Synlett 2020; 31(03): 224-229
DOI: 10.1055/s-0039-1690007
© Georg Thieme Verlag Stuttgart · New York

Selective Functionalization of Aliphatic Amines via Myoglobin-Catalyzed Carbene N–H Insertion

Viktoria Steck
Gopeekrishnan Sreenilayam
Rudi Fasan
Department of Chemistry, University of Rochester, 120 Trustee Road, Rochester, NY 14627, USA   Email:
› Author Affiliations
This work was supported in part by the U.S. National Institutes of Health grant GM098628 and in part by the U.S. National Science Foundation grant CHE-1609550. Mass spectrometry instrumentation is supported by the U.S. National Science Foundation grant CHE-0946653.
Further Information

Publication History

Received: 07 May 2019

Accepted after revision: 01 July 2019

Publication Date:
11 July 2019 (online)

Published as part of the Cluster Biocatalysis


Engineered myoglobins have recently gained attention for their ability to catalyze a variety of abiological carbene transfer reactions including the functionalization of amines via carbene insertion into N–H bonds. However, the scope of myoglobin and other hemoprotein-based biocatalysts in the context of this transformation has been largely limited to aniline derivatives as the amine substrates and ethyl diazoacetate as the carbene donor reagent. In this report, we describe the development of an engineered myoglobin-based catalyst that is useful for promoting carbene N–H insertion reactions across a broad range of substituted benzylamines and α-diazo acetates with high efficiency (82–99% conversion), elevated catalytic turnovers (up to 7,000), and excellent chemoselectivity for the desired single insertion product (up to 99%). The scope of this transformation could be extended to cyclic aliphatic amines. These studies expand the biocatalytic toolbox available for the selective formation of C–N bonds, which are ubiquitous in many natural and synthetic bioactive compounds.

Supporting Information

  • References and Notes

  • 1 Present address: Department of Chemistry, Valdosta State ­University, 1500 North Patterson Street, Valdosta, GA 31698, USA
    • 2a Gillingham D, Fei N. Chem. Soc. Rev. 2013; 42: 4918
    • 2b Moody CJ. Angew. Chem. Int. Ed. 2007; 46: 9148
    • 2c Zhu SF, Zhou QL. Acc. Chem. Res. 2012; 45: 1365
    • 3a Yates P. J. Am. Chem. Soc. 1952; 74: 5376
    • 3b Moyer MP, Feldman PL, Rapoport H. J. Org. Chem. 1985; 50: 5223
    • 3c Garcia CF, McKervey MA, Ye T. Chem. Commun. 1996; 1465
    • 3d Morilla ME, Diaz-Requejo MM, Belderrain TR, Nicasio MC, Trofimenko S, Perez PJ. Chem. Commun. 2002; 2998
    • 3e Bachmann S, Fielenbach D, Jorgensen KA. Org. Biomol. Chem. 2004; 2: 3044
    • 3f Fructos MR, Belderrain TR, de Fremont P, Scott NM, Nolan SP, Diaz-Requejo MM, Perez PJ. Angew. Chem. Int. Ed. 2005; 44: 5284
    • 3g Aviv I, Gross Z. Synlett 2006; 951
    • 3h Aviv I, Gross Z. Chem. Eur. J. 2008; 14: 3995
    • 3i Liu B, Zhu SF, Zhang W, Chen C, Zhou QL. J. Am. Chem. Soc. 2007; 129: 5834
    • 3j Baumann LK, Mbuvi HM, Du G, Woo LK. Organometallics 2007; 26: 3995
    • 3k Lee EC, Fu GC. J. Am. Chem. Soc. 2007; 129: 12066
    • 3l Deng QH, Xu HW, Yuen AW, Xu ZJ, Che CM. Org. Lett. 2008; 10: 1529
    • 3m Hou Z, Wang J, He P, Qin B, Liu X, Lin L, Feng X. Angew. Chem. Int. Ed. 2010; 49: 4763
    • 3n Mbuvi HM, Klobukowski ER, Roberts GM, Woo LK. J. Porphyrins Phthalocyanines 2010; 14: 284
    • 3o Ren YY, Zhu SF, Zhou QL. Org. Biomol. Chem. 2018; 16: 3087
    • 3p Anding BJ, Woo LK. Organometallics 2013; 32: 2599
    • 3q Zhou PX, Zhou ZZ, Chen ZS, Ye YY, Zhao LB, Yang YF, Xia XF, Luo JY, Liang YM. Chem. Commun. 2013; 49: 561
    • 3r Ramakrishna K, Sivasankar C. Org. Biomol. Chem. 2017; 15: 2392
    • 3s Liu G, Li J, Qiu L, Liu L, Xu G, Ma B, Sun J. Org. Biomol. Chem. 2013; 11: 5998
    • 3t Arredondo V, Hiew SC, Gutman ES, Premachandra ID, Van Vranken DL. Angew. Chem. Int. Ed. 2017; 56: 4156
    • 4a Antos JM, Francis MB. J. Am. Chem. Soc. 2004; 126: 10256
    • 4b Ho CH, Zhang JL, Zhou CY, Chan OY, Yan JJ, Zhang FY, Huang JS, Che CM. J. Am. Chem. Soc. 2010; 132: 1886
    • 4c Tishinov K, Schmidt K, Häussinger D, Gillingham DG. Angew. Chem. Int. Ed. 2012; 51: 12000
  • 5 Sreenilayam G, Fasan R. Chem. Commun. 2015; 51: 1532
  • 6 Wang ZJ, Peck NE, Renata H, Arnold FH. Chem. Sci. 2014; 5: 598
    • 7a Coelho PS, Brustad EM, Kannan A, Arnold FH. Science 2013; 339: 307
    • 7b Bordeaux M, Tyagi V, Fasan R. Angew. Chem. Int. Ed. 2015; 54: 1744
    • 7c Bajaj P, Sreenilayam G, Tyagi V, Fasan R. Angew. Chem. Int. Ed. 2016; 55: 16110
    • 7d Tyagi V, Bonn RB, Fasan R. Chem. Sci. 2015; 6: 2488
    • 7e Kan SB. J, Lewis RD, Chen K, Arnold FH. Science 2016; 354: 1048
    • 7f Dydio P, Key HM, Nazarenko A, Rha JY, Seyedkazemi V, Clark DS, Hartwig JF. Science 2016; 354: 102
    • 7g Sreenilayam G, Moore EJ, Steck V, Fasan R. Adv. Synth. Catal. 2017; 359: 2076
    • 7h Vargas DA, Tinoco A, Tyagi V, Fasan R. Angew. Chem. Int. Ed. 2018; 57: 9911
    • 7i Tyagi V, Fasan R. Angew. Chem. Int. Ed. 2016; 55: 2512
    • 7j Weissenborn MJ, Low SA, Borlinghaus N, Kuhn M, Kummer S, Rami F, Plietker B, Hauer B. ChemCatChem 2016; 8: 1636
    • 7k Tyagi V, Sreenilayam G, Bajaj P, Tinoco A, Fasan R. Angew. Chem. Int. Ed. 2016; 55: 13562
    • 7l Chen K, Huang XY, Kan SB. J, Zhang RK, Arnold FH. Science 2018; 360: 71
    • 8a Wei Y, Tinoco A, Steck V, Fasan R, Zhang Y. J. Am. Chem. Soc. 2018; 140: 1649
    • 8b Tinoco A, Wei Y, Bacik J.-P, Carminati DM, Moore EJ, Ando N, Zhang Y, Fasan R. ACS Catal. 2019; 9: 1514
    • 8c Khade RL, Zhang Y. J. Am. Chem. Soc. 2015; 137: 7560
    • 8d Sharon DA, Mallick D, Wang BJ, Shaik S. J. Am. Chem. Soc. 2016; 138: 9597
  • 9 Wolf MW, Vargas DA, Lehnert N. Inorg. Chem. 2017; 56: 5623
  • 10 Moore EJ, Steck V, Bajaj P, Fasan R. J. Org. Chem. 2018; 83: 7480
  • 11 Xu F, Li C, Tao Z, Pan Y. Adv. Synth. Catal. 2015; 357: 3341
  • 12 Brotzel F, Chu YC, Mayr H. J. Org. Chem. 2007; 72: 3679
  • 13 Experimental Procedures.Reagents and Analytical Methods: All chemicals and reagents were purchased from commercial suppliers (Sigma–Aldrich, Acros Organics, Alfa Aesar, TCI Chemicals) and used without further purification, unless otherwise stated. The diazo compounds isopropyl diazoacetate (3a) and cyclohexyl diazoacetate (5a) were prepared according to a reported procedure (see ref. 8b). All moisture- or oxygen-sensitive reactions were carried out under argon atmosphere in oven-dried glassware with magnetic stirring using standard gas-tight syringes, cannulae and septa. 1H and 13C NMR spectra were measured with a Bruker DPX-400 instrument (operating at 400 MHz for 1H and 100 MHz for 13C) or a Bruker DPX-500 instrument (operating at 500 MHz for 1H and 125 MHz for 13C). Tetramethylsilane (TMS) served as the internal standard (0 ppm) for 1H NMR, CDCl3 was used as the internal standard (77.0 ppm) for 13C NMR. Silica gel chromatography purifications were carried out using AMD Silica Gel 60 Å 230–400 mesh. Thin-layer chromatography (TLC) was carried out using Merck Millipore TLC silica gel 60 F254 glass plates. UV/Vis measurements were performed with a Shimadzu UV-2401PC UV/Vis spectrometer. Gas chromatography (GC) analyses were carried out with a Shimadzu GC-2010 gas chromatograph equipped with a FID detector and a Chiral Cyclosil-B column (30 m × 0.25 mm × 0.25 mm film). Separation method: 1 mL injection, injector temp.: 250 °C, detector temp.: 300 °C. Gradient: column temperature set at 140 °C for 3 min, then to 160 °C at 1.8 °C/min, then to 165 °C at 1 °C/min, then to 245 °C at 25 °C/min. Total run time was 28.31 min. HPLC analyses were performed with a Shimadzu LC-2010A-HT equipped with a VisionHT C18 column and a UV/Vis detector. Injection volume: 20 μL. Flow rate: 1 mL/min. Gradient: 40% acetonitrile (0.1% TFA) in water (0.1% TFA) for 3 min, then increased to 90% over 15 min.Protein Expression and Purification: Wild-type Mb and engineered Mb variants were cloned and expressed in E. coli BL21(DE3) or E. coli C41(DE3) cells as described previously (see ref 7b). Briefly, cells were grown in TB medium (ampicillin, 100 mg L−1) at 37 °C (180 rpm) until OD600 reached 0.6. Cells were then induced with 0.25 mM β-d-1-thiogalactopyranoside (IPTG) and 0.3 mM δ-aminolevulinic acid (ALA). After induction, cultures were shaken at 27 °C (180 rpm), harvested after 20 h by centrifugation (4,000 rpm, 20 min, 4 °C) and resuspended in Ni-NTA Lysis Buffer (50 mM KPi, 250 mM NaCl, 10 mM histidine, pH 8.0). Resuspended cells were frozen and stored at –80 °C. Cell suspensions were thawed at room temperature, lysed by sonication, and clarified by centrifugation (14,000 rpm, 50 min, 4 °C). The clarified lysate was transferred to a Ni-NTA column equilibrated with Ni-NTA Lysis Buffer. The protein was washed with Ni-NTA Wash Buffer (50 mM KPi, 250 mM NaCl, 20 mM histidine, pH 8.0). Proteins were eluted with Ni-NTA Elution Buffer (50 mM KPi, 250 mM NaCl, 250 mM histidine, pH 7.0). After buffer exchange (50 mM Kpi, pH 7.0), the proteins were stored at +4 °C. Myoglobin concentration was determined by UV/Vis spectroscopy using an extinction coefficient of ε 410 = 157 mM−1 cm−1.N–H Insertion Reactions: Under standard reaction conditions, reactions were carried out at a 400 μL scale using 1 or 20 μM myoglobin, 5–20 mM amine, 2.5–10 mM diazo compound, and 10 mM sodium dithionite. In a typical procedure, in an anaerobic chamber, a solution containing the desired myoglobin variant was mixed with a solution of sodium dithionite in argon-purged potassium phosphate buffer (50 mM, pH 8.0). Reactions were initiated by addition of amine (400 mM stock solution in EtOH) followed by the addition of diazo compound (200 or 400 mM stock solution in EtOH), and the reaction mixtures were stirred in the chamber for 12 h at room temperature.Product analysis: The reactions were analyzed by adding 8 μL of internal standard (fluorenone, 50 mM in DMSO) to the reaction mixture, followed by extraction with 400 μL of dichloromethane and analysis by GC-FID for reactions with substrates 1a, 7a18a (see the Supporting Information Reagents and Analytical Methods section for details on GC analyses). For reactions with substrates 3a6a, the organic layer was removed via evaporation and the residue was dissolved in 300 μL methanol, filtered through 0.22 μm syringe filters, and analyzed by HPLC (see the Supporting Information Reagents and Analytical Methods section for details on HPLC analyses). Calibration curves for quantification of the different N–H insertion products were constructed using authentic standards prepared as described in the Supporting Information Synthetic Procedures. All measurements were performed at least in duplicate. For each experiment, negative control samples containing either no enzyme or no reductant were included.Synthetic Procedures: Detailed procedures for the synthesis of 3b18b, relevant double insertion products as well as the precursors 1920 are provided in the Supporting Information.Synthesis of Ethyl Benzylglycinate (1b): To a flame-dried round-bottom flask under argon, equipped with a stir bar, was added benzylamine 1a (1 equiv) and Rh2(OAc)4 (1 mol%) in toluene (2–3 mL). To this solution was added a solution of ethyl diazoacetate (1 equiv) in toluene (1–2 mL) over 30 minutes at 0 °C. The resulting mixture was heated at 80 °C for another 15–18 h. The solvent was removed under vacuum and the crude mixture was purified by flash column chromatography (20% ethyl acetate/hexanes) to provide the title compound 1b (82% yield) as a pale-yellow oil. Rf  = 0.18 (20% EtOAc/hexanes). GC-MS: m/z (%) = 193 (1.4), 120 (66.3), 106 (31.2), 91 (100), 77 (2.0), 65 (15.0). 1H NMR (CDCl3, 500 MHz): δ = 7.21–7.18 (m, 2 H), 6.76 (t, J = 7.5 Hz, 1 H), 6.62 (d, J = 8.0 Hz, 2 H), 3.80 (s, 2 H), 1.50 (s, 9 H). 13C NMR (CDCl3, 125 MHz): δ = 170.3, 147.2, 129.2, 118.0, 113.0, 81.9, 45.5, 28.1