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Site-selective enzymatic C‒H amidation for synthesis of diverse lactams

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Science  10 May 2019:
Vol. 364, Issue 6440, pp. 575-578
DOI: 10.1126/science.aaw9068
  • Fig. 1 Hemeprotein-catalyzed intramolecular C‒H amidation.

    (A) Design of acyl-protected hydroxamate 1 as nitrene precursor, inspired by naturally occurring nitrene precursors in the biosynthesis of benzastatins (30) (Ac, acetyl; Piv, pivaloyl). (B) Reaction scheme and proposed catalytic cycle of intramolecular C‒H amidation catalyzed by the P411 variant. (C) Crystal structure of a variant closely related to P411 E10 (Protein Data Bank ID: 5UCW) (27), with mutated residues marked in blue. (D) Directed evolution of E10FA to LSsp3 for the synthesis of β-lactam 2a and further improvement of TTN by process optimization. Experiments were typically performed at analytical scale using suspensions of E. coli cells expressing E10FA variants [optical density at 600 nm (OD600) = 2.5] in KPi (0.1 M, pH 8.0) buffer, 50 mM substrate 1a, 5 volume % organic cosolvents, and 400 μl of reaction volume at room temperature under anaerobic conditions for 36 hours. See supplementary materials for further details. TTN is the total desired product as quantified by comparison to standards by gas chromatography or HPLC, divided by total cytochrome P411. Single-letter amino acid abbreviations (here and in Fig. 3): A, Ala; E, Glu; F, Phe; G, Gly; L, Leu; M, Met; N, Asn; P, Pro; R, Arg; T, Thr; V, Val; Y, Tyr.

  • Fig. 2 Scope of β-lactam products.

    (A) Synthesis of β-lactams from corresponding hydroxamate substrates using LSsp3. Experiments were typically performed at analytical scale using LSsp3-expressing E. coli cells resuspended to OD600 = 2.5 or 3.0 in KPi (0.1 M, pH 8.0) buffer, 25 mM or 50 mM substrate 1, 5 volume % organic cosolvents, and 400 μL of reaction volume at room temperature under anaerobic conditions for 36 hours. (B) Gram-scale synthesis of β-lactam 2s using cell lysate of E. coli expressing LSsp3. See supplementary materials (table S5) for details and more scale-up reactions. In all cases, rr > 20:1.

  • Fig. 3 Engineering lactam synthases for regiodivergent intramolecular C‒H amidation.

    (A) Evolutionary trajectory of variants LSsp2, LSsp3, LSβ, and LSγ. (B) Selectivity and scope of LSsp2- and LSsp3-catalyzed intramolecular C‒H amidation. (C) Selectivity and scope of LSβ and LSγ. (D) Regiodivergent amidation of aliphatic, homobenzylic, and benzylic C(sp3)‒H bonds catalyzed by LSβ, LSγ, and LSsp3. Regioisomeric ratio (rr) indicates the mole ratio of major product to combined minor regioisomers. See supplementary materials (tables S6 to S8) for details.

Supplementary Materials

  • Site-selective enzymatic C—H amidation for synthesis of diverse lactams

    Inha Cho, Zhi-Jun Jia, Frances H. Arnold

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods 
    • Tables S1 to S8
    • NMR Spectra
    • References 
     
    Correction (29 October 2019): In section VI (page 37), the concentration of the stock solution has been corrected; in section IX (pages 63 to 67), the sequence information for final variants and their amino acid sequences have been revised. These corrections do not affect the conclusions of the paper.
    The original version is accessible here.

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