Targetable conformationally restricted cyanines enable photon-count limited applications

: Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to parent species in a range of contexts including their use on a DNA-nano-antenna, in single-molecule Förster resonance energy transfer (FRET) applications, far-red fluorescence lifetime imaging microscopy (FLIM), and single-molecule localization microscopy. These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance. G.B. and M.S. thank the European Regional Development Fund (EFRE project “Center for Personalized Molecular Immunotherapy”) and the Deutsche Forschungsgemeinschaft (SFB/TR 240, project number 374031971, A07) for financial support. Z.H. is grateful for a CSC PhD scholarship and N.Z. thanks the Alexander von Humboldt foundation for a postdoctoral fellowship. Work in the lab of T.C. was funded by the European Commission (ERC-StG No. 638536 SM-IMPORT). VG acknowledges European Union’s research and innovation the Marie Skłodowska-Curie actions (grant agreement No. 840741) and the by Humboldt Research Fellowships from the Alexander von Humboldt Foundation. PT thanks the Deutsche Forschungsgemeinschaft (excellence cluster e-conversion under Germany’s Excellence Strategy - EXC2089/1–390776260; Project-ID 201269156 – SFB1032).

Abstract: Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to parent species in a range of contexts including their use on a DNA-nano-antenna, in single-molecule Förster resonance energy transfer (FRET) applications, far-red fluorescence lifetime imaging microscopy (FLIM), and single-molecule localization microscopy. These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance.
Modern fluorescence-based methods put intense demands on fluorescent probes. [1][2][3][4][5] Cyanine dyes, especially the pentamethine cyanines Cy5 and Alexa Fluor 647, are invaluable probes in a range of contexts, despite modest quantum yields. [7] We recently introduced the novel pentamethine cyanine Cy5B where excitedstate trans-to-cis photoisomerization is blocked by installation of a conformationally restraining ring system. [6] Relative to conventional cyanines, these compounds exhibit improved fluorescence quantum yield and longer fluorescence lifetimes. Small molecule conjugates underwent efficient recovery from hydride reduction (reductive caging) to enable high quality single-molecule localization microscopy (SMLM) in oxygenated buffer. [7] However, immunofluorescent and other biomolecule targeted applications were not possible due to the propensity of these compounds to form non-fluorescent aggregates. Here we report the optimized synthesis of di-sulfonated and tri-sulfonated derivatives and detail the application of these probes in several settings.
The physical properties of fluorescent probes is crucial to the advanced imaging applications, and sulfonation is an exceptionally versatile strategy in this context. [8][9][10][11] Scheme 1 details the synthesis of di-and tri-sulfonated conformationally restrained pentamethine cyanines. Indolinine 3, bearing a functionalizable methyl ester, was accessed through Fisher indole synthesis and alkylation from 1. Similar to prior efforts, we found that conventional cyanine formation with 3 and previously reported 4 afforded the desired mixed cyanine in modest yield (14%) after a complex purification process. [13] Consequently, we then examined alternative approaches to unsymmetrical cyanine formation, as several have been reported. [14][15][16][17] Notably, solid phase methods proved difficult to implement on significant scale. By contrast, a solution-phase sequential heterocycle addition sequence, based on a report by Wolf and coworkers, provide scalable reproducible access to rac-6 on up to gram scales. [14] This approach involved addition of 4 to the cyanine precursor, malonaldehyde bis(phenylimine) monohydrochloride, with microwave irradiation (130 °C , 30 min) to first form acylated hemicyanine 5. This unpurified intermediate was then reacted with 3 at r.t. to provide unsymmetrical pentamethine cyanine rac-6, in good yield (58% from 3) with only trace formation of other cyanine products. While installation of the a-b unsaturated aldehyde via cross-metathesis reaction was unsuccessful initially due to the poor solubility of 3 in organic solvent, we ultimately found that the addition of a phase transfer catalyst, tetrabutylammonium bromide, enabled the reaction to proceed efficiently. The key tetracyclization reaction was carried out using BBr3 in CH2Cl2, again with tetrabutylammonium bromide acting as a critical solubilityenhancing additive. As observed previously, [6] the initial cyclization reaction formed a mixture of diastereomers which were equilibrated using protic acidic conditions to form the syn product rac-7, which is a mixture of diastereomers around the pyran ring due to the additional chiral center. The mixture rac-7 could be converted to the corresponding NHS-ester, Cy5B-disulfo-NHS, through a saponification/TSTU sequence. Alternatively, the tertiary amide, Cy5B-trisulfo-NHS, could be formed via saponification and coupling with amine 8, followed by saponification/TSTU NHS-ester formation.
We then assessed the optical properties of these compounds, as well as their antibody conjugates ( Table 1). As a comparison, we used a disulfonated pentamethine cyanine (Cy5). [18] As free dyes, both conformationally restrained dyes Cy5B-disulfo and Cy5B-trisulfo have higher quantum yield (FF) and longer fluorescence lifetimes (tF) as Cy5, albeit with moderately reduced extinction coefficients (e) in an aqueous environment (Table 1). These values are quite similar to the monosulfonated derivative we Scheme 1: Synthesis of Cy5B-disulfo and -trisulfo NHS Esters.  reported previously. [6] We then prepared goat anti-mouse antibodies labeled on average with a degrees of labeling (DOL) of 1 and 3 using conventional NHS-ester labeling conditions (Fig. S1). Several observations emerge from the spectroscopic properties of the resulting conjugates. First, with unrestrained Cy5, FF decreased substantially, i.e. from 0.21 to 0.11 for DOL 3.0 and tF decreased only slightly, which is in line with prior observations (Table 1). [18][19] With the Cy5B-disulfo-mAb conjugates, FF is similar to the free dye, but moderately reduced (by 31%) at higher labeling density. By contrast, the trisulfo derivative, exhibits nearly identical FF at both  This results suggest that this simple amide modification strategy improves the properties of the resulting conjugates. Overall, these studies illustrate that cyanine conformational restraint, when combined with optimized sulfonation, can result in fluorophore antibody conjugates with excellent optical properties. To investigate the difference in photophysical properties of Cy5B-trisulfo and Alexa Fluor 647 (AF647), a Cy5 derivative with two additional sulfonate groups, [18] we employed fluorescence correlation spectroscopy (FCS) as an analytical tool for short-lived transient species. Using the well-established ROXS buffer [19] and appending both probes to DNA-origami structures (Fig. 1A), AF647 exhibits a dark state in the microsecond time scale, which is consistent with trans-cis isomerization reported previously. [20] By contrast, Cy5B-disulfo provides a stable fluorescence signal both in the millisecond and microsecond time regime. Of note, the residual autocorrelation term in the Cy5B-disulfo can be attributed to fast blinking not completely removed by ROXS [21] and is present in both samples. The lack of the photoisomerization process not only provides a "cleaner" fluorescent signal up to the microsecond time range but also increases the overall brightness of the molecule, since the dark cis-state can no longer be populated. This becomes particularly relevant when excitation rates are very high, as observed in plasmonic nanostructures. [22] Therefore, we placed the fluorophores in the plasmonic hotspot formed by two 100 nm gold nanoparticles using DNA origami nano-antennas (Fig. S2) [23] and extracted spot-wise fluorescence intensity values from confocal microscopy scans. When plotted as a function of normalized excitation intensity, Cy5B-disulfo is significantly brighter than AF647 at all excitation intensities. While both samples show a degree of saturation, [24] Cy5B-disulfo starts saturating only at much higher excitation intensities (Fig. 1B). At 1 µW excitation intensity, Cy5Bdisulfo is more that 3-fold brighter than AF647. These findings indicate that these probes are well suited for experiments requiring high temporal resolution (i.e. high excitation intensities). [25] Diffusion-based single-molecule FRET (smFRET) has become a key method to study the structural dynamics of biomacromolecules under realistic conditions, [26] yet dye performance can be severely impacted by photo-induced blinking (e.g., triplet-state formation or trans-cis isomerization) and irreversible photobleaching. [27] This limits various aspects of smFRET experiments including the quality of the resulting FRET efficiency histograms, temporal resolution and distance accuracy. We evaluated Cy5B and Cy5B-trisulfo dyes using microsecond alternating excitation (ALEX) with maltose binding protein as a model system ( Figure 2C-F, Figure S3-S6). The conformationally restrained sulfonated cyanine dyes were benchmarked against the state-of-the-art dye combinations used in the smFRET field (AF555 with AF647 and ATTO647N). The protein model systems was an established double-cysteine variant of MalE (Thr36Cys-Ser352Cys) that allows to study the conformational changes of MalE via stochastic labelling with maleimide fluorophores ( Figure S4, See SI for the synthesis of Cy5B-trisulfo maleimide). [28][29][30] The labelling positions were chosen such that the apo conformation (absence of maltose) displays lower FRET efficiency in comparison to the holo state (100 µM maltose) of the protein ( Figure S5). All doublelabeled MalE with AF555 as donor dye visualized the expected conformational changes upon addition of maltose ( Figure 1C). This meant that the protein adopts the low FRET open conformation in the absence of ligand, which is altered into the closed-liganded conformation when 100 µM maltose were added. Due to the spectral similarity between the acceptor dyes, all dye-pairs showed similar, yet not identical, mean FRET efficiencies and width with overall high histogram quality. In all cases, we also found a similar response of the FRET assays upon addition of 1.5 µM maltose (fraction closed of 0.3-0.4), a concentration close to the dissociation constant Kd for the protein-substrate interaction ( Figure S4, S5). [28] To further benchmark the quantitative performance of the dyes against each other, we compared their acceptor photon count rates at 60 µW green and 25 µW red laser excitation intensity. Strikingly, we found a maltose dependent fluorophore brightness, where all dyes were similarly bright in the holo state, yet the Cy5B dyes were slightly less bright (by 5-10 kHz) in comparison with AF647 and ATTO647N in the apo state ( Figure 2D). This observation suggests that less FRET bursts are retained for AF555-Cy5B-monosulfo and AF555-Cy5Btrisulfo in comparison to other two fluorophore pairs ( Figure 1F). Nevertheless, the Cy5B derivatives showed a unique advantage over both AF647 and ATTO647N ( Figure 1E). Due to the absence of cis/trans isomerization (in comparison to AF647) we found in a laser power dependence in the experiments of the apo state ( Figure  1E, Figure S6) that the Cy5B dye had strongly reduced bridge artifacts caused by the acceptor dye and retained the mean E* position and width sE of the population over the range of excitation powers studied ( Figure 1E). [30] Also Cy5-trisulfo had only a small change of mean peak position, yet substantial change of the population width, which was similar to ATTO647N. These observations establish Cy5B and Cy5B-trisulfo as a useful alternative to AF647 and ATTO647N, particular when the goal of an experiment is to maintain correct mean FRET efficiencies and reduced bleaching/blinking artefacts, e.g., when higher laser powers are required than usual.
Having established the utility of these probes for in vitro singlemolecule applications, we evaluated them for cellular applications. We first asked if the difference in fluorescence lifetimes of the sulfonated Cy5B and Cy5 antibody conjugates could be used for two-color fluorescence lifetime imaging microscopy (FLIM). [31][32][33] We performed FLIM measurements of Cy5 immunolabeled β-tubulin and detected an average fluorescence lifetime of 0.91 ns (Fig. 1G-J), whereas cells immunolabeled solely for clathrin with Cy5Btrisulfo showed a fluorescence lifetime of ~1.75 ns (Figs. 1a,c) similar to the values measured in ensemble TCSPC experiments (Table 1). Notably, we were able to obtain composite FLIM images of COS7 cells immunolabeled with both dyes based on the different fluorescence lifetimes of Cy5 and Cy5B-trisulfo labeled antibodies (Fig. 1J).
Finally, we evaluated the performance of the new redabsorbing dyes in single-molecule localization microscopy (SMLM) experiments. We immunostained β-tubulin of COS7 cells with Cy5 and Cy5B-trisulfo, as this is a well-established reference structure, to investigate the spatial resolution of super-resolution microscopy methods. Microtubules are hollow tubes with an outer diameter of 25 nm and 60 nm, respectively, after immunostaining with primary and secondary antibodies. [34] Direct stochastic optical reconstruction microscopy (dSTORM) [35] imaging of Cy5 (Fig. 1K) was performed in standard photoswitching buffer (100 mM MEA, pH 7.5, without enzymatic oxygen depletion whereas Cy5B-trisulfo was reduced over 30 min in NaBH4/PBS and measured in the same buffer by TIRF microscopy using solely 640 nm irradiation (Fig. 1L). Removal of residual reductant NaBH4 and imaging in PBS provided a similar image quality (Fig. S7). Additional irradiation at 405 nm did not significantly change the SMLM image quality (Fig. S8). For both dyes, Cy5 and Cy5B-trisulfo we could reconstruct high quality super-resolved microtubule images but for Cy5B-trisulfo we detected twice as many photon counts per ON-event (~1.800) as for Cy5 (~770), which translates into localization precisions of 6.6±3.3 nm (s.d.) for Cy5B-trisulfo and 8.4±4.4 nm (s.d.) for Cy5. [36] Fourier ring correlation analysis [12] of microtubule SMLM images corroborated these numbers and provided spatial resolutions of ~13 nm for Cy5B-trisulfo and ~19 nm for Cy5.
In conclusion, we report the optimized synthesis of conformationally restricted di-and tri-sulfonated pentamethine cyanines and their evaluation in various experiments were photon count is crucial. As we have shown for bio-orthogonal labeling and small molecule conjugates, we now demonstrate that this strategy provides exceptionally bright protein conjugates. [6,37] The unique properties of these probes enable single-molecule applications including high photon-fluxes in DNA nanoantennas, FRET, and SMLM experiments. Going forward, these results suggest the use constrained pentamethine cyanines for live SMLM-imaging as imaging is performed in thiol-free and deoxygenated PBS. Additionally, conjugation using labeling strategies to further exploit their enhanced brightness is of significant interest. Broadly, these studies provide a new set of well characterized far-red probes with utility in demanding fluorescence-based applications.