Thioethers

The [Cys–Arg–Glu–Lys–Ala] peptide was synthesized using standard Fmoc-mediated solid phase peptide synthesis methods on rink Amide resin (Anaspec, Fremont, CA, USA) using an automated PS3 Benchtop Peptide Synthesizer (Protein Technologies, Tucson, AZ, USA). Peptides were cleaved and deprotected with 94:2.5:2.5:1 by volume trifluoroacetic acid: 1,2-ethanedithiol:H₂O:triisopropylsilane and were precipitated and washed several times with cold diethyl ether, dissolved in water, lyophilized, and stored as lyophilized powders at 20 °C. Crude, peptide mixtures were purified by reverse-phase HPLC (Prominence, Shimadzu, Columbia, MD, USA) on a C8 column (Waters, Milford, MA) at 55 °C using 0.1% trifluoroacetic acid in acetonitrile/water mixtures and characterized by MALDI-TOF/TOF mass spectral analysis (Biflex III, Bruker, Billerica, MA, USA). Cysteine-containing peptides were conjugated via a thioether linkage to DSPE-PEG(2000)-maleimide (Avanti Polar Lipids, Alabaster, AL, USA) by adding a 10% molar excess of the lipid to peptide in water. After reaction at RT for 24 h, the resulting product was purified and characterized as described above. Cy7 was conjugated via a peptide bond to DSPE-PEG(2000)-amine (Avanti Polar Lipids, Alabaster, AL, USA) by adding an equal molar equivalent of Cy7 mono-N-hydroxysuccinimide ester (GE Healthcare Life Sciences, Pittsburgh, PA, USA) to the lipid dissolved in 10 mM aqueous sodium carbonate buffer (pH 8.5). After reaction at RT for 24 h, the mixture was also purified on a C4 column and characterized as described above.
CREKA micelles were assembled by dissolving the Cy7 and peptide containing DSPE-PEG(2000) amphiphiles in methanol (10:90 molar ratio), mixing the components, and evaporating the mixed solution under nitrogen. The resulting film was dried under vacuum O/N, and then hydrated at 80 °C for 30 min in water or PBS and allowed to cool to RT. Non-targeting micelles were assembled by combing DSPE-PEG(2000)-Cy7 and DSPE-PEG(2000)-maleimide.

Based on the AspH-TPR-Ox:hFX crystal structure, stable cyclic peptides comprising the core ring residues (aa 101–110) of the hFX EGF1_39mer-substrate with a thioether replacing the Cys3–4 disulfide were synthesized. The D-stereochemistry of the N-terminal amino acid (aa 101) aligns the peptide side chain with the main chain of the original substrate.
Commercial Fmoc-protected amino acids (AGTC Bioproducts; Alfa Aesar; CSBio; Iris Biotech; Novabiochem; Sigma-Aldrich; TCI) were used as received. SPPS was performed using a CSBio CS336X automated peptide synthesizer following standard Fmoc-strategy: linear peptides were synthesized from the C- to N-termini on a 0.1 mmol scale using a Rink amide linker using N,N-diisopropylcarbodiimide/1-hydroxybenzotriazole for coupling.
To obtain cyclic peptides, the N-terminal Fmoc-protecting group was cleaved on the resin, and the resin suspended in DMF (4 mL) containing N-chloroacetylsuccinimide^{73 (link)} (150 mg). The mixture was gently shaken for 3 h, filtered, and the dried resin treated with a solution of trifluoroacetic acid, triisopropylsilane, and water (4 mL; 95/2.5/2.5). After 3 h, the mixture was filtered and the solution diluted with 45 mL cold diethyl ether. The suspension was centrifuged (4000 rpm, 10 min, 4 °C), decanted, and then taken up in 1.5 mL aqueous triethylammonium acetate buffer (1 M, pH 8.5, pH readjusted with trimethylamine) and heated for 10 min at 100 °C in a microwave reactor (Biotage Initiator). Directly afterwards, the crude cyclic peptides were purified by semi-preparative HPLC (DionexTM UltiMate^® 3000, Thermo Scientific) using a reverse phase column (Grace Vydac^® 218TP101522) and a gradient of acetonitrile in milliQ water (each containing 0.1%_v/v trifluoroacetic acid) specified in Supplementary Fig. 14.

To assess the S oxidation activity, the aromatic thioethers methyl-p-tolyl sulfide sulfide (Cat. # 275956-5G, Sigma-Aldrich) and the bulky benzyl phenyl sulfide (Cat. # 8415660025, Sigma-Aldrich) were tested^{52 (link),53 (link)}. For the N oxidation activity, benzydamine (Cat. # B5524-5G, Sigma-Aldrich) and tamoxifen (Cat. # T5648-1G, Sigma-Aldrich) were selected^{54 (link),55 (link)}. To follow the BV activity the aliphatic hepta-2-one (Cat. # 537683-100 ML, Sigma-Aldrich), the alicyclic cyclohexanone (Cat. # 29140-100 ML, Sigma-Aldrich) and the aromatic phenylacetone (Cat. # 135380-100G, Sigma-Aldrich) were selected^{56 ,57} .
Substrate conversions were done in duplicates, using 1.0–5.0 mM substrate (1% methanol), 0.10 mM NADPH, 2.0 µM enzyme, 5.0 µM phosphite dehydrogenase (PTDH, produced in-house⁵⁸ ), and 20 mM sodium phosphite. The last two components were used as a regeneration system for NADPH and the control did not contain any tAncFMO. All compounds were prepared in storage buffer (50 mM KPi, 250 mM NaCl, 0.05% Triton^Tm X-100 reduced, pH 7.5) the final reaction volume was adjusted to 1.0 ml and put into 4 ml vials before being incubated at 30 °C, with shaking, for 16 h. Conversions of phenylacetone, heptan-2-one, cyclohexanone, benzyl phenyl sulfide and methyl-p-tolyl sulfide were analyzed by GC–MS while benzydamine and tamoxifen conversions were monitored by HPLC. Due to their poor solubility, tamoxifen, benzydamine, and benzyl phenyl sulfide conversions were done using 1.0 mM substrate while the remaining substrates were tested at 5.0 mM.

Thioether-stabilized
Ru nanoparticles were generated using ethyl sulfide, butyl sulfide,
and octyl sulfide. RuCl₃·H₂O (0.1 g, 0.49
mmol; 0.49 mmol; and 0.49 mmol) was dissolved in 20 mL of ethanol
in three Erlenmeyer flasks. The stabilizing ligands ethyl sulfide,
butyl sulfide, and octyl sulfide (0.427 g, 4.7 mmol; 0.713g, 4.8 mmol;
and 1.255 g, 4.8 mmol) were added to the solutions, respectively.
All solution colors were dark orange at the beginning of the reaction.
The flasks were capped with a rubber septum and stored in a vacuum
desiccator. After 5 h, a 1 μL aliquot of each solution was removed
to make a grid for TEM imaging.

To a DCM solution of NCBrS2 (30 mg, 0.087 mmol), a suspension of Ni(COD)₂ (24 mg, 0.087 mmol) was added. The mixture was stirred at room temperature for 5 min. The solution was filtered through celite to remove Ni black. The desired product was crashed out as a reddish brown powder by adding an excess amount of Et₂O. Yield: 13 mg, 54%. X-ray quality crystals of [(NCS2)Ni(μ-Br)]₂ were obtained by slow diffusion of Et₂O diffusion into a dichloromethane solution at ‒35 °C
UV–vis (DCM; λ nm (ε, M⁻¹ cm⁻¹)): 335, 470 nm.
Elemental Analysis: found, C 41.35%, H 3.15%, N 3.19%; calculated C₃₀H₂₈Br₂N₂Ni₂S₄·CH₂Cl₂, C 41.06, H 3.33, N 3.09%.
MALDI-TOF MS (m/z): 820.8760; calcd. for [(NCS2)Ni(µ-Br)]₂ + H⁺, C₃₀H₂₈Br₂N₂Ni₂S₄: 820.8267.
To a DCM solution of [(NCS2)Ni(µ-Br)]₂, TlPF₆ was added in a MeCN solution. The color changes from red to yellow-brown. It was stirred for two hours, and the solution was filtered through a syringe filter to separate the TlBr. The dark brown solution was reduced in volume and a brown powder was crashed out by adding excess diethyl ether. Despite several attempts using different counterions, no crystal of 2 could be grown. But it was characterized by IR, ESI-MS, EA, and UV–vis. A solution magnetic moment of 2.68 μ_B, corresponding to two unpaired electrons is suggestive of an octahedral geometry in solution.
IR (ν_CN): 2331, 2303 cm⁻¹UV–vis (MeCN; λ nm (ε, M⁻¹ cm⁻¹)): 273 (280), 308 (60)
ESI-MS (m/z): 518.9871; calcd for [(NCS2)Ni(MeCN)(OTf)]-H, C₁₈H₁₆F₃N₂NiO₃S₃: m/z 518.9629.
Evan’s Method: µ_eff = 2.68 µ_BWhen attempting to recrystallize 2 out of a MeCN/Et₂O solution, we often found crystals of [(NCHS2)Tl][PF₆], which points towards the proclivity of the soft thioether ligands to coordinate to the soft Tl⁺.
Elemental Analysis: found, C 38.38%, H 3.38%, N 6.46%; calculated C19H20F6N3NiPS2·0.1 TlPF₆, C 38.48, H 3.40, N 7.08%.