2-cyanopyridine derivatives enable N-terminal cysteine bioconjugation and peptide bond cleavage of glutathione under aqueous and mild conditions

Inspired by the chemical reactivity of apalutamide, we have developed an efficient method for N-terminal cysteine bioconjugation with 2-cyanopyridine derivatives. Systematic investigations of various 2-cyanopyridines revealed that 2-cyanopyridines with electron-withdrawing groups react efficiently with cysteine under aqueous and mild conditions. Moreover, the highly reactive 2-cyanopyridines enable the peptide bond cleavage of glutathione. The utility of our method is demonstrated by its application to the cysteine-selective chemical modification of bioactive peptides.


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General procedure for the reaction between 2-cyanopyridines and cysteine General procedure: To a solution of 2-cyanopyridine 1 (0.3 mmol, 1.0 equiv.),L-cysteine methyl ester hydrochloride 2 (0.6 mmol, 2.0 equiv.) in THF (0.3 mL) and 0.5 M TCEP (pH = 7.0) aqueous solution (2.4 mL, 4.0 equiv.) was added DIPEA (105 µL, 2.0 equiv.).After stirring at 40 °C (silicone oil bath) for 1 h, the reaction was quenched with saturated NaHCO3 aq.The aqueous layer was extracted with AcOEt and the combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo.The crude product was purified by flash column chromatography on silica gel to afford the corresponding products 3.The obtained thiazolines 3a, 3d and 3i did not show optical activity and the single-crystal X-ray diffraction analysis of thiazoline 3i confirmed that the thiazoline products were obtained as a racemic form.In the case of 2-cyanopyridine 1a, a trace amount of thioimidate intermediate 3a' was isolated.
In the case of 2-cyano-5-nitropyridine 1f , the reaction resulted a messy mixture and the desired product 3f could not be detected.When the reaction with 3-nitro-2-cyanopyridine, the reaction also resulted a messy mixture but a trace amount of the reduction product was obtained as described below.
Its structure was confirmed by 1 H NMR and X-ray crystal structure analysis.This result suggests that the nitro group could be reduced to amino group under the reaction conditions, which is probably the main decomposition pathway of 1f.

The reaction between various amino acids and 2-cyanopyridine 1d
To a solution of 2-cyanopyridine 1d (0.3 mmol, 1.0 equiv.)and amino acids (0.6 mmol, 2.0 equiv.) in THF (0.3 mL) and H2O (2.4 mL) was added DIPEA (2.0-4.0 equiv.).After stirring at 40 °C (silicone oil bath) for 1 h, the reaction was quenched with saturated NH4Cl aq.The aqueous layer was extracted with AcOEt and the combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo.The crude product was purified by flash column chromatography on silica gel.
Table S1.Screening of the reaction between various amino acids and 1d.
a) The reactions were conducted without DIPEA.

The reaction between 2-cyanopyridine 1b and glutathione
Following the representative procedure described above, the progress of the reaction between 2cyanopyridine 1b and glutathione was monitored by ESI-MS analysis at 48 h and 72 h.

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The reaction between 2-cyanopyridine 1c and glutathione Following the representative procedure described above, the progress of the reaction between 2cyanopyridine 1c and glutathione was monitored by ESI-MS analysis at 24 h and 72 h.

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The reaction between 2-cyanopyridine 1d and glutathione Following the representative procedure described above, the progress of the reaction between 2cyanopyridine 1d and glutathione was monitored by ESI-MS analysis at 24 h and 96 h.

The reaction between 2-cyanopyridine 1e and glutathione
Following the representative procedure described above, the progress of the reaction between 2cyanopyridine 1e and glutathione was monitored by ESI-MS analysis at 48 h and 72 h.
Bioconjugation efficiency was evaluated by analytical RP-HPLC, and all RP-HPLC was performed with a linear gradient of 10-40% acetonitrile and H2O containing 0.1% (v/v) TFA over 30 min with a flow rate of 1.0 mL min -1 at room temperature.The eluting products were detected by UV at 220 nm and the mass spectra were acquired by ESI-MS in positive detection mode.To a solution of peptide (1 mg, 1 µmol, 4.3 mM) in 0.5 M TCEP (pH 7.0) aqueous solution (62 µL, 134 mM) was added a solution of 1d (1 mg, 8.2 µmol, 35 mM) in THF (10 µL) and water (160 µL).

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for a linear gradient of 10-95% acetonitrile and H2O containing 0.1% (v/v) TFA over 85 min with a flow rate of 1.0 mL min -1 .The observed trace peaks were confirmed not to be derived from oxytocin.In the case of lypressin, the desulphurization product 15 was detected.This result indicates that the thiazoline products from bioactive peptides could be potentially unstable under the reductive conditions and the main decomposition pathway may be the desulphurization reaction.

N-Labeling experiments
To a solution of L-cysteine-15 N (1.0 mg, 8.25 µmol) in dehydrated MeOH (66 µL) was added SOCl2 (6 µL, 10 equiv.) at room temperature.After stirring at room temperature for 5 h, the reaction mixture was concentrated in vacuo.The resulting residue was dissolved in THF (16 µL) and 0.5 M TCEP (pH 7.0) aqueous solution (165 µL, 10 equiv.) and 3-fluoro-2-cyanopyridine 1d (1.0 mg, 1.0 equiv.) was added.After stirring at 40 °C (silicone oil bath) for 15 h, the reaction was quenched with water.The aqueous layer was extracted with CHCl3 and the combined organic layers were washed with brine, dried over MgSO4, filtered and concentrated in vacuo.The crude product was purified by preparative RP-HPLC to afford the thiazoline product.Preparative RP-HPLC was performed with a Shim-pack PREP-ODS column (20 × 250 mm).HPLC condition: 0.1% TFA (v/v) in water, 0.1% TFA (v/v) in acetonitrile, 15% in 40 min, 10 mL min -1 flow rate, detected by UV at 254 nm.Product yield was not determined because the reaction scale was too small to allow accurate calculation.The entire obtained product was used for 15 N-NMR and HRMS experiments. 15N-NMR and HRMS analysis confirmed that the thiazoline product 3d-15 N contained 15 N-labeled nitrogen.

Figure S3 .
Figure S3.ESI-MS analysis of the reaction between 1d and glutathione.
ray diffraction data of thiazoline product 3i was collected by a Rigaku XtalLAB Synergy Custom (Custom-made machine).CCDC-2310511 for 3i contain the supplementary crystallographic data for this paper.This data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. and Fachinformationszentrum Karlsruhe Access Structures service.

bond cleavage of glutathione with activated 2-cyano pyridine derivatives Representative procedure for the reaction between 2-cyanopyridines and glutathione
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