Bisazide Cyanine Dyes as Fluorogenic Probes for ... - ACS Publications

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Bisazide Cyanine Dyes as Fluorogenic Probes for BisCyclooctynylated Peptide Tags and as Fluorogenic Cross-Linkers of Cyclooctynylated Proteins Orsolya Demeter,#,† Attila Kormos,#,† Christine Koehler,‡ Gábor Mező,§ Krisztina Németh,† Eszter Kozma,† Levente B. Takács,† Edward A. Lemke,‡ and Péter Kele*,†

Bioconjugate Chem. 2017.28:1552-1559. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 08/18/18. For personal use only.

†

“Lendület” Chemical Biology Research Group, Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2, H-1117, Budapest, Hungary ‡ Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117, Heidelberg, Germany § MTA-ELTE Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Pázmány Péter sétány 1a, H-1117, Budapest, Hungary S Supporting Information *

ABSTRACT: Herein we present the synthesis and fluorogenic characterization of a series of double-quenched bisazide cyanine probes with emission maxima between 565 and 580 nm that can participate in covalent, two-point binding bioorthogonal tagging schemes in combination with biscyclooctynylated peptides. Compared to other fluorogenic cyanines, these double-quenched systems showed remarkable fluorescence intensity increase upon formation of cyclic dye− peptide conjugates. Furthermore, we also demonstrated that these bisazides are useful fluorogenic cross-linking platforms that are able to form a covalent linkage between monocyclooctynylated proteins.

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background fluorescence of nonspecifically bound probes is readily reduced by applying fluorogenic (often called smart or turn-on) probes. The characteristic feature of fluorogenic probes is that they remain practically dark until they react with target structures. Since the unreacted forms of such probes still exist in their nonemissive form, minimal or practically zero background fluorescence originates from these species even when bound unspecifically.5−7 Among biomolecules, mainly proteins are subjected to fluorescent manipulation schemes. Genetic alteration by means of fusion proteins or genetic code expansion (GCE) allows specific and site-selective labeling of the POI. While fusion proteins, e.g., small enzyme tags, or self-labeling peptide tags8 (e.g., Cys4) can mostly be used for terminal modification of POIs, recent advances in GCE by means of amber suppression enable site-specific incorporation of noncanonical amino acids in selected proteins virtually at any location.9−14 The combination of GCE and bioorthogonal chemistry led to the emergence of versatile bioorthogonal labeling schemes that are valuable surrogates for fusion proteins. Among bioorthogonal reactions the most frequently used ones are

INTRODUCTION Site-specific labeling schemes have greatly facilitated the exploration of intra- and extracellular biological processes through fluorescent imaging of proteins, nucleic acids, lipids, and other biomolecules.1−4 Most techniques are based on the use of fusion proteins such as tailored fluorescent proteins (FPs), tags (e.g., Halo, SNAP, Cys4), and small engineered enzymes (e.g., lipoic acid ligase). Besides being restricted only to the fluorescent modifications of proteins (mostly at the termini), the photophysical properties of the existing FPs often have limitations. Moreover, in many cases fusion protein tags may alter the original function of the protein of interest (POI) due to their comparable size. Alternatively, fluorescent manipulation of a wider range of biomolecules is possible by means of biorthogonal chemistry in combination with small molecule labels. Besides enabling fluorescent labeling of nucleic acids, carbohydrates, lipids, and small metabolites, photophysical characteristics and chemical reactivities of such small synthetic dyes can be synthetically tailored.3 The greatest limitations of using small synthetic dye-based labeling schemes are associated with autofluorescence in the case of shortwavelength excitable dyes and background fluorescence due to nonspecific dye−biomolecule interactions. While autofluorescence is efficiently suppressed by dyes that are either excitable in the red regime of the spectrum or have large Stokes-shifts, © 2017 American Chemical Society

Received: March 31, 2017 Revised: April 20, 2017 Published: April 25, 2017 1552

DOI: 10.1021/acs.bioconjchem.7b00178 Bioconjugate Chem. 2017, 28, 1552−1559

Article

Bioconjugate Chemistry

octynylated peptide tags, high yielding expression of doubleamber suppressed proteins is more difficult than single amber suppression and not yet routine, but efforts into this direction have been made.33−35 However, another feature of bisazides is to create a unique platform as potential fluorogenic crosslinkers to covalently link monocyclooctynylated proteins. Such cross-linking can create static, covalently linked species useful in studying protein−protein interactions. Interesting findings along this line are known, for example, FlAsH-based crosslinkers to investigate protein−protein interactions 36 or oligomeric states37 and bipartite tetracysteine display.38−40 We aimed to further explore the covalent approach, demonstrating the cross-linking ability of bis-quenched fluorogenic probes as cross-linking moieties between genetically altered model proteins.

the strain-promoted azide−alkyne cycloaddition (SPAAC) between cyclooctynes and azides15 and the inverse electron demand Diels−Alder reactions of trans-cyclooctenes and tetrazines.16 There are many examples where strained alkyneor alkene-modified noncanonical amino acids (ncAAs) are genetically encoded into various proteins by means of orthogonal pyrrolysine (Pyl) tRNA/tRNA synthetase (RS) pair from Methanosarcina species.17−20 Lately, our group reported a series of azide substituted fluorogenic dyes that could be used for bioorthogonal labeling in the biological environment. The azide group acts as a bioorthogonal handle on one hand and as an efficient quencher of fluorescence through opening a nonradiative relaxation pathway on the other. Upon transformation of the azide to a triazole in a reaction with a cyclooctyne, the fluorescence reinstates.21,22 Just very recently, we introduced a double-quenched bisazide fluorogenic probe that can participate in covalent, two-point binding bioorthogonal tagging schemes in combination with biscyclooctynylated short peptide sequences.23 Although the bisazides presented therein were UV-excitable, thus not fully compatible with live-cell imaging schemes, demonstration of the concept highlighted that such biscyclooctynylated peptide tags can be good surrogates for Cys4 tags for several reasons: (i) they do not require a reducing environment to ensure the presence of free SH-groups, (ii) bioorthogonalized labels can be used instead of arsenic-based probes, and (iii) there is no competing reaction with nonspecific thiols (e.g., GSH). In order to get a better insight into the nature of such quenched bisazide probes and to develop new probes with photophysical characteristics more suitable for biological imaging, we set forth a study that addresses design, synthesis, and fluorogenic characterization of further bisazide fluorogenic dyes. We opted for the cyanine scaffold since the “CyDye” range of fluorophores has a rich history in terms of chemical synthesis and application in life sciences. Their remarkable photostability, large absorption cross sections, and compatibility with common lasers also made these dyes (especially indocarbocyanines Cy3 and Cy5) a prime choice for fluorescence applications.24 Due to the structural fact that common chromophore Cy3 can exist in many conformations, it is capable of undergoing numerous rotational and translational modes of vibration which causes the lowering of quantum yields. Waggonner et al. introduced rigidified cyanine Cy3B, a trimethyne cyanine modified with a rigid backbone that locks the molecule into a fixed conformation, thus increasing the fluorescent output upon excitation.25,26 Cy3B has been used, e.g., routinely in fluorescence polarization assays, as monomers in solid-phase oligonucleotide synthesis,27 or as fluorescent derivative of serotonine.28 Notwithstanding these remarkable advantages, bioorthogonally applicable fluorogenic cyanines, with the exception of one biarsenical probe (AsCy3),29 do not exist. Indeed, in the case of AsCy3, the biarsenical moiety is built on a Cy3 scaffold and the AsCy3-Cys4-tag combinations proved to be valuable tools in super resolution fluorescence imaging,30 in diatom-based biosensors31 or dithiol oxidation in bacteria.32 The present study has therefore been undertaken to investigate the synthesis and fluorogenic features of bisazide monomethine, trimethine, and rigidified trimethine cyanine probes in combination with bis-cyclooctynylated peptide sequences. While we envision our bisazides also as surrogates of biarsenical probes in combination with double cyclo-

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RESULTS AND DISCUSSION A. Synthesis of Bisazide Probes. In this study we have chosen monomethine cyanine (Cy1, Figure 1, 1) and

Figure 1. Cyanine bisazide dyes (1−5) involved in this study.

trimethine cyanine (Cy3, Figure 1, 2−5) dyes consisting of azido-benzothiazol and azido-indole units. We have designed frameworks involving two benzothiazoles or two indoles and mixed indole-benzothiazole cyanines. The rationale behind the involvement of a Cy3B indole framework (Figure 1, 3) was the aforementioned spectral advantage granted by the presence of a rigid polymethine linker. Synthesis of bisazide cyanines 1 and 2 started with the nitration of 2-methylbenzo[d]thiazol (Scheme 1, 6). The resulting 2-methyl-6-nitrobenzo[d]thiazol (7) was turned into the corresponding quaternary iodide 8 with ethyl iodide. Reaction of 8 with half equivalent of N-nitrosodiphenylamine gave dinitro substituted cyanine-1 (9), while treatment of 8 with triethyl othoformate furnished cyanine-3 derivative, 10, in medium yields. Dinitro compounds 9 and 10 were then reduced to obtain the corresponding diamines (11 and 12) in good yields. Subsequent diazotization of 11 and 12 and treatment with sodium azide gave target bisazides 1 and 2 in medium yields (Scheme 1). To prepare 3 the synthesis started with the nitration of 13 (Scheme 2), followed by the reduction of the resulting nitro compound to the corresponding amine 15 that was subsequently protected by acetylation with Ac2O. Acetamide 16 was N-alkylated with 2-(2-iodoethyl)-1,3-dioxolane to furnish 17 in medium to good yields. Iodoethyl-dioxolane was prepared by the exact reproduction of the process published by Hauck.41 Compound 17 was treated with 1553


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Bioconjugate Chemistry Scheme 1. Synthesis of Bisazido Cyanines 1 and 2a

(a) HNO3, H2SO4, 0 °C, 2 h, 94%; (b) EtI, 130 0 °C, 67%; (c) N-nitrosodiphenylamine, Ac2O, Δ, 15 min, 68%; (d) (EtO)3CH, Ac2O, Δ, 15 min 77%; (e) SnCl2·2H2O, ccHCl, rt, 4 h, 75% for n = 0 90% for n = 1; ( f) NaNO2, HCl, 0 °C, 10 min, then NaN3, 0 °C → rt, 6 h, 48% for n = 0 58% for n = 1.

a

Scheme 2. Synthesis of Cyanine 3a

(a) KNO3, H2SO4, 1 h, 0 °C, 92%; (b) SnCl2·2H2O, ccHCl, 1.5 h, Δ, 88%; (c) Ac2O, MeCN, rt, 10 min, 92%; (d) 2-(2-iodoethyl)-1,3-dioxolane, MeCN, 100 °C, 48 h, N2, 33−80%; (e) (EtO)3CH, EtOH, Δ, 24 h, N2, 54%; (f) DCM/H2SO4, rt, 30 min, 75−80%; (g) BF3·OEt2, MeOH, 50 °C, 2 h, N2, 60%; (h) NaNO2, HCl, 0 °C, 30 min, then NaN3, 0 °C → rt, 1 h, 55%. a

triethylorthoformate to result in cyanine 18. Treatment of 18 with sulfuric acid resulted in intramolecular cyclization to give 19 in 80% yield. Intermediate 19 was reacted with BF3·Et2O to remove acetyl protecting groups,42 deprotection was followed by a diazotization−azide substitution sequence to access 3 in medium yield (Scheme 2). During the preparation of the aromatic acetamide cores (23 and 24) of nonsymmetrical dye 4 (Scheme 3) we followed the nitration-reduction-acetylation-alkylation sequence-based syn-

thetic strategy with good yields. Compound 24 was reacted with N,N-diphenylformamidine to yield hemicyanine 25. The combination of acetamide 25 with 23 and triethyl-orthoformate resulted in cyanine 26. After the removal of amino-protecting groups with BF3·OEt2, diamine 27 was converted into target dye 4 (Scheme 3). Synthesis of 5 started from acetamido derivative 24 (Scheme 4) that was treated with triethyl-orthoformate to yield 28. Subsequent removal of the acetyl groups with BF3·OEt2 gave 1554


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Bioconjugate Chemistry Scheme 3. Synthesis of Bisazide Dye 4a

a (a) SnCl2, HCl, 1.5 h, Δ, 80%; (b) Ac2O, MeCN, rt, 10 min, 96%; (c) EtI, 120 °C, 12 h, 59%; (d) EtI, MeCN, Δ, 24 h, 84%; (e) N,N-diphenylformamidine, (EtO)3CH, EtOH, Δ, 24 h, 82%; (f) compound 23, Ac2O, pyridine, Δ, 2 h, 67%; (g) BF3·OEt2, MeOH, 50 °C, 12 h, N2, 78%; (h) NaNO2, HCl, 0 °C, 30 min, then NaN3, 0 °C → rt, 1 h, 56%.

Scheme 4. Synthesis of Bisazide Dye 5a

(a) HC(OEt)3, pyridine, 12 h, Δ, 62%; (b) BF3·OEt2, MeOH, 50 °C, 12 h, N2, 86%; (c) NaNO2, HCl, 0 °C, 30 min, then NaN3, 0 °C → rt, 1 h, 64%.

a

Figure 2. Increase of emission of bisazide dye 1 (left) and dyes 2−5 (right) when reacted with 2 equiv of BCN (in PBS pH = 7.4, c = 0.625 μM, at room temperature).

Table 1. Photophysical Properties and Fluorescence Enhancement Caused by Double-Click Reactions with BCN of Dyes 1−5a

29 in good yield. Diazotization of 31 and treatment with sodium azide allowed target compound 5 (Scheme 4). B. Fluorescence and Fluorogenicity Studies. With the target compounds in hand, we have first established the main photophysical properties and fluorogenicity of the new probes upon strain-promoted click reaction with a commercially available cyclooctyne (bicyclo[6.1.0]non-4-yn-9-yl-methanol, BCN). Compound 1 showed very weak emission at around 490 nm when excited at 410 nm. Cy3 congeners 2−5 on the other hand had more intense intrinsic fluorescence in the biologically more advantageous yellow−orange regime (i.e., 565−580 nm) upon excitation at 550−560 nm (Figure 2, Table 1). The more intense fluorescence of the latter ones suggests that the azide-mediated quenching works less efficiently with extended conjugation. The most intense fluorescence was measured, as expected, with rigid Cy3B-bisazide 3. Relative

bisazide dye

IBCN/Ibisazideb

λexc [nm]

λem [nm]

Φbisazide

ΦBCNc

1 2 3 4 5

14 7 29 22 10

410 560 560 550 555

487 575 571 565 569

0.00065 0.0195 0.065 0.011 0.0125

0.0073 0.057 0.73 0.13 0.082

In PBS pH = 7.4, c = 0.625 μM, at room temperature. bCalculated at the emission maxima of the products. cQuantum yields relative to Coumarin 153 (in the case of dye 1) or to Rhodamine B (dyes 2−5). a

fluorescence quantum yields of dyes 1−5 and of bis-clicked products with BCN were also determined (Table 1). 1555


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Bioconjugate Chemistry During the synthetic procedures, we have observed that in protic solutions all of the bisazides prepared are sensitive to indoor lighting, not surprisingly, since the tendency of aromatic azides to decompose upon thermal or photolytic conditions is a well-known phenomenon.43,44 In our case, the fact that our dyes contain two azide groups added an extra layer of complexity to the possible decomposition pathways and we were unable to identify them; however, none of the products were fluorescent. Further exploration of this interesting feature revealed that exposure of aqueous solutions of bisazides 1−5 with a common household compact fluorescent lamp (CFL, 20W, 800 lm) completely diminished fluorescence within 5 min. We were thus curious whether click-reaction products of 1−5 with BCN possesses this photosensitivity. To this end we have allowed bisazides 1−5 to react with 2 equiv of BCN for 2 h upon which time the fluorescence intensities reached their maxima. Following this we have exposed the solutions to light for 5 min. To our delight minimal or no decrease of fluorescence was observed in each case indicating that the click products are photostable. We concluded that our bisazide dyes were therefore indeed fluorogenic and that, if needed, unreacted bisazides could be photobleached leaving only the highly fluorescent bis-clicked products behind. Next, we aimed at testing fluorogenicity of the probes with short peptide tags bearing two cyclooctyne (BCN) units. In our preceding account23 we identified a hexapeptide sequence, AcK(BCN)AEAAK(BCN)-NH2 (Peptide-1) that formed a fluorescent, cyclic bis-clicked product with a bisazide fluorogenic dye. Thus, we first chose compound 1 and allowed it to react with Peptide-1. Within 2 h the reaction went to completion and no further increase of the fluorescence intensity was observed. The fluorescence enhancement was 13-fold compared to the fluorescence intensity of 1 (Table 2, Figure 3).

Figure 3. Fluorescence spectra of bisazide dye 1 and monoazide dye 37, and fluorescence enhancement of dye 1 when reacted with biscyclooctynylated hexapeptide, Peptide-1.

solution, we synthesized the monoazide-monotriazole derivative 37 from separately prepared triazole 32 (Scheme 5). Comparison of the fluorescence spectra of 1 and 37 showed that the fluorescence of the Cy1 framework was also quenched in monoazide 37, however to a less extent. Compound 37 had ca. 2 times higher fluorescence intensity than bisazide 1 (Figure 3). Allowing 37 to react with 1 equiv BCN resulted in a 6-fold fluorescence enhancement (Figure 3). This observation supports that in the reaction of 1 with Peptide-1 a bis-clicked (cyclic) product was formed. We also tested dyes 2−5 with Peptide-1; however, with the exception of 2, which showed 3.5fold increase with respect to the fluorescence of the bisazide, none of them gave appreciable fluorescence increase. We assumed that Peptide-1 was too short to react efficiently with Cy3 derivatives 2−5, a hypothesis that was supported by the fact that no m/z value corresponding to the cyclic products was detected during MS measurements. Starting from the excellent comparative work by Schepartz et al.45 we designed Peptide-2 with sequence Ac-K(BCN)AEAADAEAAK(BCN)-NH2 and reacted it with dyes 2−5 until no further increase of fluorescence intensity was observed (typically 2 h). Satisfyingly all dyes reacted with Peptide-2 and gave 12-, 28-, 39-, and 11-fold increase relative to the original fluorescence of 2−5, respectively (Figure 4 and Table 2, calculated at the emission maxima of the bis-clicked products). LC-MS measurements showed single products with m/z values corresponding to the cyclic peptide−dye conjugates. To the best of our knowledge no cyanine-type dye in this emission regime possesses such high fluorescence enhancements. We also investigated the stability of these bisazide probes in the presence of various thiols as possible reducing agents under physiological conditions. Azide-quenched dyes are often prone to thiol mediated reduction that can lead to fluorescent products. Thus, the fluorescence intensity is a good indicator of any thiol-driven side reactions. We found that the weak fluorescence intensity of the bisazides did not change substantially in reducing media such as excessive amounts of ethanethiol, cysteine, or glutathione especially in comparison to the intensive signal evolution upon SPAAC reaction with strained alkyne reaction partners. C. Cross-Linking of BCN-Tagged Proteins. To further demonstrate the potential of our bisazides, we thought to explore cross-linking of site-specifically modified proteins. To this end, we first expressed GFP-6His harboring a single TAG (Amber mutation) at position 39 (GFPY39→TAG) in the presence of a pyrrolysine (Pyl) RSAF/tRNAPyl pair from Methanosarcina mazei (PylRS with mutationsY306F Y384F)

Table 2. Photophysical Properties and Fluorescence Enhancement Caused by Double-Click Reactions with Biscyclooctynylated Peptidesa bisazide dye d

1 1e 2e 3e 4e 5e

Ibis‑clicked/Ibisazideb

λexc [nm]

λem [nm]

Φbis‑clickedc

13 34 12 28 39 11

410 410 560 560 550 555

487 487 577 573 565 569

0.0065 0.018 0.16 0.85 0.21 0.14

In PBS pH = 7.4, c = 0.625 μM, at room temperature, after 2 h of stirring in the dark. bCalculated at the emission maxima of the products. cQuantum yields relative to Coumarin 153 (in case of dye 1) or to Rhodamine B (dyes 2−5). dWith Peptide-1. eWith Peptide-2. a

LC-MS analyses of the reaction mixture of 1 with AcK(BCN)AEAAK(BCN)-NH2 indicated the formation of a sole product with m/z of 716 ([M + H]2+) ruling out the formation of products with a stoichiometry other than 1:1. In order to verify that indeed a bis-clicked product was formed we intended to compare fluorescence intensities of 1 with its mono- and bis-clicked products. It was, however, impossible to obtain a monotriazolic product even by adding only half an equivalent of BCN to 1. This was in line with our earlier observations, i.e., the monoclicked product of 1 was more reactive than the starting bisazide23 and the formation of a cyclic conjugate was exceedingly favored. As an alternate 1556


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Bioconjugate Chemistry Scheme 5. Synthesis of Mono-Clicked Cyanine-1 Dye (6)a

a

Conditions: Et3N, EtOH, rt, reflux, 2 h. See SI for full synthesis of intermediates 32 and 36.

Figure 4. Comparative fluorescence enhancements of dyes 2−5 when reacted with bis-cyclooctynylated Peptide-2.

Figure 5. Fluorescent (left) and corresponding Coomassie stained SDS-PAGE (right) of GFPY39→BCN, the mix (1:1 mixture of GFPY39→BCN and GFPWT) and GFPWT cross-linking reactions with compounds 2, 3, and 5. TAMRA-azide (A) was used as control. The first lane (−) for each sample shows the protein without dye. The black arrow indicates the GFP dimer on the gel.

in E. coli.19 In the presence of commercially available BCN-Lys in the growth medium, this leads to site-specifically incorporated BCN (GFPY39→BCN). The protein was purified via N-terminal 6His-tag using Immobilized Metal Affinity Chromatography. Second, the purified protein was labeled with 2-fold excess of dyes 2, 3, and 5. Dyes 1 and 4 were excluded from this experiment due to the high tendency for nonspecific sticking to proteins and insufficient solubility. In each case an intensely fluorescent band appeared in SDS PAGE gel analysis at a size double of GFP, whereas control experiment with TAMRA-azide showed only the formation of the monomeric labeled species (Figure 5). Interestingly, in the case of dyes 2

and 3 the main product was the monolabeled GFP as suggested by the fluorescence intensities at the detection channel of the dyes. In the case of dye 5, however, mainly the cross-linked GFP was formed and the monolabeled species was only faintly fluorescent. This suggests that the bisazides, in particular, dye 5 can indeed be used as fluorogenic cross-linkers. In the case of wild-type GFP none of the samples showed fluorescence. A mixture (mix) of GFPY39→BCN and GFPWT protein (1:1) was also examined, resulting in half of the intensity of the cross-linked band compared to the intensities observed for GFPY39→BCN alone. 1557


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Bioconjugate Chemistry

(6) Shieh, P., Dien, V., Beahm, B., Castellano, J., Wyss-Coray, T., and Bertozzi, C. (2015) CalFluors: A Universal Motif for Fluorogenic Azide Probes across the Visible Spectrum. J. Am. Chem. Soc. 137, 7145−7151. (7) Cserép, G. B., Baranyai, Z., Komáromy, D., Horváti, K., Bő sze, Sz. B., and Kele, P. (2014) Fluorogenic tagging of peptides via Cys residues using thiol-specific vinyl sulfone affinity tags. Tetrahedron 70, 5961−5965. (8) Pomorski, A., and Krężel, A. (2011) Exploration of Biarsenical Chemistry-Challenges in Protein Research. ChemBioChem 12, 1152− 1167. (9) Liu, C., and Schultz, P. (2010) Adding New Chemistries to the Genetic Code. Annu. Rev. Biochem. 79, 413−444. (10) Elliott, T. S., Bianco, A., and Chin, J. W. (2014) Genetic code expansion and bioorthogonal labeling enables cell specific proteomics in an animal. Curr. Opin. Chem. Biol. 21, 154−160. (11) Nikić, I., Plass, T., Schraidt, O., Szymański, J., Briggs, J. A., Schultz, C., and Lemke, E. A. (2014) Minimal Tags for Rapid Dual Color Live Cell Labeling and Super Resolution Microscopy. Angew. Chem., Int. Ed. 53, 2245−2249. (12) Lang, K., and Chin, J. W. (2014) Cellular Incorporation of Unnatural Amino Acids and Bioorthogonal Labeling of Proteins. Chem. Rev. 114, 4764−4806. (13) Kozma, E., Nikić, I., Varga, B., Aramburu, I., Kang, J., Fackler, O., Lemke, E., and Kele, P. (2016) Hydrophilic Trans-Cyclooctenylated Noncanonical Amino Acids For Fast Intracellular Protein Labeling. ChemBioChem 17, 1518−1524. (14) Plass, T., Nikić, I., Aramburu, I. V., Koehler, C., Gillandt, H., Lemke, E., and Schultz, C. (2015) Highly Stable trans-Cyclooctene Amino Acids for Live-Cell Labeling. Chem. - Eur. J. 21, 12266−12270. (15) Dommerholt, J., Rutjes, F., and van Delft, F. (2016) StrainPromoted 1,3-Dipolar Cycloaddition of Cycloalkynes and Organic Azides. Top. Curr. Chem. 374, 16. (16) Kozma, E., Demeter, O., and Kele, P. (2017) Bio-Orthogonal Fluorescent Labeling Of Biopolymers Through Inverse-ElectronDemand Diels-Alder Reactions. ChemBioChem 18, 486. (17) Borrmann, A., Milles, S., Plass, T., Dommerholt, J., Verkade, J. M., Wiessler, M., Schultz, C., van Hest, J. C., van Delft, F. L., and Lemke, E. A. (2012) Genetic Encoding of a Bicyclo[6.1.0]nonyneCharged Amino Acid Enables Fast Cellular Protein Imaging by MetalFree Ligation. ChemBioChem 13, 2094−2099. (18) Plass, T., Milles, S., Koehler, C., Schultz, C., and Lemke, E. A. (2011) Genetically encoded copper-free click chemistry. Angew. Chem., Int. Ed. 50, 3878−3881. (19) Plass, T., Milles, S., Koehler, C., Szymanski, J., Mueller, R., Wiessler, M., Schultz, C., and Lemke, E. A. (2012) Amino acids for Diels-Alder reactions in living cells. Angew. Chem., Int. Ed. 51, 4166− 4170. (20) Lang, K., Davis, L., Wallace, S., Mahesh, M., Cox, D. J., Blackman, M. L., Fox, J. M., and Chin, J. W. (2012) Genetic Encoding of Bicyclononynes and trans-Cyclooctenes for Site-Specific Protein Labeling in Vitro and in Live Mammalian Cells via Rapid Fluorogenic Diels−Alder Reactions. J. Am. Chem. Soc. 134, 10317−10320. (21) Herner, A., Estrada Girona, G., Nikić, I., Kállay, M., Lemke, E. A., and Kele, P. (2014) New Generation Of Bioorthogonally Applicable Fluorogenic Dyes With Visible Excitations And Large Stokes Shifts. Bioconjugate Chem. 25, 1370−1374. (22) Herner, A., Nikić, I., Kállay, M., Lemke, E. A., and Kele, P. (2013) A New Family Of Bioorthogonally Applicable Fluorogenic Labels. Org. Biomol. Chem. 11, 3297−3306. (23) Demeter, O., Fodor, E., Kállay, M., Mező, G., Németh, K., Szabó, P., and Kele, P. (2016) A Double-Clicking Bis-Azide Fluorogenic Dye For Bioorthogonal Self-Labeling Peptide Tags. Chem. - Eur. J. 22, 6382−6388. (24) Levitus, M., and Ranjit, S. (2011) Cyanine dyes in biophysical research: the photophysics of polymethine fluorescent dyes in biomolecular environments. Q. Rev. Biophys. 44, 123−151. (25) Waggoner, A. S., and Mujumdar, R. (2001) Rigidized Trimethine Cyanine Dyes, U.S. Patent 6133445A.

In conclusion, we have synthesized a series of new, doublequenched bisazide fluorogenic cyanine probes with emission maxima between 565 and 580 nm that can participate in covalent, two-point binding bioorthogonal tagging schemes in combination with bis-cyclooctynylated peptides. The probes exhibited good fluorescence enhancements upon formation of cyclic dye−peptide conjugates. To the best of our knowledge, these enhancement values are outstanding within fluorogenic cyanine dyes. We have also demonstrated that these bisazides are useful fluorogenic cross-linking platforms that are able to covalently attach monocyclooctynylated GFPs. Such fluorogenic, bifunctional species could be used to follow protein− protein interactions using single amino acid mutated, thus minimally perturbed engineered proteins. We believe that these probes can be used in highly specific and fluorogenic, two-point binding labeling schemes with proteins bearing two genetically encoded cyclooctyne motifs in the future. This work is currently being investigated in our laboratories and results will be reported in due course.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.7b00178. Details of synthetic procedures, characterization data for small molecules, peptide and protein labeling experiments (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Attila Kormos: 0000-0002-4378-4699 Péter Kele: 0000-0001-7169-5338 Author Contributions #

The first two authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The present work was supported by the Hungarian Scientific Research Fund (OTKA, NN-116265) and the “Lendület” Program of the Hungarian Academy of Sciences (LP2013-55/ 2013). E.A.L. acknowledges funding from the SPP1623.

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REFERENCES

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