Synlett 2020; 31(17): 1701-1706
DOI: 10.1055/s-0040-1707220
letter
© Georg Thieme Verlag Stuttgart · New York

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

Julian Tu
,
Minghao Xu
,
R.M.F. gratefully acknowledges financial support from the Huntsman Cancer Institute (GU Center Pilot Grant) and the American Cancer Society (129785-IRG-16-190-01-IRG). J.T. gratefully acknowledges financial support from a University of Utah Skaggs Graduate Fellowship and from an American Foundation for Pharmaceutical Education Pre-Doctoral Fellowship.
Further Information

Publication History

Received: 10 June 2020

Accepted after revision: 03 July 2020

Publication Date:
31 July 2020 (online)


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.

Supporting Information

 
  • References and Notes

  • 1 Current address: School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.
    • 2a Sletten EM, Bertozzi CR. Angew. Chem. Int. Ed. 2009; 48: 6974
    • 2b McKay CS, Finn MG. Chem. Biol. 2014; 21: 1075
    • 2c Carell T, Vrabel M. Top. Curr. Chem. 2016; 374: 9
    • 2d Wu H, Devaraj NK. Top. Curr. Chem. 2016; 374: 3
    • 2e Oliveira BL, Guo Z, Bernardes GJ. L. Chem. Soc. Rev. 2017; 46: 4895
    • 3a Versteegen RM, Rossin R, ten Hoeve W, Janssen HM, Robillard MS. Angew. Chem. Int. Ed. 2013; 52: 14112
    • 3b Matikonda SS, Orsi DL, Staudacher V, Jenkins IA, Fiedler F, Chen J, Gamble AB. Chem. Sci. 2015; 6: 1212
    • 3c Wu H, Alexander SC, Jin S, Devaraj NK. J. Am. Chem. Soc. 2016; 138: 11429
    • 3d Xu M, Tu J, Franzini RM. Chem. Commun. 2017; 53: 6271
    • 3e Carlson JC. T, Mikula H, Weissleder R. J. Am. Chem. Soc. 2018; 140: 3603
    • 3f Versteegen RM, ten Hoeve W, Rossin R, de Geus MA. R, Janssen HM, Robillard MS. Angew. Chem. Int. Ed. 2018; 57: 10494
    • 3g Sarris AJ. C, Hansen T, de Geus MA. R, Maurits E, Doelman W, Overkleeft HS, Codee JD. C, Filippov DV, van Kasteren SI. Chem. Eur. J. 2018; 24: 18075
    • 3h Lukasak B, Morihiro K, Deiters A. Sci. Rep. 2019; 9: 1470
    • 3i Li Y, Lou Z, Li H, Yang H, Zhao Y, Fu H. Angew. Chem. Int. Ed. 2020; 59: 3671
    • 3j Tu J, Svatunek D, Parvez S, Eckvahl HJ, Xu M, Peterson RT, Houk KN, Franzini RM. Chem. Sci. 2020; 11: 169
    • 3k Wang Q, Wang Y, Ding J, Wang C, Zhou X, Gao W, Huang H, Shao F, Liu Z. Nature 2020; 579: 421
    • 4a Li J, Chen PR. Nat. Chem. Biol. 2016; 12: 129
    • 4b Devaraj NK. ACS Cent. Sci. 2018; 4: 952
    • 4c Tu J, Xu M, Franzini RM. ChemBioChem 2019; 20: 1615
    • 4d Ji X, Pan Z, Yu B, De La Cruz LK, Zheng Y, Ke B, Wang B. Chem. Soc. Rev. 2019; 48: 1077
  • 5 Deb T, Franzini RM. Synlett 2020; 31: 938
  • 6 Tu J, Xu M, Parvez S, Peterson RT, Franzini RM. J. Am. Chem. Soc. 2018; 140: 8410
    • 7a Imming P, Mohr R, Müller E, Overheu W, Seitz G. Angew. Chem., Int. Ed. Engl. 1982; 21: 284
    • 7b Stockmann H, Neves AA, Stairs S, Brindle KM, Leeper FJ. Org. Biomol. Chem. 2011; 9: 7303
  • 8 Tu J, Svatunek D, Parvez S, Liu AC, Levandowski BJ, Eckvahl HJ, Peterson RT, Houk KN, Franzini RM. Angew. Chem. Int. Ed. 2019; 58: 9043
  • 9 Chen Y, Wu KL, Tang J, Loredo A, Clements J, Pei J, Peng Z, Gupta R, Fang X, Xiao H. ACS Chem. Biol. 2019; 14: 2793
  • 10 Xu M, Deb T, Tu J, Franzini RM. J. Org. Chem. 2019; 84: 15520
  • 11 Lelieveldt L, Eising S, Wijen A, Bonger KM. Org. Biomol. Chem. 2019; 17: 8816
  • 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; Rf = 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; Rf = 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, Rf = 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, Rf = 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.
  • 15 Litwinowicz M, Kijeński J. Sustainable Chem. Processes 2015; 3: 1
  • 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, Rf = 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, Rf = 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.
    • 18a Dijkgraaf I, Beer AJ, Wester HJ. Front. Biosci. 2009; 14: 887
    • 18b Simecek J, Notni J, Kapp TG, Kessler H, Wester HJ. Mol. Pharm. 2014; 11: 1687
  • 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.