Bioorthogonally Applicable Fluorogenic Cyanine-Tetrazines for No

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BIOORTHOGONALLY APPLICABLE FLUOROGENIC CYANINETETRAZINES FOR NO-WASH SUPER-RESOLUTION IMAGING Gergely Knorr, Eszter Kozma, Judith Schaart, Krisztina Németh, György Török, and Peter Kele Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00061 • Publication Date (Web): 12 Feb 2018 Downloaded from http://pubs.acs.org on February 13, 2018

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Bioconjugate Chemistry

BIOORTHOGONALLY APPLICABLE FLUOROGENIC CYANINE-TETRAZINES FOR NO-WASH SUPER-RESOLUTION IMAGING

Gergely Knorr,[a] Eszter Kozma,[a] Judith M. Schaart,[a] Krisztina Németh,[a] György Török,[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] Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2, H-1117, Budapest, Hungary

CONTACT Péter Kele: [email protected]

GRAPHICAL ABSTRACT

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 The synthesis, fluorogenic characterization and labeling application of four tetrazinequenched cyanine probes with emission maxima in the red - far red range is reported. Fluorescence of the cyanine-cores is quenched via through-bond-energy-transfer (TBET) exerted by a bioorthogonal tetrazine unit. Upon bioorthogonal labeling reaction with cyclooctyne tagged proteins, the quenching effect ceases, thus the fluorescence reinstates, resulting in an increase in fluorescence intensity. As a rare example amongst indocyanines, one of our new probes was found suitable in STED-based super-resolution imaging. The applicability of this fluorogenic Tet-Cy3 probe was therefore further demonstrated in the bioorthogonal labeling of cytoskeletal protein, actin, with subsequent super-resolution microscopy (STED) imaging even under no-wash conditions.

INTRODUCTION Chemical biology applies synthetic chemical tools for the small-molecular manipulation of biological systems.1,2 Such modification of biomolecules mostly involves installation of signaling markers, very frequently fluorescent labels. Selective labeling of biomatter has greatly facilitated the exploration of intra- and extracellular biological processes.3–8 Besides labeled antibodies or affinity probes, selective labeling of proteins is also possible by means of fusion proteins such as tailored fluorescent proteins (FPs), tags (e.g. Halo, SNAP, Cys4) or small engineered enzymes (e.g. lipoic acid ligase).3–8 However, these latter techniques are limited to N- or C-terminal modifications and in many cases these tags perturb the original function of the protein of interest due to their comparable size. Minimal perturbation became possible with advances in genetic code expansion techniques by means of amber codon suppression, which enables implementation of a single non-canonical amino acid, virtually at


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any position.9-12 Genetic code expansion technology in combination with bioorthogonal chemistry, has brought substantial changes into chemical biology driven studies, lately.9-12 Following implementation of the fluorescent markers, detection of the signal is often associated with problems such as autofluorescence of naturally occurring fluorophores and background fluorescence arising from non-specific dye-biomolecule interactions.13 In order to improve signal-to-noise ratio, the use of red / far-red excitable probes or probes having large Stokes-shifts, efficiently minimize autofluorescence. Reduction of background fluorescence, on the other hand, is best accomplished by the use of fluorogenic probes that become fluorescent only upon specific reaction.14-20 Within the past years, substantial progress was made in the field of super-resolution microscopy (SRM) and several techniques emerged to overcome the limitations due to diffraction in fluorescence microscopy.21-24 These techniques enabled visualization of cellular structures at resolutions comparable to their real size. Super-resolution microscopy methods are typically based either on the use of tailored GFP variants or small-sized synthetic dyes. Synthetic fluorescent probes have the advantage of possessing smaller size not to mention that their chemical reactivity and photophysical parameters can be synthetically tailored. Due to recent developments in fluorescent microscopy it is the unavailability of dyes suitable for sitespecific labeling of proteins that is recently considered as the biggest limitation in superresolution techniques. New, fluorescent SRM probes harboring a selectively reacting e.g. bioorthogonal motif are therefore highly demanded. Such probes in combination with sitespecifically encoded ncAAs or targeted affinity probes bearing a cognate bioorthogonal function can even lead to improved resolution in the imaging of intracellular structures. We therefore aimed at designing and synthesizing bioorthogonally applicable, fluorogenic probes that are excitable towards the red range of the electromagnetic spectrum.



RESULTS AND DISCUSSION Indocyanine dyes are frequently used in fluorescence microscopy applications including a wide range of SRM techniques.25,26 Their popularity can be attributed to their high brightness and excellent photon yields together with their relatively easy synthetic access. Besides these, the propensity of these polymethines to exist in emissive and dark states in blinking buffers makes them even more attractive especially in stochastic methods. Few examples, however, are found in the literature that applies cyanines in stimulated emission depletion (STED) microscopy. This is due to the relatively low photostability of cyanines under the applied conditions. Some examples exist though, but these apply mixed cyanines e.g. coumarylindocianine.27 Although indocyanines possess relatively lower quantum yields in aqueous media (less than 30%) due to non-radiative deactivation through cis-trans isomerization processes, which can be somewhat suppressed by more rigid frames such as in Cy3B, the high radiative rates result in high photon yields.28,29 Most commercially accessible cyanine derivatives are negatively charged due to the presence of multiple sulfonate substituents. While negatively charged sulfonate moieties facilitate aqueous solubility they render these cyanines membrane impermeant. Membrane permeable dyes are usually positively charged or have zero net charge. In light of these, we designed indocyanines that bear only one sulfonate function in order to enhance water solubility but result in a zero net charge. To deliver bioorthogonality and fluorogenicity, we have planned to incorporate a tetrazine moiety via a conjugated but electronically decoupled linker such as phenylene or vinylene. In these setups tetrazines keep on acting as a bioorthogonal handle and a quencher of fluorescence via through-bond energy-transfer process.18-20,30 Along these considerations, we have designed Cy3 and Cy5 dyes 1 and 2 (Figure 1).


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Figure 1. Structures of bioorthogonally applicable, fluorogenic cyanines 1 and 2.

A. Synthesis of tetrazine-cyanine probes In order to access precursors of the final products p-iodoaniline (3) was first converted to 4iodophenylhydrazine (4) in a diazotization-reduction sequence. The hydrazine was then converted to indole 5 in the presence of 3-methylbutan-2-one. Treatment of indole 5 with EtI provided iodoindolium 6. Parallel to this, sulfoindolium 9 was also prepared from 4hydrazinylbenzenesulfonate 7 through Fischer indole synthesis of 8 and subsequent alkylation with EtI. Treatment of 9 with 10a or 10b in a mixture of acetic acid and acetic anhydride gave access to hemicyanines 11a and 11b. Subsequent reaction of the hemicyanines with iodoindolium 6 in pyridine provided halogenated sulfocyanines 12a and b. (Scheme 1).



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O I

I

1. HCl, NaNO 2 0°C, 90 min

I

2. SnCl2, concd. HCl 0°C, 60 min NH2 55-78% 3

EtI

AcOH 120°C, 3 h 42-65%

NH-NH2

120°C, 30 min MW 55-72%

N

4

I N

5

I-

6

O SO 3SO 3K

NH-NH2

1. AcOH 120°C, 18 h 2. KOH/iPrOH 56-60%

120°C, 30 min MW 70-77%

N

7

SO3 -

EtI N+

8

9

SO3SO3

H N () N n

+

N+

-

AcOH, Ac 2 O N (

120°C, 1 h

)n N+

O 10a n = 1 10b n = 3

11a n = 1 11b n = 2

9

SO3I N

pyridine, Ac2 O

+ I-

N (

)n N+

rt, 18 h

N

(

)n N +

12a : 18-34% 12b : 12-27% for 2 steps

O 6

SO 3-

I

11a n = 1 11b n = 2

12a n = 1 12b n = 2

Scheme 1. Synthesis of cross-coupling precursors iodo-Cy3 and iodo-Cy5, 12a,b.

Precursors 12a and b were then subjected either to Heck-type of cross-coupling reaction with 13 or Suzuki-coupling with 14 to furnish target compounds 1 and 2, respectively. (Scheme 2).


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Scheme 2. Synthesis of tetrazine-cyanines 1 and 2.

B. Fluorescence and fluorogenicity studies Next we have established the main photophysical properties and fluorogenicity of the new Cy3 and Cy5 probes upon strain-promoted click reaction with a commercially available dienophile, (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol, BCN.31 Excitation maxima were at around 570 and 660 nm for the a and b series, respectively. Emission spectra peaked at around 590 in case of the Cy3 and 685 nm for the Cy5 derivatives. Upon reaction with BCN, small changes were observed in spectral maxima. In each case the fluorescence intensity increased as the dye-BCN conjugate was formed (Figure 2 and Table 1) suggesting fluorogenic potential of the probes. Probe 1a had considerably higher enhancement compared to the rest of the dyes (14 vs. 3-5 fold).



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Figure 2. A) Absorption and emission spectra of BCN conjugates of 1a and 2a and B) 1b, 2b (the red and purple lines represents the wavelength of common depletion lasers) C) Fluorescence intensity enhancements of dyes 1 and 2 at emission maxima of BCN conjugates. Spectra were acquired in PBS (pH=7.4 containing 0.1% SDS, c = 1.0 µM, at room temperature, λexc = 510 nm and 600 nm, for the a and b series respectively). Table 1. Main photophysical properties and fluorescence enhancement values of dyes 1-2 and their conjugates with BCNa

Probe λem (nm) 594 (597)

free probe c d ε Φ 148.1 0.01

+BCN d ε Φ 129.2 0.14

FE Ι/Ι0 Φ/Φ 0 14.2 14.0

675 (673)

685 (695)

214.2

0.03

196.4

0.10

5.0

3.3

566 (566)

567 (567)

587 (587)

125.9

0.05

122.1

0.18

3.5

3.6

664 (664)

665 (665)

685 (685)

176.2

0.06

164.0

0.16

2.9

2.7

b

1a

λexc (nm) 573 (574)

b

λabs (nm) 578 (574)

1b

661 (671)

2a 2b

b

a

c

e

all reactions and measurements were performed in PBS buffer (pH=7.4 containing 0.1% SDS, at room temperature), with dye concentrations between 0.3-1.5 µM. bwavelengths in parentheses belong to the conjugates. ccalculated at absorption maxima (×103 M-1cm-1). dλexc = 510 nm (1a, 2a) and 600 nm (1b, 2b) relative to Cresyl violet. emeasured at emission maxima of the BCN conjugates.


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C. Confocal and STED microscopy applications under no-wash conditions To further demonstrate the potential of our fluorogenic cyanine-tetrazines, we aimed at studying their applicability in protein labeling schemes in mammalian cells. To this end, we have synthesized BCN derivatized, actin binder affinity probe, phalloidin19 (SI, Figure S1). Fixed mammalian COS7 cells were treated with phalloidin-BCN, washed and labeled with fluorogenic cyanine-tetrazines 1-2, then imaged using confocal microscopy (SI, Figure S2). Unfortunately, Cy5-derivatives (1b, 2b) did not show any specific labeling of pre-targeted actin filaments. Reasons for this can either be explained on the basis of less advantageous photophysical properties of Cy5 dyes or their steric demand. On the contrary, both Cy3 derivatives (1a, 2a) labeled the filaments selectively, which was apparent in the confocal images. We also determined the second order rate constant for the reaction between 1a and BCN and a k2 value of 25.9 M-1s-1 was obtained (supporting information). Furthermore, in accordance with its higher fluorogenicity, which enables clear distinction between reacted and unreacted forms, 1a showed negligible background. This prompted us to demonstrate the applicability of 1a in wash-free labeling schemes. We were also curious whether our Cy3 probe is suitable for super-resolution imaging in STED microscopy. Although Cy3 is less frequently used in STED imaging, its compatibility with the commercial 660 nm depletion laser suggest its suitability (Figure 2). Thus, we allowed phalloidin-BCN tagged actin filaments to react with 3 µM 1a for 30 min and subjected it to STED imaging using a 552 nm laser for excitation and a 660 nm laser for stimulated depletion. Figure 3a-d shows confocal and STED images of 1a labeled actin filaments. Line analysis of the actin meshwork showed subdiffraction resolution in STED images (Figure S4) with full width at half maximum (FWHM) value of 163 nm compared to 356 nm available with confocal imaging (Figure S5ad). These values are in good agreement with previously reported data.32,33 Super-resolution imaging of the fine actin meshwork is quite challenging using phalloidin, however, besides



microfilament bundles, microfilament networks were also visible in our imaging setup (Figure 1, S4, S5).33 When tested the applicability of 1a under no-wash conditions, to our delight, we observed no substantial difference between wash and no-wash conditions. Both confocal and STED images gave similar resolutions to images acquired under wash conditions (368 nm for confocal image, 168 nm for STED image) (Figure 3e-h, S5e-h). Moreover, improved resolution was observed when individual actin filaments were detected (Figure S6). It should also be noted that no loss of signal-to-noise ratio was observed for three cycles of STED imaging with >90% STED intensity. Further cycles, however, resulted in faded images due to bleaching of the cyanine dye.


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CONCLUSION In conclusion, we have synthesized a series of bioorthogonally applicable tetrazine-quenched fluorogenic cyanine probes. Fluorogenicity of the new dyes was assessed upon reaction with a strained cyclooctyne, BCN. Probe 1a showed an order of magnitude higher fluorescence enhancement compared to the rest of the dyes, anticipating its superior performance in labeling applications. We have tested the specific labeling potentials of the dyes using phalloidin-BCN tagged actin filaments. While Cy5 dyes, 1b and 2b showed no specific staining, Cy3 derivatives 1a and 2a specifically labeled actin filaments. Furthermore, probe 1a showed only negligible background, which prompted us to further test it upon no-wash conditions. To our satisfaction, no substantial difference was observed between the images acquired under wash or no-wash conditions. This suggests that the over 10-fold fluorescence enhancement enables efficient distinction between reacted and unreacted forms with a good signal-to-noise ratio. Although indocyanines are not preferred in STED-based superresolution techniques, we have tested the applicability of 1a in STED imaging of actin filaments, using a 660 nm continuous wave laser for depletion. To our delight, 1a was found to be suitable in STED and resulted in subdiffraction imaging of the labeled actin meshwork at resolutions similar to reported values. We believe that upon further improvement of such site-specifically reactive, fluorogenic probes, superresolution imaging of live cells becomes possible even with improved resolution. Further testing of our dyes in terms of membrane permeability using genetically modified intracellular proteins is in progress in our laboratory and results will be reported in due course.



Figure 3: Confocal (a, e) and STED images (b, f) of actin filaments in mammalian COS7 cells. Actin filaments were tagged with phalloidin-BCN and labeled with 1a (3 µM). Images were taken after (a, b) or without washing out (e, f) the dye after 30 min incubation. Enlargement of the boxed areas are shown at c, g (confocal, corresponding to a, e) and d,h panels (STED, corresponding to b,f). λexc = 552 nm with 660 nm depletion laser for STED images. Color encodes the intensity values of each pixel.


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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). G.K. acknowledges support by the ÚNKP-17-3-IV-ELTE-274 New National Excellence Program of the Ministry of Human Capacities.

Supporting Information Available: Details of synthetic procedures, characterization data for small molecules and cellular protein labeling experiments are provided. This material is available free of charge via the Internet at http://pubs.acs.org.

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Bioorthogonally Applicable Fluorogenic Cyanine-Tetrazines for No

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