A Stable Precursor for Bioorthogonally Removable 3-Isocyanopropyloxycarbonyl (ICPrc) Protecting Groups

Abstract

Studies have established 3-isocyanopropyloxycarbonyl (ICPrc) moieties as bioorthogonally removable protecting groups. However, reagents to prepare ICPrc-protected amines are unstable, which critically limits the practical implementation of this chemistry. Here we report 3-isocyanopropyl (pentafluorophenyl) carbonates as bench-stable precursors for the synthesis of ICPrc-protected primary and secondary amines. The utility of the chemistry for bioconjugation applications is demonstrated by reversibly masking a lysine residue on a bioactive peptide.

Key words

bioorthogonal - isonitrile - isocyanide - protecting groups - tetrazine

• References and Notes

• 1 Current address: School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.
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• 12 Isocyano (Pentafluorophenyl) Carbonates; General Procedure To an oven-dried two-neck flask equipped with a stir bar was added the appropriate alcohol (3-isocyanopropan-1-ol or 3-isocyano-2-phenylpropan-1-ol, prepared as previously described, respectively)⁶,¹⁰ (1 equiv) and solid bis(pentafluorophenyl) carbonate (1.5 equiv) as a single portion in anhydrous CH₂Cl₂ [0.06 M]. To the stirring solution at room temperature was added triethylamine (5 equiv) dropwise, after which the solution developed a purple color. Upon completion of the reaction (1 h for ICPr-PFC; 3 h for ICPPr-PFC), the mixture was dry loaded with silica gel for purification by silica column chromatography. 3-Isocyanopropyl (pentafluorophenyl) carbonate (ICPr-PFC) Yield: 33 mg (92%); off-white oil; R_f = 0.5 (CH₂Cl₂/hexane = 1:1 v/v; I₂ stain). ¹H NMR (400 MHz, CDCl₃): δ = 4.50 (t, J = 5.9 Hz, 2 H), 3.61 (t, J = 6.6 Hz, 2 H), 2.23–2.11 (m, 2 H). ¹³C NMR (100 MHz, CDCl₃): δ = 157.91-157.63 (m), 151.05 (s), 142.6-142.38 (m), 141.25-141.00 (m), 140.05-139.83 (m), 139.30-138.94 (m), 138.73-138.46 (m), 136.79-136.43 (m), 66.28 (s), 38.11-37.95 (m), 28.14 (s). ¹⁹F NMR (375 MHz, CDCl₃): δ = –153.05, –153.10, –156.92, –161.63, –161.64, –161.69. HRMS (ESI): m/z [M + H]⁺ calcd for C₁₁H₇F₅NO₃: 296.0341; found: 296.0351. 3-Isocyano-2-phenylpropyl (Pentafluorophenyl) Carbonate (ICPPr-PFC) Yield: 8 mg (70%); yellow oil; R_f = 0.5 (CH₂Cl₂/hexane = 3:1 v/v; I₂ stain). ¹H NMR (400 MHz, CDCl₃): δ = 7.46–7.32 (m, 3 H), 7.28 (d, J = 1.8 Hz, 2 H), 4.69–4.58 (m, 2 H), 3.83 (dd, J = 6.4, 3.3 Hz, 2 H), 3.44 (quin, J = 6.5 Hz, 1 H). ¹³C NMR (100 MHz, CDCl₃): δ = 157.79, 151.05, 142.56, 141.13, 140.04, 139.13, 138.60, 136.65, 136.59, 135.72, 129.24, 128.56, 127.58, 69.76, 43.84-43.66 (m), 43.65-43.37 (m) ¹⁹F NMR (375 MHz, CDCl₃): δ = 152.99, –153.00, –153.01, –153.02, –153.06, –153.07, –153.08, –153.09, –156.88, –156.94, –157.00, –161.61, –161.66, –161.67, –161.72. HRMS (ESI): m/z [M + H]⁺ calcd for C₁₇H₁₁F₅NO₃: 372.0654; found: 372.0666.
• 13 3-Isocyanopropyl Benzylcarbamate (ICPr-Bn) To an oven-dried two-neck flask equipped with a stir bar was added ICPr-PFC (15 mg, 51 μmol) and benzylamine (11 mg, 102 μmol) in anhydrous CH₂Cl₂ [0.1 M]. To the stirring solution was added triethylamine (15 mg, 152 μmol) dropwise and the mixture was allowed to stir for 3 h (reaction monitored by TLC) before being quenched by the addition of water (10 mL) and extracted with CH₂Cl₂ (3 × 10 mL). The combined organic layers were washed with brine (5 mL), dried over MgSO₄, filtered, and concentrated for purification by silica column chromatography (CH₂Cl₂, R_f = 0.3; I₂ stain) to afford the desired product as an off-white oil (9 mg, 81%). ¹H NMR (400 MHz, CDCl₃): δ = 7.40–7.26 (m, 5 H), 4.99 (br s, 1 H), 4.37 (d, J = 5.9 Hz, 2 H), 4.25 (t, J = 5.9 Hz, 2 H), 3.50 (s, 2 H), 2.02 (br s, 2 H). ¹³C NMR (100 MHz, CDCl₃): δ = 156.87, 156.06, 138.21, 128.72, 127.59, 127.54, 61.10, 45.12, 38.52, 28.89. MS (ESI): m/z [M + H]⁺ calcd for C₁₂H₁₅N₂O₂: 219.11; found: 219.07. 3-Isocyanopropyl Pyrrolidine-1-carboxylate (ICPr-Pyrr) To an oven-dried two-neck flask equipped with a stir bar was added ICPr-PFC (15 mg, 51 μmol) and pyrrolidine (7 mg, 102 μmol) in anhydrous CH₂Cl₂ [0.1 M]. To the stirring solution was added triethylamine (15 mg, 152 μmol) dropwise and the reaction mixture was stirred for 3 h (reaction monitored by TLC). The reaction was quenched by the addition of water (10 mL) and extracted with CH₂Cl₂ (3 × 10 mL). The combined organic layers were washed with brine (5 mL), dried over MgSO₄, filtered, and concentrated for purification by silica column chromatography (CH₂Cl₂/MeOH = 50:1 v/v, R_f = 0.4; I₂ stain) to afford the desired compound as an off-white oil (8 mg, 86%). ¹H NMR (400 MHz, CDCl₃): δ = 4.22 (t, J = 5.9 Hz, 2 H), 3.51 (ddd, J = 6.7, 4.8, 1.9 Hz, 2 H), 3.38 (t, J = 6.5 Hz, 2 H), 3.33 (t, J = 6.5 Hz, 2 H), 2.03 (tt, J = 5.7, 2.5 Hz, 2 H), 1.86 (dt, J = 11.6, 5.8 Hz, 4 H). ¹³C NMR (100 MHz, CDCl₃): δ = 156.51-156.26 (m), 154.66 (s), 61.13 (s), 46.24 (s), 45.81 (s), 38.92-38.39 (m), 29.01 (s), 24.91 (s). MS (ESI): m/z [M + H]⁺ calcd for C₉H₁₅N₂O₂: 183.11; found: 183.08.
• 14 3-Isocyanopropyl (4-Methoxyphenyl)carbamate Upon following the procedure outlined for the synthesis of ICPr-Bn (Ref. 13), no desired product formation was found upon TLC analysis or by LCMS. Refluxing in dichloromethane did not provide the product either and instead resulted in loss of the starting material; ICPr-PFC was observed by TLC analysis.
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• 16 N-(3-Isocyanopropyl-1-carbamoyl)doxorubicin (ICPr-dox) To an oven-dried two-neck flask equipped with a stir bar was added doxorubicin·HCl (10 mg, 17 μmol) in anhydrous DMF [0.03 M]. Triethylamine (4.4 mg, 43 μmol) was added followed by ICPr-PFC (10 mg, 34 μmol) and the mixture was allowed to stir for 16 h before being diluted with CH₂Cl₂ (20 mL). The mixture was washed with water (2 × 15 mL) followed by brine (10 mL), dried with MgSO₄, filtered, and concentrated for purification by preparatory TLC (CH₂Cl₂/hexane = 10:1 v/v, R_f = 0.4) to afford the desired product as a red solid (2 mg, 18%). HRMS (ESI): m/z [M + Na]⁺ calcd for C₃₂H₃₄N₂O₁₃Na: 677.1959; found: 677.1920. The NMR data is in agreement with spectra reported in the literature.⁶
• 17 3-Isocyano-2-phenylpropyl Benzylcarbamate (ICPPr-Bn) To an oven-dried two-neck flask equipped with a stir bar was added ICPPr-PFC (5 mg, 13 μmol) and benzylamine (3 mg, 27 μmol) in anhydrous CH₂Cl₂ [0.1 M]. To the stirring solution was added triethylamine (4 mg, 40 μmol) dropwise and the mixture was allowed to stir for 12 h (reaction monitored by TLC) before being quenched by the addition of water (5 mL) and extracted with CH₂Cl₂ (3 × 5 mL). The combined organic layers were washed with brine (5 mL), ran through a short Celite plug, and concentrated for purification by preparatory TLC (CH₂Cl₂, R_f = 0.2; I₂ stain) to afford the desired product as a yellow oil (2.4 mg, 60%). ¹H NMR (400 MHz, CDCl₃): δ = 7.45–7.15 (m, 10 H), 5.82 (dd, J = 8.0, 5.4 Hz, 1 H), 5.08 (s, 1 H), 4.37 (dt, J = 11.6, 6.0 Hz, 2 H), 3.42 (dd, J = 28.0, 6.6 Hz, 2 H), 2.23 (dd, J = 46.8, 6.6 Hz, 2 H). ¹³C NMR (100 MHz, CDCl₃): δ = 157.14, 155.53, 139.40, 138.24, 128.91, 128.59, 127.76, 126.24, 73.67, 45.33, 38.33, 36.26. HRMS (ESI): m/z [M + H]⁺ calcd for C₁₈H₁₉N₂O₂: 295.1441; found: 295.1461.
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• 19 Brief procedure for peptide labeling in an aqueous environment using ICPr-PFC: A stock solution of 5 mg/mL c(RGDfK) (AdooQ Bioscience, USA) in 0.1 M sodium bicarbonate buffer at pH 9 and a stock solution of 10 mg/mL of ICPr-PFC in anhydrous DMSO was prepared. Next, 0.05 mL of the ICPr-PFC stock solution was added slowly to 0.2 mL of the peptide stock solution. The final labeling solution contains 20% anhydrous DMSO in 0.1 M sodium bicarbonate buffer and was allowed to react for 1 hour at room temperature. After the allotted time, the reaction mixture was diluted 10-fold with milli-Q water for LCMS analysis.
• 20 Brief procedure for removal of the ICPrc group from a peptide in an aqueous environment: To the 10-fold diluted peptide solution, obtained as described above,¹⁹ was added a tetrazine stock solution prepared in anhydrous DMSO to obtain a final deprotection solution containing approximately a 4-fold excess of tetrazine and 20% (w/v) DMSO in a 10 mM sodium bicarbonate buffer. The solution was warmed to 37 °C and allowed to react for 12 hours. After the allotted time, the reaction mixture was cooled, filtered, and analyzed by LCMS.