Deuterated rhodamines for protein labelling in nanoscopy

Rhodamine molecules are setting benchmarks in fluorescence microscopy. Herein, we report the deuterium (d12) congeners of tetramethyl(silicon)rhodamine, obtained by isotopic labelling of the four methyl groups, which improves photophysical (i.e. brightness, lifetimes) and chemical (i.e. bleaching) properties. We explore this finding for SNAP- and Halo-tag labelling, and highlight enhanced properties in several applications, such as Förster resonance energy transfer, fluorescence activated cell sorting, fluorescence lifetime microscopy and stimulated emission depletion nanoscopy. We envision deuteration as a generalizable concept to improve existing and develop new Chemical Biology Probes.

We next turned to cellular labelling and imaging and aimed to determine the behavior of our dyes on targets that are mainly expressed on the membrane, as well as targets that are found intracellularly, both regions that have been addressed for SNAP-tag labelling. [3] First, we employed CHO-K1 cells stably expressing SNAP-tagged glucagon-like peptide 1 receptor (SNAP-GLP1R:CHO-K1) [12] , a cell line intensely used to study the physiology of this blockbuster class B G protein-coupled receptor (GPCR) involved in glucose homeostasis and targeted in diabetes treatment [13,14] , as a benchmark for d12 performances. As such, cells were labelled with 1 μM BG-TMR/SiR(-d12) for 30 min, before washing and live imaging by confocal microscopy, which revealed staining of SNAP-GLP1R with all deuterated and parental dyes tested ( Fig. 2A). Having established labelling on the outer plasma membrane, we secondly investigated intracellular staining in HeLa cells that stably express SNAP-tagged Cox8A (SNAP-Cox8A:HeLa), located in the inner mitochondrial membrane (Fig. 2B), which has been used to study mitochondrial ultrastructure in live cells. [15,16] As for SNAP-GLP1R, we observed clean labelling with all dyes, and for both colors with an observable increase in brightness for the d12 derivatives. With this enhanced performance in microscopy, we wanted to quantify brightness by fluorescence activated cell sorting (FACS) to obtain robust values over large sample sizes. Accordingly, we labelled SNAP-GLP1R:CHO-K1 and SNAP-Cox8A:HeLa cells with both, BG-TMR(-d12) and BG-SiR(-d12) to compare red and far-red color intensities by subsequent sorting (Fig. 2C). Histograms of SNAP-GLP1R:CHO-K1 cells labelled showed a right-shift in fluorescence intensity when dyes were deuterated (Fig. 2C, left). In contrast, SNAP-Cox8A:HeLa cells were labelled more homogeneously with a pronounced shift to higher intensities for SiR-d12 compared to its nondeuterated congener (Fig. 2C, right), while TMR(d12) only displayed a subtle change. By normalizing intensities and comparison, we calculate higher mean intensities for our deuterated dye versions (Fig. 2D). While no large increase was observed in SNAP-Cox8A:HeLa cells for TMR-d12 (2%), mean intensity was markedly increased in SNAP-GLP1R:CHO-K1 cells (28%).
Furthermore, SiR-d12 outperformed SiR on SNAP-GLP1R and SNAP-Cox8A with an intensity increase of 43% and 50%, respectively, which compares well with in vitro increase in brightness (35% and XX%). These results demonstrate that rhodamines with CD3 groups on the amines are not only the applicable to live cells but even outperform non-deuterated fluorophores, which is in line with our in vitro data of the unbound dyes.
With these encouraging results, we decided to test our deuterated probes in fluorescent lifetime confocal (FLIM) and stimulated emission by depletion (STED) microscopy, both stateof-the-art imaging techniques to reveal cellular dynamics and structures. Intriguingly, fluorescent lifetimes where higher for d12 congeners than their parent counterparts (τ(TMR) = 2.3 vs. τ(TMR-d12) = 2.6 ns; τ(SiR) = 2.9 vs. τ(SiR-d12) = 3.5 ns) (Fig. 3A). However, TMR-d12 was not as susceptible to bleaching as TMR, while SiR and SiR-d12 exhibited similar trends of not being prone to fast bleaching in this setup (Fig. 3B). Lastly, and with SiR being one of the most successful far-red dyes for nanoscopy, we investigated super-resolution images acquired in live SNAP-Cox8A:HeLa cells. After incubation with 1 μM BG-SiR or BG-SiR-d12, we recorded images of mitochondrial cristae under the same imaging conditions (Fig. 3C). While both dyes displayed labelling, SiR-d12 was able to resolve cristae sharper with less background, demonstrating its power in nanoscopy.
In our study, we synthesized and tested deuterated fluorophores. We install labelling moieties for SNAP-and Halo-tag conjugation and apply them in different experimental setups, i.e. in vitro FRET, and in cellulo confocal microscopy, FACS, FLIM and STED. We found that our fluorescent xanthene-based dyes TMR-d12 and SiR-d12 are improved by hydrogen-deuterium exchange on their methyl groups, enhancing photophysical parameters, such as brightness and lifetime, while reducing critical chemical parameters, such as bleaching. While the exact reason for this is unknown, we argue the following: i) affecting the rotation around the aromatic carbonnitrogen bond (in our case due to higher mass of the CD3 groups) has marked effects on fluorescent properties [17] , which could suppress non-radiative decays and in turn enhances quantum yield and lifetime [6] ; and ii) a lower zero-point energy of the C-D vs. C-H bond results in slower reaction kinetics, as an higher energy barrier has to be overcome [18] , and this could reduce bleaching through for example generated reactive oxygen species. While these explanations need more experimentation, ideally in combination with in silico calculations, we showcase novel deuterated dyes that outperform their parent molecules in multiple experiments, ranging from in vitro FRET, to live cellular labelling and sorting, in lifetime and super-resolution microscopy. We anticipate this concept i) to be generalizable to more xanthenes (e.g. fluoresceins, rhodols, SNARFs, and quenchers like QSYs), and other dye scaffolds (e.g. coumarins, cyanines, BODIPYs, EDANS, NBDs, Hoechst) including quenchers at any C-H bond positions for improving and fine-tuning spectroscopic properties; ii) to be further explored with other isotopes, such as 13 C, 15 N or even radioactive 3 H; iii) to be used in different labelling approaches, such as the attachment to sulfonated BG (SBG) scaffolds allowing the separation of SNAP-tagged receptor pools [19] , to biomolecule targeting probes [20][21][22] , to "click chemistry" reagents (e.g. cyclopropenes, cyclooctenes) [23] or to photoswitchable ligands [24,25] including black hole quenchers (BHQs), and iv) to serve as multimodal dyes for confocal fluorescence and Raman microscopy [26] . These efforts are of ongoing interest in our laboratories.

Synthesis
Chemical synthesis (Supporting Scheme 1) and characterization of compounds is outlined in the Supporting Information. Purity of all dyes was determined to be of >95% by UPLC-UV/Vis traces at 254 nm and dye specific λmax that were recorded on a Waters H-class instrument equipped with a quaternary solvent manager, a Waters autosampler, a Waters TUV detector and a Waters Acquity QDa detector with an Acquity UPLC BEH C18 1.7 μm, 2.1 x 50 mm RP column (Waters Corp., USA).

SNAPf and SNAP-Halo expression and purification
SNAPf was expressed and purified as described previously [16] and complete amino acid sequences