Bisazide Cyanine Dyes as Fluorogenic Probes for Bis

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BISAZIDE CYANINE DYES AS FLUOROGENIC PROBES FOR BISCYCLOOCTYNYLATED PEPTIDE TAGS AND AS FLUOROGENIC CROSSLINKERS OF CYCLOOCTYNYLATED PROTEINS Orsolya Demeter, Attila Kormos, Christine Koehler, Gábor Mezõ, Krisztina Németh, Eszter Kozma, Levente Takacs, Edward A. Lemke, and Peter Kele Bioconjugate Chem., Just Accepted Manuscript • Publication Date (Web): 25 Apr 2017 Downloaded from http://pubs.acs.org on April 26, 2017

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Bioconjugate Chemistry

Bisazide Cyanine Dyes as Fluorogenic Probes for Bis-cyclooctynylated Peptide Tags and as Fluorogenic Crosslinkers of Cyclooctynylated Proteins

Orsolya Demeter,#[a] Attila Kormos,#[a] Christine Koehler,[b] Gábor Mező,[c] Krisztina Németh,[a] Eszter Kozma,[a] Levente B. Takács,[a] Edward A. Lemke,[b] Péter Kele*[a]

[a] “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 [b] Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, D-69117, Heidelberg, Meyerhofstrasse 1, Germany; [c] MTA-ELTE Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Pázmány Péter sétány 1a, H-1117, Budapest, Hungary #

equally contributing authors

CONTACT Péter Kele: [email protected]

ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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


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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 biomolecules1–4. 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 tailored3. The greatest limitations of using small synthetic dye-based labeling schemes are associated with autofluorescence in case of short-wavelength excitable dyes and background fluorescence due to non-specific 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, background fluorescence of non-specifically 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 unspecifically5–7. Amongst 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 non-canonical amino acids in selected proteins virtually at any location9–14. The combination of GCE and bioorthogonal chemistry led to the emergence of versatile bioorthogonal labeling schemes that are valuable surrogates for fusion proteins. Amongst bioorthogonal reactions the most frequently used ones are the strain-promoted azide-alkyne cycloaddition (SPAAC) between cyclooctynes and azides15 and the inverse electron demand Diels–Alder reactions of trans-cyclooctenes and tetrazines16. There are many examples where strained alkyne- or alkene-modified non-canonical amino acids (ncAAs) are genetically encoded into various proteins by means of orthogonal pyrrolysine (Pyl) tRNA/tRNA synthetase (RS) pair from Methanosarcina species17–20. Lately, our group reported a series of azide substituted fluorogenic dyes that could be used for bioorthogonal labeling in biological environment. The azide group acts as a bioorthogonal handle on the one hand and as an efficient quencher of fluorescence through opening a non-radiative relaxation pathway on the other. Upon transformation of the azide to a triazole in a reaction with a cyclooctyne the fluorescence reinstates21–22. 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 sequences23. 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 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 non-specific thiols (e.g. GSH).


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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 aims the 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, compatibility with common lasers also made these dyes (especially indocarbocyanines Cy3 and Cy5) a prime choice for fluorescence applications24. 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 excitation25–26. Cy3B has been used e.g. routinely in fluorescence polarization assays, as monomers in solid-phase oligonucleotide synthesis27 or as fluorescent derivative of serotonine28. 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 imaging30, in diatom-based biosensors31 or dithiol oxidation in bacteria32. 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 cyclooctynylated peptide tags, high yielding expression of double-amber suppressed proteins is more difficult than



single amber suppression and not yet routine, but efforts into this direction have been made33– 35

. However, another feature of bisazides is to create a unique platform as potential

fluorogenic crosslinkers to covalently link mono-cyclooctynylated proteins. Such crosslinking 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 interactions36 or oligomeric states37 and bipartite tetracysteine display38–40. We aimed to further explore the covalent approach, demonstrating the crosslinking ability of bis-quenched fluorogenic probes as crosslinking moieties between genetically altered model proteins.

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


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

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 Nnitrosodiphenylamine 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).



Scheme 1. Synthesis of bisazido cyanines 1 and 2 (a) HNO3, H2SO4, 0 °C, 2 h, 94 % (b) EtI, 130 0 °C, 67 % (c) N-nitrosodiphenylamine, Ac2O, ∆, 15 min, 68%; (d) (EtO)3CH, Ac2O, ∆, 15 min 77%; (e) SnCl2x2H2O, ccHCl, rt, 4h, 75% for n=0 90 % for n=1; (f) NaNO2, HCl, 0 °C, 10 min, then NaN3, 0 °C → rt, 6h, 48 % for n=0 58 % for n=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-(2iodoethyl)-1, 3-dioxolane to furnish 17 in medium to good yields. Iodoethyl-dioxolane was prepared by the exact reproduction of the process published by Hauck41. Compound 17 was treated with 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 groups42, deprotection was followed by a x

diazotization-azide substitution sequence to access 3 in medium yield (Scheme 2).


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Scheme 2. Synthesis of cyanine 3 (a) KNO3, H2SO4, 1h, 0 ⁰C, 92 % (b) SnCl2x2H2O, ccHCl, 1.5 h, ∆, 88 % (c) Ac2O, MeCN, rt, 10 min, 92 % (d) 2-(2-Iodoethyl)-1,3-dioxolane, MeCN, 100 ⁰C, 48 h, N2, 33-80 %; (e) (EtO)3CH, EtOH, ∆, 24 h, N2, 54 % (f) DCM/H2SO4, rt, 30 min, 75-80 %; (g) BF3*OEt2, MeOH, 50 ⁰C, 2 h, N2, 60 %; (h) NaNO2, HCl, 0°C, 30 min, then NaN3, 0°C → rt, 1h, 55 %.

During the preparation of the aromatic acetamide cores (23 and 24) of non-symmetrical dye 4 (Scheme 3) we followed the nitration-reduction-acetylation-alkylation sequence-based synthetic

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).



Scheme 3. Synthesis of bisazide dye 4 (a) SnCl2, HCl, 1.5 h, ∆, 80 % (b) Ac2O, MeCN, rt, 10 min, 96 % (c) EtI, 120 ⁰C, 12 h, 59 % (d) EtI, MeCN, ∆, 24 h, 84 % (e) N, N-diphenylformamidine, (EtO)3CH, EtOH, ∆, 24 h, 82 % (f) compound 23, Ac2O, pyridine, ∆, 2 h, 67 %. (g) BF3*OEt2, MeOH, 50 ⁰C, 12 h, N2, 78 %; (h) NaNO2, HCl, 0°C, 30 min, then NaN3, 0 °C → rt, 1h, 56 %.

Synthesis of 5 started from acetamido derivative 24 (Scheme 4) that was treated with triethylorthoformate to yield 28. Subsequent removal of the acetyl groups with BF3*OEt2 gave 29 in good yield. Diazotization of 31 and treatment with sodium azide allowed target compound 5 (Scheme 4).


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Scheme 4. Synthesis of bisazide dye 5. (a) HC(OEt)3, pyridine, 12 h, ∆, 62 % (b) BF3*OEt2, MeOH, 50 ⁰C, 12 h, N2, 86 % (c) NaNO2, HCl, 0 °C, 30 min, then NaN3, 0 °C → rt, 1h, 64 %. 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-9ylmethanol, 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 fluorescence quantum yields of dyes 1–5 and of bis-clicked products with BCN were also determined (Table 1).



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Figure 2. Increase of emission of bisazide dye 1 (left) and dyes 2-5 (right) when reacted with two equivalents 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 1–5. (in PBS pH=7.4, c = 0.625 µM, at room temperature) Bisazide Dye 1 2 3 4 5

a

IBCN / Ibisazide 14 7 29 22 10

λexc [nm] 410 560 560 550 555

λem [nm] 487 575 571 565 569

b

Φbisazide 0.00065 0.0195 0.065 0.011 0.0125

b

ΦBCN 0.0073 0.057 0.73 0.13 0.082

a

Calculated at the emission maxima of the products; bQuantum yields relative to Coumarin 153 (in case of dye 1) or to Rhodamine B (dyes 2–5)

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 phenomenon43, 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


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exposure of aqueous solutions of bisazides 1–5 with a common household compact fluorescent lamp (CFL, 20W, 800 lumen) completely diminished fluorescence within 5 minutes. 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 equivalents of BCN for 2 hours upon which time the fluorescence intensities reached their maxima. Following this we have exposed the solutions to light for 5 minutes. 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 bisclicked 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, Ac-K(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 hours 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). LC-MS analyses of the reaction mixture of 1 with Ac-K(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 mono-triazolic product even by adding only half an equivalent of BCN to 1. This was in line with our earlier observations i.e. the mono-clicked product of 1 was more reactive than the starting bisazide23 and the formation of a cyclic conjugate was



exceedingly favored. As an alternate solution, we synthesized the monoazide-monotriazole derivative 37 from separately prepared triazole 32 (Scheme 5).

Scheme 5. Synthesis of mono-clicked cyanine-1 dye (6), conditions: Et3N, EtOH, rt, reflux, 2h. See ESI for full synthesis of intermediates 32 and 36.

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 equivalent 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


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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 cyaninetype dye in this emission regime possess such high fluorescence enhancements.

Table 2. Photophysical properties and fluorescence enhancement caused by double-click reactions with biscyclooctynilated peptides (in PBS pH=7.4, c = 0.625 µM, at room temperature, after 2 hours of stirring in the dark) Bisazide dye c

1 1d 2d 3d 4d 5d

a

Ibis-clicked / Ibisazide

λexc [nm]

λem [nm]

13 34 12 28 39 11

410 410 560 560 550 555

487 487 577 573 565 569

b

Φbis-clicked

0.0065 0.018 0.16 0.85 0.21 0.14

a

Calculated at the emission maxima of the products; bQuantum yields relative to Coumarin 153 (in case of dye 1) or to Rhodamine B (dyes 2–5); cwith Peptide-1; dwith Peptide-2

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



Figure 4. Comparative fluorescence enhancements of dyes 2-5 when reacted with biscyclooctynylated Peptide-2.

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. Crosslinking of BCN-tagged proteins To further demonstrate the potential of our bisazides, we thought to explore crosslinking 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


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(Pyl) RSAF/tRNAPyl pair from Methanosarcina mazei (PylRS with mutationsY306F Y384F) in E.coli19. 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 non-specific 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 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 case of dye 5, however, mainly the crosslinked 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 crosslinkers.

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 crosslinking 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 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 crosslinked band compared to the intensities observed for GFPY39→ BCN alone.

In conclusion, we have synthesized a series of new, double-quenched bisazide fluorogenic cyanine probes with emission maxima between 565–580 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 crosslinking 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.

ACKNOWLEDGEMENTS 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). EAL acknowledges funding from the SPP1623.


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Supporting Information Available: Details of synthetic procedures, characterization data for small molecules, peptide and protein labeling experiments are provided. This material is available free of charge via the Internet at http://pubs.acs.org. The authors declare no conflict of interest.

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GRAPHICAL ABSTRACT


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Bisazide Cyanine Dyes as Fluorogenic Probes for Bis

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