General Strategy
Nguyet Mahrenholz
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The carbon skeleton of any organic molecule serves as the foundation for its three-dimensional structure, playing a pivotal role in determining its physical and biological properties.1As such, taxane diterpenes are one of the most well known natural product families, primarily owing to the success of their most prominent compound, paclitaxel, an effective anti-cancer therapeutic for more than 25 years.2-6 In contrast to classical taxanes, the bioactivity of cyclotaxanes (also referred to as complex taxanes) remains significantly underexplored. The carbon skeletons of these two groups of taxanes differ significantly, and so would typically their own distinct synthetic approaches. Here, we report a versatile synthetic strategy based on the interconversion of complex molecular frameworks, providing general access to the wider taxane diterpene family. A range of classical and cyclotaxane frameworks was prepared including, among others, the first total syntheses of taxinine K (2), canataxapropellane (5) and dipropellane C from a single advanced intermediate. The synthetic approach deliberately eschews biomimicry, emphasizing instead the power of stereoelectronic control in orchestrating the interconversion of polycyclic frameworks.
Fluorescence imaging is an invaluable tool to study biological processes and further progress depends on the development of advanced fluorogenic probes that reach intracellular targets and label them with high specificity. Excellent fluorogenic rhodamine dyes have been reported, but they often require long and low-yielding syntheses, and are spectrally limited to the visible range. Here we present a general strategy to transform polymethine compounds into fluorogenic dyes using an intramolecular ring-closure approach. We illustrate the generality of this method by creating both spontaneously blinking and no-wash, turn-on polymethine dyes with emissions across the visible and near-infrared spectrum. These probes are compatible with self-labelling proteins and small-molecule targeting ligands, and can be combined with rhodamine-based dyes for multicolour and fluorescence lifetime multiplexing imaging. This strategy provides access to bright, fluorogenic dyes that emit at wavelengths that are more red-shifted compared with those of existing rhodamine-based dyes.
Fluorescence microscopy is crucial to study the structure and function of cells. Fluorescent protein tags allow for the dynamic observation of proteins in living cells, but their brightness and photostability are often inferior to those of small-molecule fluorophores1. Fluorogenic dyes conjugated to self-labelling protein tags such as HaloTag2 or SNAP-tag3 combine the excellent photophysical properties of small-molecule dyes with the precise labelling of genetically encoded tags and have been widely used for fluorescence microscopy and nanoscopy.
We next investigated whether we could induce fluorescence turn-on upon conjugation of dye 4b to a self-labelling protein tag. Given that HaloTag has been thoroughly optimized for rhodamine-based dyes2,41, we chose to work with SNAP-tag42, thereby providing an orthogonal system that could be used in multiplexing studies with rhodamines and HaloTag. We synthesized the azide-modified SNAP-tag ligand 10 using the diazotizing reagent FSO2N3 (ref. 43) and combined it with alkyne-modified Cy5 dye 4c in a click reaction to generate probe 11 (Fig. 2d and Methods). This dye displayed a 10-fold turn-on in absorbance and a 21-fold turn-on in fluorescence when incubated with purified SNAP-tag protein (Fig. 2e and Methods), demonstrating its fluorogenicity upon binding to a self-labelling tag. Live-cell experiments using SNAP-tag fused to a fragment of histone H2B demonstrated that although the cellular uptake of compound 11 is not enhanced compared to its 5-endo-trig derivative, it labels SNAP-tag more efficiently and display much less non-specific signal (Supplementary Fig. 3).
Carbocyanine dyes can cover a large spectral range by varying the number of conjugated carbon atoms in between the two indoleninium moieties. We therefore explored whether the 5-exo-trig fluorogenic strategy could be extended to Cy3 and Cy7 dyes, providing fluorophores for two extra imaging channels. We suspected that the Cy3 derivative would have a higher LUMO energy than Cy5, whereas the opposite would be true for the Cy7 derivative; thus, we expected the Cy3 dyes to be more likely to adopt the open form than Cy5 dyes, whereas Cy7 dyes would tend to adopt the closed form. To balance these trends, we added a CF3 moiety on the capping indoleninium to favour the closed form of Cy3. Similarly, we replaced the nucleophilic N-methyl amide with an electron-deficient amide to facilitate ring-opening in our Cy7 design. From a synthetic point of view, compared with the preparation of Cy5 derivatives, we only changed the commercially available linker and adjusted the temperatures during the microwave-assisted protocol (Supplementary Information).
We demonstrated the versatility of our turn-on strategy by using the self-labelling protein tag SNAP-tag, as well as jasplakinolide or Hoechst 33342 dye, to drive fluorescence turn-on. The mechanism of fluorescence turn-on of 5-exo-trig to such varied macromolecular targets remains to be fully elucidated. Preliminary modelling results suggest that ring-opening could be triggered by specific interactions (for example, hydrogen bonds) between the lactam rings and ubiquitous amides in proteins or phosphate groups in nucleic acids (Supplementary Discussion 2). More detailed studies including molecular dynamics simulations and site-directed mutagenesis could shed further light on the mechanism of fluorescence turn-on for specific 5-exo-trig polymethine dyes.
Given the high modularity of polymethine dyes, the spectral range can be further extended into the green (for example, Cy1 dyes50) as well as into the shortwave infrared (for example, Cy9 dyes51) wavelengths. Furthermore, the photophysical properties and fluorogenicity of polymethine dyes could be further tuned by varying the substituents on the indolenines or the linker13. We envision that this simple, yet general, method will be used to develop improved fluorogenic probes, facilitating new bioimaging experiments.
Absorbance spectra were recorded in 96-well plates (Corning) on the Multiskan SkyHigh microplate reader and fluorescence measurements were performed on the FS5 Spectrofluorometer (Edinburgh Instruments) equipped with a SC-40 plate holder. All spectroscopic measurements were carried out in triplicates and at room temperature.
Confocal imaging was performed with a Nikon W1 spinning disk microscope operated with NIS Elements AR software equipped with a CMOS camera (Photometrix). Brightfield imaging was performed with a white LED. Laser lines and filters were set up for the appropriate channel as described in Supplementary Table 5.
For TD-DFT calculations, we first performed a systematic conformational search at the B3LYP/DGTZVP level of theory with an implicit solvation model (IEFPCM) by varying all rotatable bonds in 60 steps. We then applied the Boltzmann distribution to the set of low-energy minima obtained by using the free energy differences, and considered the structures above the 0.1% population threshold for the TD-DFT calculation. Time-dependent density functional theory calculations were performed at the CAM-B3LYP/DGTZVP level of theory with IEFPCM as the solvation model.
All data supporting this paper, including coordinates for all calculated structures, are available through Zenodo53. X-ray crystallographic datasets used for modelling are available from the PDB under accession nos. 6Y8P and 1DNH. Samples of small-molecule probes are available from the authors on reasonable request. Source Data are provided with this paper.
This work was supported by EPFL (SViPhD internal grant, P.R.-F.), University of Zurich, and the European Research Council (ERC Starting Grant HDPROBES no. 801572 to P. R.-F.). We thank S. Emmert for assistance with FLIM, L. Blatti (University of Zurich) for the synthesis of some indoleninium building blocks, K. Gademann for access to a polarimeter, and P. Gnczy and L. Reymond for valuable discussions. This work made use of infrastructure services provided by SCITAS, the Scientific IT and Application Support of EPFL, and the Service and Support for Science IT (S3IT) at the University of Zurich. Samples of jasplakinolide and Hoechst 33342 ligands were donated by Spirochrome AG ( ). FLIM was performed at the Center for Microscopy and Image Analysis of the University of Zurich. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
A.M. and P.R.-F. conceived the method, wrote the manuscript, perfomed computational modelling and analysed the results. A.M. performed all of the experiments. P. R.-F. acquired funding and supervised the project.
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