Green- to far-red-emitting fluorogenic tetrazine probes – synthetic access and no-wash protein imaging inside living cells† †Electronic supplementary information (ESI) available: Synthetic procedures and spectroscopic data, details of cell experiments and imaging. See DOI: 10.1039/c6sc03879d Click h

Fluorogenic probes for bioorthogonal labeling chemistry are highly beneficial to reduce background signal in fluorescence microscopy imaging.


Introduction
Synthetic uorophores represent a valuable alternative to uorescent proteins for the labeling of biomolecules in living cells and organisms. They are much smaller in size and in general exhibit superior photophysical properties such as higher brightness and photostability. 1 However, the benecial photophysical properties come with the disadvantage that applications of synthetic uorophores in live cell experiments oen suffer from unwanted background signal due to unbound uorophore and unspecic localization. To overcome this problem, uorophores can be equipped with a uorescence-quenching moiety that is either chemically converted or eliminated in the labeling process. This leads to an uorescence increase upon binding to the biomolecular target, which is why such molecules are referred to as uorogenic probes. 2,3 In addition to reducing background signal, uorogenic probes have the potential to be used in uorescence microscopy experiments that do not tolerate excessive washing of the imaging sample. Fluorogenic probes are highly desirable when a specic biomolecule is to be selectively labeled in the presence of a plethora of biomolecules.
To date, a number of methods for selective biomolecule labeling have been reported including the use of protein tags, 4-6 peptide tags 6-8 and unnatural amino acids. 9,10 They oen involve bioorthogonal chemical reactions like the copper catalyzed (CuAAC) 11,12 and strain-promoted azide alkyne cycloaddition (SPAAC) 13 as well as the inverse electron demand Diels-Alder cycloaddition (DA inv ) 14,15 between 1,2,4,5-tetrazines and strained olens. Fluorogenic variants exist for all of those reactions. [16][17][18][19][20][21] Tetrazine-based uorogenic probes [16][17][18][19][20] are particularly interesting as the tetrazine moiety within these molecules serves as reactive group and uorescence quencher at the same time. Hence, conversion of the tetrazine in DA inv results in the loss of its quenching properties. Existing reports of extracellular or intracellular biomolecule labeling with tetrazine uorogenic probes were either conducted in xed cells, 18 on cell surfaces 17 or needed additional dye wash-out steps in live cells. 16,22 In short, although impressive uorescence enhancements have been reported in vitro, the ultimate purpose of uorogenic probes, namely their application inside living cells under nowash conditions, remains a challenge.
Herein, we report the synthesis of highly water-soluble uorogenic xanthene tetrazine derivatives covering a wide emission range from green (uorescein, Oregon Green) over yellow (tetramethylrhodamine) to far-red (Si-rhodamine). The novel tetrazine dyes were prepared via two highly modular synthetic routes. Furthermore, we successfully applied the new uorogenic probes in selective protein labeling experiments. We demonstrate the usefulness of their uorogenic properties by uorescence microscopy imaging of the actin cytoskeleton in xed cells as well as mitochondrial and nuclear proteins in living cells. To our knowledge, this is the rst report of DA inv -mediated no-wash protein imaging inside living cells utilizing uorogenic tetrazines. rst, the distance between the tetrazine, a chromophore itself, and the uorophore is crucial for efficient quenching and the resulting uorescence turn-on upon conversion in DA inv . Therefore we developed new synthetic strategies to access uorophore tetrazine probes with a minimal interchromophore distance. Tetrazine uorogenic probes with ultra-short interchromophore distance have been reported for coumarins 18 and boron dipyrromethene (BODIPY) dyes. 17 However, coumarin and BODIPY uorophores are not ideally suited for live cell imaging. Coumarins suffer from low brightness and the need of UV-light excitation. BODIPY dyes have limitations like low Stokes shis and oen poor aqueous solubility. Second, we aimed to focus on uorophore classes that are preferentially used for intracellular live cell imaging. A common synthetic scheme to access tetrazine uorophores involves the construction of the tetrazine moiety in the last synthetic step by converting nitriles with hydrazine in a classical Pinner-type reaction. Yet, this approach implies certain drawbacks. The harsh conditions of the Pinner tetrazine synthesis can result in product mixtures that are difficult to purify. Hence, our third aim was to nd new synthetic methods to link tetrazines to uorophores under mild conditions.
The derivatization of 1,2,4,5-tetrazines by various Pd-catalyzed cross-coupling reactions such as Stille, [23][24][25] Suzuki, 26,27 Sonogashira 25,28 and Heck 19 reactions as well as Buchwald-Hartwig aminations 27 has been described in literature. There are only few examples of C-C-bond formation at the 3-or 6position of the tetrazine ring. Pop et al. have described the Stille coupling of 3,6-dichloro-1,2,4,5-tetrazine with an electron-rich tetrathiofulvalene tributylstannane. 29 This encouraged us to employ the Stille cross-coupling for the derivatization of tetrazines at the 3-and 6-positions. While being compatible with the base-sensitive tetrazine moiety, this chemistry further allows substitution directly at the tetrazine core, thereby giving access to a variety of tetrazine-based uorogenic probes with minimal interchromophore distance. We identied 3-bromo-6-methyl-1,2,4,5-tetrazine 5 (ref. 30) as suitable building block for Stille cross-coupling chemistry. 5 can be synthesized in 5 steps starting from carbon disulde and hydrazine monohydrate (Scheme S1 †). Next, the uorophore organotin derivatives for coupling with 5 were prepared from the corresponding aryl iodides or bromides. Halogen-substituted uorescein and Oregon Green derivatives were MOM-protected (7,11,15) and converted to the organotin derivatives 8, 12 and 16. The critical cross-coupling of the uorophore stannanes with 5 yielded the MOM-protected tetrazine probes 9, 13 and 17 (Fig. 1, route I) which were deprotected to tetrazine dyes Fl-5-Tz, Fl-6-Tz, OG-5-Tz and OG-6-Tz. When transferred to rhodamine and Sirhodamine substrates, the Stille coupling procedure described above did not give satisfactory results. Therefore, we established an alternative synthetic strategy to gain access to more red-shied tetrazine uorophore probes. In literature, both the reaction of diarylethers 31 and diarylsilanes 32 with acid chlorides and aldehydes respectively to obtain (Si-)rhodamines have been described. We focused on aldehydes as electrophilic component and synthesized the required tetrazine benzaldehyde derivatives in a two-step procedure. First, commercially available or easily accessible hydroxymethyl benzonitrile derivatives were converted to the corresponding tetrazine benzylic alcohols 18, 19 and 20. These were mildly oxidized to the tetrazine benzaldehyde derivatives 21, 22 and 23 using Dess-Martin periodinane. The aldehydes were subsequently reacted with diarylether 24 or diarylsilane 25 in a Friedel-Cras alkylation. Subsequent oxidation with chloranil afforded the rhodamine tetrazines Rh-5-Tz, Rh-6-Tz and Si-rhodamine tetrazines SiRh-5-Tz, SiRh-6-Tz and MeSiRh-5-Tz (Fig. 1, route II). All newly synthesized tetrazine uorogenic probes were photophysically characterized (Table 1, Fig. 2a), which required removal of uorescent trace impurities by HPLC-purication. The uorogenic properties were determined by conversion with transcyclooct-4-en-1-ol (TCO) in DA inv (Table 1, Fig. 2c).
Fluorescence turn-on ratios of up to 109-fold for Fl-6-Tz were measured. As a general trend, the turn-on ratios were found to decrease with increasing emission wavelength. Though exhibiting quantum yields of the same magnitude as the yellowemitting dyes, the Si-rhodamine tetrazines showed rather modest turn-on ratios (Table 1). Fluorescence quenching can be due to e.g. resonance energy transfer (RET) or Dexter interaction. Both energy transfer mechanisms are well known to be dependent on donor-acceptor spectral overlap. 33 The presence of such a process might explain the observed wavelengthdependent variation of the quenching efficiency for the herein presented tetrazine dyes. By monitoring the uorescence increase over time, we found the cycloaddition to be nished within 10-15 min at equimolar reactant concentrations (Fig. 2b). The excellent aqueous solubility of all nine dyes makes the new uorogenic tetrazines ideal candidates for application in biological environment. Having shown the potential of the synthesized dyes as uorogenic probes, we were eager to evaluate their performance in cellular protein labeling experiments. First, we turned our focus to xed cells and tested the probes for actin cytoskeleton staining. We used phalloidin-TCO to target the DA inv -reactive TCO to actin laments. Fixed HeLa cells were incubated with phalloidin-TCO and subsequently treated with either OG-5-Tz, Rh-6-Tz or MeSiRh-5-Tz and subjected to confocal imaging without previous dye wash-out. Phalloidin-specic labeling of lamentous structures was observed for all three uorogenic dyes thereby covering a wide range of the visible spectrum (Fig. 3). Even though we observed low uorescence turn-on in vitro for Si-rhodamine tetrazines, they proved to be well-suited for no-wash imaging (Fig. 3c). This indicates that there might be a cellular environmental effect on the Si-rhodamines' uorescence properties, which has been previously reported by Lukinavičius et al. 34 for spirolactoneforming Si-rhodamines. When reacted with a TCO-modied model protein, SiRh-5-Tz and MeSiRh-5-Tz showed turn-on ratios that were up to three times higher than with TCO alone (Table 1, ESI †). This effect might be even more pronounced in cellular environment. In the absence of phalloidin-TCO no specic staining of actin laments was observed (Fig. S5g-i †) with the tested tetrazine dyes.
To date, there are only few reports on tetrazine-mediated labeling inside living cells. 16,22,[34][35][36] Yet successful target visualization required washing steps aer addition of the tetrazine uorophore. The herein reported new uorogenic tetrazines enable no-wash imaging experiments inside living cells. We assumed that the OG-Tz dyes represent the best candidates for application in live cell labeling, as they combine high uorescence turn-on rates in DA inv with excellent photostability. Thus, we converted OG-5-Tz to its cell-permeable dipivalate ester derivative OG-5-Tz(piv) 2 , which is hydrolyzed to the parent dye by endogenous esterases aer having passed the cell membrane (Fig. 4a). For the labeling experiments HeLa cells expressing either mitochondrial Tom20-eDHFR (E. coli dihydrofolate reductase) or nuclear H2B-eDHFR were cultivated. We use the   (Fig. S5 †). We also surveyed the stability of OG-5-Tz by incubation in PBS, cell lysate and in presence of glutathione and found the probe to be highly stable within the timescale of DA inv (Fig. S1 †). Labeling of the target structures was conrmed by colocalization with commercially available MitoTracker Red CMXRos (mitochondria) and Hoechst 33342 (nuclei), respectively. We also recorded the live cell labeling of nuclear H2B-eDHFR in real-time and observed good signal-to-noise ratio within 30 min (Fig. 4e). OG-5-Tz(piv) 2 allows intracellular live cell labeling of POI fused to non-covalent domain tags (eDHFR tag) as well as POI carrying a covalent domain tag like HaloTag 39 (Fig. S4 †).

Conclusions
In conclusion, we have described two novel synthetic routes giving access to a variety of uorogenic xanthene tetrazine derivatives ideally suited for applications in cell biology. The highly water-soluble dyes exhibit strong uorescence enhancement upon conversion in DA inv and cover a broad visible emission range going from green to far-red. We have demonstrated their potential in no-wash protein imaging in xed and most notably inside living cells.
Notes and references § Regioisomer of TCO with an improved stability towards trans/cis-isomerization