably, the emission maximum of 4-cyanotryptophan is in the blue visible region (λem = 405 nm) and it has a high quantum yield (~80%), good photostability and a long fluorescence lifetime (~13.7 ns), which makes it an interesting building block for spectroscopic and microscopic measurements of proteins, such as the study of peptide-membrane interactions 31 . The structural similarity of cyanotryptophans to tryptophan also makes them an attractive platform to explore enzymebased reactions, facilitating the preparation of enantiopure tryptophan analogues by derivatization at multiple aromatic positions, using, for example, tryptophan synthase S (TrpS) 32 or variants of its β-subunit (TrpB) 33 .…”