Synthetic Host–Guest Assembly in Cells and Tissues: Fast, Stable, and Selective Bioorthogonal Imaging via Molecular Recognition

Bioorthogonal strategies are continuing to pave the way for new analytical tools in biology. Although a significant amount of progress has been made in developing covalent reaction based bioorthogonal strategies, balanced reactivity, and stability are often difficult to achieve from these systems. Alternatively, despite being kinetically beneficial, the development of noncovalent approaches that utilize fully synthetic and stable components remains challenging due to the lack of selectivity in conventional noncovalent interactions in the living cellular environment. Herein, we introduce a bioorthogonal assembly strategy based on a synthetic host–guest system featuring Cucurbit[7]uril (CB[7]) and adamantylamine (ADA). We demonstrate that highly selective and ultrastable host–guest interaction between CB[7] and ADA provides a noncovalent mechanism for assembling labeling agents, such as fluorophores and DNA, in cells and tissues for bioorthogonal imaging of molecular targets. Additionally, by combining with covalent reaction, we show that this CB[7]–ADA based noncovalent interaction enables simultaneous bioorthogonal labeling and multiplexed imaging in cells as well as tissue sections. Finally, we show that interaction between CB[7] and ADA fulfills the demands of specificity and stability that is required for assembling molecules in the complexities of a living cell. We demonstrate this by sensitive detection of metastatic cancer-associated cell surface protein marker as well as by showing the distribution and dynamics of F-actin in living cells.


Synthesis of CB[7]
-OH: CB [7] has been prepared and purified by following literature reported protocols 1,2 and characterized by 1 H NMR and HRMS. Next, mono-hydroxylation of CB [7] has been done by following a literature reported procedure with some modification 3 . CB [7] (250 mg, 0.215 mmol) was taken in a 50 ml quartz tube. It was dissolved in 40 ml solution of miliQ water and 10 M  Fig. S4.
2. The concentration of the antibody was measured and used for the conjugation with tetrazine.
6. After that tetrazine conjugated antibody was purified by Zeba spin column (pre-equilibrated with PBS).
2. The concentration of the antibody was measured and used for the conjugation with TCO .
9. TCO conjugated antibody was then purified by Zeba spin column (pre-equilibrated with PBS) and characterized by MALDI mass spectrometry. MALDI mass spectrum has been shown in

MALDI−MS analysis for characterization of antibody-CB[7] conjugate
Antibodies (1 µl) after desalting through Zeba spin column (~ 1 mg.ml -1 in miliQ water) was taken in micro centrifuge tube. 1 µl of Sinapinic acid (10 mg.ml -1 in 50:50 water (0.1% TFA)/acetonitrile) was mixed with the antibodies. The mixed solution was placed onto a MALDI plate and allowed to dry at room temperature for analysis. MALDI−MS analysis was performed using a Bruker autoflex system. Mass shift after conjugation was used to calculate the degree of CB [7] conjugation. We estimated an average of six CB [7] molecules attached with a single antibody. Mass shift after conjugation was used to calculate the degree of TCO conjugation. We estimated an average of fifteen numbers of TCO was attached with a single antibody.
3. 30.34 µl Tetrazine-NHS ester 9 (from a stock solution of 10 mg ml −1 in DMF, 0.7615 µmol) was added to it and the reaction was stirred at room temperature for 3 h.
4. After the reaction, the reaction mixture was diluted using 120 µl of mili Q water and injected to HPLC for purification using water/acetonitrile containing 0.1% TFA as eluent. The purity of the isolated compound was again analysed by HPLC (see Fig. S8). 0.076 µmol) was added to it and stirred at room temperature for overnight to prepare CB [7] conjugated phalloidin .

After purification
7. CB [7] conjugated phalloidin was then used for imaging of F-actin. S-14
3. 3.6 µl DBCO NHS ester (from a stock solution of 25 mg ml −1 in DMF, 0.1270 µmol) was added to it and the reaction was stirred at room temperature for 3 h. 4. After the reaction, the reaction mixture was diluted using 80 µl of mili Q water and injected to HPLC for purification using water/acetonitrile containing 0.1% TFA as eluent. The purity of the isolated compound was again analysed by HPLC (see Fig. S9).

Synthesis of compound 1
Mono tosylated tetraethylene glycol was synthesized following reported protocol with some modifications. 10 Tetra ethylene glycol (10 g, 51.22mmol) was dissolved in 130 ml acetonitrile in 250 ml RB Flask and Triethyl amine (7.13 ml, 51.22mmol) was added to it. Tosyl chloride (9.76 g, 51.22mmol), dissolved in 20 ml acetonitrile was added drop wise from dropping funnel over 1 h, keeping the reaction mixture at 0 o C. After addition, the reaction mixture was stirred at room temperature for 14h. During the reaction, a white precipitate of Triethyl ammonium hydrochloride was formed. After completion of reaction, the precipitate was filtered and washed with acetonitrile.

Synthesis of compound 2
A solution of NaOH (0.864 g, 21.6 mmol) in water (8 ml) was added to a solution of triphenylmethanethiol (3.98 g, 14.402 mmol) in a mixture of EtOH/toluene (1:1 v/v, 50 ml). The

S-17
mono tosylated product (compound 1, 5.019 g, 14.402mmol) was dissolved in a second solution of EtOH/toluene (1:1 v/v, 50 ml), which was then added to the Triphenylmethanethiol mixture in one portion. The reaction mixture was stirred for 18 hours at RT. After completion of reaction (monitored by TLC) the reaction mixture was poured into a saturated solution of NaHCO 3 (20 ml) and extracted with Et 2 O (3 × 40 ml). The combined organic layers were washed with brine (3 × 40 ml), dried over Na 2 SO 4 and solvent was removed under reduced pressure to give a pale−yellow oil. The crude product was purified by silica column (60-120 mess size) chromatography (eluent: hexane/EtOAc, 2:1→1:3) to give Trt-TEG-OH as a pale-yellow oil ( Fig. S13.

Synthesis of compound 4
Adamantane amine hydrochloride (176.2 mg, 0.9386 mmol) and potassium carbonate (260 mg, 1.8772 mmol) and Trt-TEG-Oms (500 mg, 0.9386 mmol) were dissolved in 2 ml of DMF placed in a 10 ml RB flask. The reaction mixture was heated to 85 o C and stirred for 12h. After completion of reaction (monitored by TLC), the reaction mixture was cooled down at room temperature. 10 ml Methanol was added to the reaction mixture and the solid ppt was filtered off and filtrate was concentrated using rotary evaporator. 20 ml water was added to the crude product and product was extracted using ether (3x2 ml) from aqueous layer. Organic layer was dried over Na 2 SO 4 and purified S-18 by Flash silica column chromatography (Eluent: gradient eluent of 0-5% methanol in DCM v/v).  Fig. S15 and S16 respectively.      dissolved in 100 µL DMF was added to it and the reaction mixture was stirred at room temperature for 12 h. After that the reaction mixture was diluted to 1 ml using Mili Q water and purified by reverse phase column chromatography using Biotage @ SNAP C18 cartridge (eluent: a gradient elution using acetonitrile/water from 0 to 100%). The blue colored eluent from the column was collected, concentrated [M+H] + . HRMS spectrum of Cy5-maleimide is shown in Fig. S17. µmol) dissolved in 100 µL DMF was added to it and the reaction mixture was stirred at room temperature for 12 h. After that the reaction mixture was diluted to 1 ml using Mili Q water and purified by reverse phase column chromatography using Biotage @ SNAP C18 cartridge (eluent: a gradient elution using acetonitrile/water from 0 to 100%). The blue colored eluent from the column was collected, concentrated and lyophilized to yield Rhodamine maleimide (1.4 mg, 68%, a blue HRMS spectrum of Rhodamine-maleimide is shown in Fig. S19.

DNA-PEG 2 -maleimide
DNA-NH 2 (10 nmol, 10 µl from 1 mM stock in water) was taken in a micro centrifuge tube. 1.3 µl 10× PBS was added to it. Maleimide-PEG 2 -succinimidyl ester (42.54 µg, 100 nmol, 1.7 µl from 25 mg.ml -1 stock in DMF) was added to it and stirred at room temperature for 3 h. The conjugated product was purified by reversed phase HPLC after passing through Zeba spin column. The purified product was lyophilized and finally dissolved in PBS for ADA conjugation.

DNA-ADA conjugate
DNA-PEG 2 -maleimide (5 nmol, 10 µl from 500 nM stock in PBS) was taken in a micro centrifuge tube. ADA-PEG-thiol (34.35 µg, 100 nmol, 17.18 µl from 2 mg.ml -1 stock in PBS) was added to it and stirred at room temperature for 12 h. The conjugated product was purified by reversed phase HPLC. The purified product was lyophilized and characterized by MALDI mass spectrometry (Fig.   S25).

Synthesis of DNA-Atto 655 conjugate
DNA-NH 2 (15 nmol, 15 µl from 1 mM stock in water) was taken in a micro centrifuge tube and diluted to 55 µl using water. 8 µl 1M NaHCO 3 was added to it. Atto 655 NHS ester (33.2 µg, 37.5 nmol, 16.6 µl from 2 mg.ml -1 stock in DMF) was added to it and stirred at room temperature for 12 h.
The conjugated product was purified by reversed phase HPLC after passing through Zeba spin column. The purified product was lyophilized and finally stored in water for DNA−PAINT imaging.

MALDI mass spectrometry analysis of DNA-ADA conjugate
ADA conjugated DNA (1 µl, 100 µM in miliQ water) was taken in a micro centrifuge tube. 4-hydroxy picolinic acid (10 mg.ml -1 dissolved in 50:50 water/acetonitrile, 1 µl) was mixed with it. The mixed solution was plated onto the MALDI plate and allowed to dry at RT from analysis.      incubation of Tz−Cy5 with TCO labeled microtubules. A significant reduction in labeling efficiency was observed from this experiment. Scale bars: 5 µm (a and b).

CB[7]-ADA mediated labeling of microtubule in variable pH conditions:
Microtubules in HeLa cells were targeted using CB [7] conjugated antibody against beta-tubulin.
CB [7] labeled cells were incubated with ADA conjugated cy5 fluorophores (Conc. = 100 nM) in different pH solutions ranging from 4.5 to 9.2. The compositions of buffers used in this study are mentioned in the following table.
Supplementary  Microtubules in HeLa cells were targeted using CB [7] conjugated antibody against beta-tubulin.

Dissection of thoracic muscle tissue and ovary tissue
Adult wild type drosophila flies were collected which were maintained in 12 h light and 12 h dark at 25°C. Once collected, flies were kept in ice for around 15 minutes for anesthetizing.
Thoracic muscle dissection 1. After flies were anesthetized, they were submerged in PBS placed dorsally on the dissection plate and were pierced with insect pins on the abdomen region.
2. Using forceps, top layer of thorax was dissected slowly, peeled out gently and bunch of clustered thoracic muscles were taken out.
4. Three differently labelled cells were transferred to 8-well chamber slide system and bright field immunofluorescence microscopic images were captured immediately using a motorized inverted microscope (IX81, Olympus, Tokyo, Japan) equipped with a CCD camera (FVII with CellP software, Olympus, Tokyo, Japan).