An Optical Probe for Real-Time Monitoring of Self-Replicator Emergence and Distinguishing between Replicators

Self-replicating systems play an important role in research on the synthesis and origin of life. Monitoring of these systems has mostly relied on techniques such as NMR or chromatography, which are limited in throughput and demanding when monitoring replication in real time. To circumvent these problems, we now developed a pattern-generating fluorescent molecular probe (an ID-probe) capable of discriminating replicators of different chemical composition and monitoring the process of replicator formation in real time, giving distinct signatures for starting materials, intermediates, and final products. Optical monitoring of replicators dramatically reduces the analysis time and sample quantities compared to most currently used methods and opens the door for future high-throughput experimentation in protocell environments.


Synthesis of compound 2b:
Compound 7 (0.10 g, 0.29 mmol) was dissolved in DCM (5.0 mL) and the solution was basified (judging by wet pH paper) with DIPEA (0.10 mL, 0.58 mmol). Coupling reagent HATU (0.22 g, 0.58 mmol) was added to the above reaction mixture and stirred for 10 min at room temperature. Then 8 (0.09 g, 0.32 mmol) was added and the reaction was continued at room temperature for another 3h under argon. After the reaction was completed (judging by silica TLC Plate, 4% methanol in DCM, R f : 0.2 (compound 7), 0.6 (product)), the solvent was removed in vacuum and the residue was dissolved in DCM (10.0 mL) and washed twice with water (20.0 mL). The organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by column chromatography (silica gel, 1.5% MeOH in DCM) to afford 9 as a yellow foamy substance (0.145 g, 77%). 1  To obtain compound 2b, the tert-butyl ester group of 9 was deprotected by adding TFA (2.0 mL) to a solution of 9 (0.10 g, 0.17 mmol) in DCM (2.0 mL) under ice cooling condition. The reaction mixture was allowed to warm up to room temperature and stirring was continued for another 3 h. After the reaction was completed, DCM and TFA were evaporated. The traces of TFA were removed by co-evaporation with DCM and the product was used in the next step without further purification.
The solvent was removed in vacuum and the residue was suspended in water (50.0 mL) and stirred for 4h followed by vacuum filtration, and dried under reduced pressure. The crude material washed with hexane and purified by column chromatography (silica gel, 75% EtOAc in hexane) to afford 12 as a white solid (0.087 g, 81%). 1 compound 14), 0.6 (product)).
After the reaction was completed (6h), the solvent was removed in vacuum and the residue was washed with hexane. The residue was dissolved in DCM (40.0 mL) and washed twice with 5% HCl (20.0 mL) and water (30.0 mL).
The organic layer was dried over anhydrous magnesium sulfate, evaporated to dryness, and the crude material was purified by column chromatography

Synthesis of compound 21:
Compound 19 (0.05 g, 0.11 mmol) was dissolved in DCM (5.0 mL) and the solution was basified (judging by wet pH paper) with DIPEA (44 µL, 0.25 mmol). Coupling reagent HATU (0.10 g, 0.25 mmol) was added to the above reaction mixture which was stirred for 10 min at room temperature.
Then compound 18 (0.10 g, 0.13 mmol) was added and reaction was continued at room temperature for another 24h under argon. After the reaction was completed (judging by silica TLC plate, 8% methanol in DCM, R f : 0.9 (compound 19), 0.7 (product)), the solvent was removed in vacuum and the residue was dissolved in DCM (10.0 mL) and washed with water (20.0 mL), brine (20.0 mL), and again with water (2 × 20.0 mL). The organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by column S12 chromatography (silica gel, 7% MeOH in DCM) to afford 20 as a deep brown solid (0.109 g, 71% and TFA were evaporated and excess diethyl ether (25.0 mL) was added and the suspension was filtered.
The residue was dried under high vacuum for 5h and used in the next step without further purification.

Synthesis of compound 22:
Compound 2b (0.05 g, 0.09 mmol) was dissolved in DCM (5.0 mL) and the solution was basified (judging by wet pH paper) with DIPEA (30 µL, 0.18 mmol). Coupling reagent HATU (0.07 g, 0.18 mmol) was added to the above reaction mixture which was stirred for 10 min at room temperature. Then

Synthesis of compound 2c:
Compound 22 (0.10 g, 0.06 mmol) was dissolved in 20% piperidine in DMF (3.5 mL). The reaction mixture was stirred for 1h at room temperature in the dark. After the reaction was completed (judging

Synthesis of compound 2a:
Compound

Synthesis of compound 24:
Compound 7 (0.10 g, 0.29 mmol) was dissolved in DCM (5.0 mL) and the solution was basified (judging by wet pH paper) with DIPEA (0.10 mL, 0.58 mmol). Coupling reagent HATU (0.22 g, 0.58 mmol) was added to the above reaction mixture which was stirred for 10 min at room temperature. Then compound 13 (0.12 g, 0.29 mmol) was added and the reaction was continued at room temperature for another 6h under argon. After
The reaction mixture was stirred for 1h at room temperature in the dark. After the reaction was completed (judging by neutral Al 2 O 3 TLC plate, 2% methanol in DCM, R f : 0.7 (compound 24), 0.4 (product)), the solvent was removed in vacuum and the residue was extracted with DCM (20.0 mL).
The organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by column chromatography (Neutral Al 2 O 3 , 7.5% MeOH in DCM) to afford compound 25 as a yellowish solid (0.065 g, 80%). 1         S26 Figure S11. UPLC traces (monitored at 254 nm) and mass spectra (bottom and top panels show theoretical and observed mass, respectively) recorded for monomers 1b, mixture of trimers-tetramers (1b) 3 /(1b) 4 , and fibers (1b) 8 prepared from building 1b using the protocol described in the methods section (main text). Figure S12. UPLC traces (monitored at 254 nm) and mass spectra (bottom and top panels show theoretical and observed mass, respectively) recorded for monomers 1c, mixture of trimers-tetramers (1c) 3 /(1c) 4 , and fibers (1c) 8 prepared from building 1c using the protocol described in the methods section (main text).