Thioesters provide a plausible prebiotic path to proto-peptides

It is widely assumed that the condensation of building blocks into oligomers and polymers was important in the origins of life. High activation energies, unfavorable thermodynamics and side reactions are bottlenecks for abiotic peptide formation. All abiotic reactions reported thus far for peptide bond formation via thioester intermediates have relied on high energy molecules, which usually suffer from short half-life in aqueous conditions and therefore require constant replenishment. Here we report plausible prebiotic reactions of mercaptoacids with amino acids that result in the formation of thiodepsipeptides, which contain both peptide and thioester bonds. Thiodepsipeptide formation was achieved under a wide range of pH and temperature by simply drying and heating mercaptoacids with amino acids. Our results offer a robust one-pot prebiotically-plausible pathway for proto-peptide formation. These results support the hypothesis that thiodepsipeptides and thiol-terminated peptides formed readily on prebiotic Earth and were possible contributors to early chemical evolution.


Materials
All chemicals and reagents were obtained from Sigma-Aldrich and used as received.

Thioglycolic-L-alanine methyl ester bisamide disulfide, 1a
Dithiodiglycolic acid (0.91 g, 5 mmol, Sigma) was suspended in 40 mL dry DCM. HOBt hydrate (1.84 g, 12 mmol) was added, followed by EDC (2.3 g, 12 mmol) and L-Ala-OMe hydrochloride (1.47 g, 10.5 mmol). Finally, triethylamine (2.09 mL, 15 mmol) was added. The initially cloudy mixture gradually cleared to a reddish solution that stirred at RT overnight. The reaction was diluted with DCM (100 mL) and washed 2x with sat. NaHCO3, 2x with sat. KHSO4, 1x with brine, and dried over MgSO4. TLC (visualized by UV) with 7:3 EtOAc/hexanes showed conversion to the product (Rf of 0.2), and the product mass was confirmed by TLC-ESI-MS (obs'd. 352.8 for M+H, obs'd. 374.7 for M+Na). The solvent was evaporated to yield a yellow oily solid (1.7 g crude weight). The product was purified by flash chromatography using 100 g silica in a 5 cm diameter column with 2% MeOH/DCM as the eluent. After evaporation of the solvent from fractions containing the product, a pale yellow solid was obtained (1.53 g, 87%).

Thioglycolic-L-alanine, 2a
The bisamide disulfide 1a was dissolved in 20% H2O/THF (19 mL). The solution was degassed by bubbling Ar for 10 min, after which Bu3P was added slowly and the mixture stirred at RT for 10 min. TLC (7:3 EtOAc/hexanes, visualized by UV) indicated the disulfide reduction was complete. The mixture was diluted with 7.5 mL ethanol, after which 9.25 mL 2 M NaOH was added. The solution stirred at RT for 1 h. The solvent was evaporated and the residue was taken up in water and extracted 3x with EtOAc. The organic layers were discarded. The aqueous layer was acidified using 1 N HCl to pH 2. The aqueous layer was evaporated to a gummy oil.

Synthesis of thioglycolic-L-alanine-L-Alanine standard
2-(tritylthio)acetic acid (535 mg, 1.6 mmol, Ambeed) and HOBt hydrate (276 mg, 1.8 mmol) were dissolved in DCM (10 mL) and placed in an ice bath. EDC (345 mg, 1.8 mmol) was added, followed by L-Ala-OMe hydrochloride (237 mg, 1.7 mmol). Triethylamine (0.28 mL, 2.0 mmol) was added and the reaction stirred and was allowed to warm to RT overnight. The solvent was removed and the residue was taken up in EtOAc. The organics were washed with saturated NaHCO3 (3x), saturated KHSO4 (3x), and brine. The organic layer was dried over MgSO4, and the solvent was evaporated to yield 0.64 g (95%) of a clear oil. The product was used without further purification. The methyl ester was removed by dissolving the compound (0.64 g, 1.5 mmol) in MeOH (4 mL) and adding a solution of LiOH monohydrate (0.21 g, 5.0 mmol) in water (4 mL).
The cloudy mixture was stirred at RT for 1 h, after with HPLC indicated completion of the reaction.
The solvent was evaporated and the residue was taken up in EtOAc and 1 N HCl. The organic layer was removed, and the aqueous layer was extracted once more with EtOAc. The combined organic layers were washed with brine and dried over MgSO4. Evaporation of the solvent yielded 0.61 g (98%) of a white foamy solid. Without further purification, the acid thus obtained was coupled to L-Ala-OMe hydrochloride. The acid (200 mg, 0.5 mmol) and HOBt hydrate (87 mg, 0.57 mmol) were dissolved in DCM (5 mL

Synthesis of thioglycolic acid-L-alanine-L-Alanine-L-Alanine standard
The compound was synthesized via standard Fmoc solid-phase peptide synthesis protocols on 0.3 mmol scale using Fmoc-Ala-Wang resin. Coupling reactions were carried out using a five-fold excess of oxyma, diisopropylcarbodiimide, and Fmoc-protected Ala or 2-(tritylthio)acetic acid (Ambeed, CAS # 34914-36-8) for 75 min. Fmoc deprotection steps were carried out by treating the resin with 25% 4-methylpiperidine/DMF twice for 10 min. The peptide was cleaved from the resin using 94:2:2:2 TFA/H2O/ethanedithiol/triethylsilane for 90 min. The cleavage solution was added dropwise to cold diethyl ether (100 mL), and the crude peptide was obtained by centrifugation. The desired compound was purified by preparative HPLC using a water/acetonitrile/TFA solvent system. Fractions containing the product were lyophilized to yield 67 mg of a white powder (73% yield based on resin loading). Purity was verified by analytical HPLC.
Next, diisopropylethylamine (1.6 mL, 9.16 mmol) was added. After 1 h, the ice bath was removed and the solution was stirred at room temperature for 14 h. The solvent was evaporated and the residue was taken up in EtOAc. The organics were washed 3 times with saturated KHSO4, once with brine, and dried over magnesium sulfate. After filtration and removal of the solvent, a white
The resulting white solid was dissolved in 2 N HCL (20 mL) and evaporated (two times) to exchange the TFA salt for the HCl salt. The resulting gummy white solid was lyophilized and used directly in the next step. Methylene chloride (80 mL) was added to the residue and the suspension was chilled to 0 C. Next were added EDC.HCl (642 mg, 3.35 mmol), HOBt.H2O (512 mg, 3.35 mmol), and diisopropylethylamine (1.07 mL, 6.14 mmol). The mixture was stirred vigourously for 3 h. The organics were washed 2 times with saturated NaHCO3, 2 times with saturated KHSO4 once with brine, and dried over magnesium sulfate. After filtration and removal of the solvent, a white solid was obtained. The solid was taken up in a minimal volume of hot acetonitrile and allowed to cool slowly to yield colorless crystals (223 mg, 51% yield).

Crystal structure determination of (S)-3-methylthiazine-2,5-dione
The single crystal X-ray diffraction studies were carried out on a Bruker D8 Venture Ultra diffractometer equipped with Mo K radiation ( = 0.71073). Crystals of the subject compound were used as received (grown from Acetonitrile). A 0.250 x 0.200 x 0.175 mm colorless block was mounted on a Cryoloop with Paratone oil.
Data were collected in a nitrogen gas stream at 100(2)K using  and  scans. Crystal-to-detector distance was 50 mm using exposure time 2s (depending on the detector 2 position) with a scan width of 0.70°. Data collection was 99.8% complete to 25.242° in . A total of 14669 reflections were collected covering the indices, -9<=h<=9, -10<=k<=10, -13<=l<=13. 2831 reflections were found to be symmetry independent, with a Rint of 0.0395. Indexing and unit cell refinement indicated a Primitive, Monoclinic lattice. The space group was found to be P21. The data were integrated using the Bruker SAINT Software program and scaled using the SADABS software program. Solution by direct methods (SHELXT) produced a complete phasing model consistent with the proposed structure.
All carbon bonded hydrogen atoms were placed using a riding model. Their positions were constrained relative to their parent atom using the appropriate HFIX command in SHELXL-2014.
The data have been deposited to CCDC as a pre-publication file (Deposition Number #2078153).

Supplementary Figure 4. Minimal oxidation occurs upon dry-down reactions under
experimental anoxic conditions. tg and L-Ala were dried at a 5:1 molar ratio (in favor of tg) at 65 ˚C for seven days and the resulting products were analyzed by hydrophobicity-based separation using C18-HPLC before (black) and after (red) incubation with 500 mM TCEP for 1 hr at RT.
Blue arrows indicate species that were reduced with TCEP.

Supplementary Figure 6. Fourier Transform Infrared Spectroscopy (FTIR) shows shifts in
the C=O stretch upon dry-down of tg, supportive of thioester formation. tg was dried at 65 ˚C for seven days and the resulting mixture was analyzed by FTIR (A-B). Panel B is an enlargement of the 1500-1800 cm -1 region. Thioesters have lower carbonyl stretching frequencies than esters (2). Upon dry-down of tg, the C=O stretch shifts from a carboxylic acid to lower frequencies, as expected following formation of thioesters (2,3). Changes were also observed in the thiol S-H stretch (~2570 cm -1 ) following the dry-down of tg (4,5).