Blue fluorescent amino acid for biological spectroscopy and microscopy Mary Rose Hilairea,1, Ismail A. Ahmedb,1, Chun-Wei Lina, Hyunil Joc, William F. DeGradoc,2, and Feng Gaia,d,2 aDepartment of Chemistry, University of Pennsylvania, Philadelphia, PA 19104; bDepartment of Biochemistry and Molecular Biophysics, University of Pennsylvania, Philadelphia, PA 19104; cDepartment of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158; and dUltrafast Optical Processes Laboratory, University of Pennsylvania, Philadelphia, PA 19104 Contributed by William F. DeGrado, April 20, 2017 (sent for review February 20, 2017; reviewed by Jacob W. Petrich and Dongping Zhong) Many fluorescent proteins are currently available for biological are potential candidates that meet the aforementioned require- spectroscopy and imaging measurements, allowing a wide range ments. However, the emission maxima of 6CN-Trp and 7CN-Trp of biochemical and biophysical processes and interactions to be in methanol are centered at 370 and 390 nm, respectively, studied at various length scales. However, in applications where a making them less attractive as visible fluorophores. As shown small fluorescence reporter is required or desirable, the choice of (Fig. 1), the absorption and emission spectra of 4-cyano-indole fluorophores is rather limited. As such, continued effort has been (4CNI), the sidechain of 4CN-Trp, are significantly shifted from devoted to the development of amino acid-based fluorophores those of indole (or Trp) and, in particular, an emission maximum that do not require a specific environment and additional time to of 405 nm suggests that 4CN-Trp could be a viable candidate for mature and have a large fluorescence quantum yield, long fluores- the aforementioned amino acid-based fluorophores. Indeed, our cence lifetime, good photostability, and an emission spectrum in results confirm that 4CN-Trp emits in the blue region of the the visible region. Herein, we show that a tryptophan analog, > 4-cyanotryptophan, which differs from tryptophan by only two atoms, visible spectrum with a large QY ( 0.8) and has a long fluo- is the smallest fluorescent amino acid that meets these requirements rescence lifetime (13.7 ns), a reasonably large molar extinction ∼ −1· −1 and has great potential to enable in vitro and in vivo spectroscopic and coefficient ( 6,000 M cm ), and good photostability. Taken together, these qualities make 4CN-Trp an attractive blue microscopic measurements of proteins. SCIENCES fluorescent amino acid (BFAA) for biological spectroscopy fluorescence protein | fluorescence probe | unnatural amino acid | imaging and microscopy. APPLIED BIOLOGICAL Results and Discussion f the amino acids that are inherently responsible for the Ofluorescence of proteins, tyrosine (Tyr), tryptophan (Trp), Fluorescence Quantum Yield Measurements. To explore the feasi- and phenylalanine (Phe), Trp is the most widely used fluores- bility of using 4CN-Trp as a biological fluorescence emitter, we cence reporter of protein structure, function, and dynamics, as its first examined the QY of its fluorophore, 4-cyano-indole (4CNI). B fluorescence quantum yield (QY) is comparatively large and is As shown (Fig. 1 ), the absorption spectrum of 4CNI in water is also sensitive to environment (1–3). However, Trp absorbs/emits broader than and red shifted from that of Trp, making it possible in the UV wavelength region and has low photostability. Com- to prepare its electronically excited and fluorescent state using λ bined, these factors render this naturally occurring fluorescent an excitation wavelength ( ex) of greater than 310 nm, without amino acid hardly useful as a fluorophore for single-molecule exciting any of the naturally occurring fluorescent amino acids. measurements (4) and imaging applications, especially under This constitutes one of the key advantages of using 4CNI-based in vivo conditions. For this reason, significant efforts have been amino acid fluorophores. Similarly, the fluorescence spectrum of devoted to identifying small unnatural fluorophores (5–11) that 4CNI in water is red shifted from that of indole (Fig. 1C), with a could overcome this limitation. So far, only naphthalene-based maximum emission wavelength of 405 nm. In addition, and fluorophores, such as prodan (8, 9) and aladan (11) have been perhaps more importantly, the integrated fluorescence intensity shown to be useful in this regard. However, these fluorophores of 4CNI is much larger than that of indole, indicating that 4CNI are not structurally based on any naturally occurring amino acids has a much larger fluorescence QY than indole. To determine and are larger in size than Trp, making them less attractive in the QY of 4CNI, we quantitatively compared its fluorescence applications where there are stringent requirements for the flu- orophore size and structure. Therefore, the development of a Significance smaller, ideally amino acid-based, fluorophore that does not require additional time to mature, have a large fluorescence QY, Herein, we report a tryptophan-based fluorophore that emits long fluorescence lifetime, good photostability, and an emission in the visible spectral region when excited with 355-nm light. spectrum in the visible range, would enhance biological research. Because this blue fluorescent amino acid has a small size and Herein, we show that a Trp analog, 4-cyanotryptophan (4CN- affords unique photophysical properties, we expect that it will Trp), meets these requirements and has great potential to ex- find use in various biological studies, especially in spectroscopic pand biological fluorescence spectroscopy and microscopy into and/or microscopic measurements where an amino acid-sized additional territory. fluorescence reporter is required. Many past studies have focused on Trp-based unnatural amino acids, including azatryptophans (5, 6, 12) and various indole-ring Author contributions: M.R.H. and F.G. designed research; M.R.H., I.A.A., C.-W.L., and H.J. substituted analogs (13–16), aiming to identify useful biological performed research; and M.R.H., W.F.D., and F.G. wrote the paper. fluorophores. Whereas some Trp analogs indeed exhibit im- Reviewers: J.W.P., Iowa State University; and D.Z., The Ohio State University. proved fluorescent properties over Trp, none of them has found The authors declare no conflict of interest. broad applications due to certain photophysical limitations. 1M.R.H. and I.A.A. contributed equally to this work. Recently, Talukder et al (15) showed that 6-cyanotryptophan 2To whom correspondence may be addressed. Email: [email protected] or gai@ (6CN-Trp) and 7-cyanotryptophan (7CN-Trp) have a signifi- sas.upenn.edu. cantly increased QY (about 0.5 in methanol) compared with that This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of Trp (about 0.15 in water), suggesting that cyanotryptophans 1073/pnas.1705586114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1705586114 PNAS Early Edition | 1of5 Downloaded by guest on September 29, 2021 and Methods, whereas the corresponding 4CN-Trp* peptide de- rivatives were prepared from the commercially available and relatively inexpensive 4CNI-3AA, which can be conveniently appended to the N-terminal end of a peptide (Fig. 1A). As shown (Fig. 1), the absorption and emission spectra of 4CN-Trp, 4CNI- 3AA, Gly-Gly-4CN-Trp, and 4CN-Trp*-Gly in water are very similar to each other, suggesting that 4CNI-3AA can be used as an alternative of 4CN-Trp. Furthermore, their absorption spectra extend further to the longer wavelengths in comparison with that of 4CNI, with a second maximum at 325 nm. These absorption characteristics are important, as they render the use of various commercially available lasers, such as the 355-nm YAG laser or the 325-nm HeCd laser commonly used inflowcytometry,toexcitethe 4CN-Trp fluorophore. As indicated (Fig. 1C), the fluorescence spectra of 4CN-Trp, 4CNI-3AA, Gly-Gly-4CN-Trp, and 4CN-Trp*-Gly have a peak wavelength of greater than 415 nm, making the 4CN-Trp chro- mophore a true blue fluorescence emitter. Perhaps more im- portantly, their fluorescence QYs in water at 25 °C are larger than 0.80 when an λex of 325 nm was used (Table 1), confirming that 4CN-Trp is a highly emissive BFAA. Further measurements on 4CN-Trp*-Gly in a 20 mM phosphate buffer (pH 7.2) at 37 °C and in tetrahydrofuran (THF), an aprotic solvent commonly Fig. 1. (A) Structures of 4CNI, 4CNI-3AA, 4CN-Trp, and 4CN-Trp*-Gly, as in- used to mimic the hydrophobic interior of proteins because of its dicated. (B) Absorption spectra of Trp, 4CNI, 4CNI-3AA, 4CN-Trp, 4CN-Trp*-Gly, low dielectric constant, show that the QY is only decreased to and Gly-Gly-4CN-Trp, as indicated. (C) Comparison of the fluorescence spec- about 0.6 (Table 1) under these condition. This is a key finding as trum of indole with those of 4CNI and 4CNI-3AA. These spectra were obtained it indicates that the strong fluorescence emission property 4CN- with an excitation wavelength of 270 nm at 25 °C and the optical densities of Trp is maintained under physiological conditions and also in a all of the samples at 270 nm were equal. (D) Comparison of the fluorescence dehydrated environment. It is well known that several amino spectrum of DPA with those of 4CNI, 4CNI-3AA, 4CN-Trp, and 4CN-Trp*-Gly. acids (19), including methionine (Met) (20), can quench Trp These spectra were obtained with an excitation wavelength of 325 nm at A 25 °C, and the optical densities of all of the samples at 325 nm were equal. fluorescence. However, as shown (Table 1 and Fig. S1 ), the QY of 4CN-Trp*-Met is similar to that of 4CN-Trp*-Gly. This result suggests that the fluorescence QY of 4CN-Trp may not be as spectrum with that of Trp or 9,10-diphenylanthracene (DPA) sensitive to nearby residues as Trp is. Taken together, the above (Fig. 1D) using the following relationship (17): results suggest that the brightness of a 4CNI fluorophore in a protein will not be significantly affected by its location and can I A n2 be attached to either a solvent-exposed or buried position.