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Evolved RebH chlorinates indoles at C5, C6 or C7
Mass-spectrometry-guided evolution of RebH produced variants that chlorinate tryptamine ortho, meta or para to the indole nitrogen without a synthetic directing group.
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What selectivity problem did RebH evolution address?
Catalyst-controlled halogenation of different indole C–H bonds without installing directing groups.
Source: Directed Evolution of RebH for Catalyst-Controlled Halogenation of Indole C-H Bonds · DOI · quote verified
How were selectivity changes screened?
Deuterium-substituted probe substrates analysed by mass spectrometry.
Source: Directed Evolution of RebH for Catalyst-Controlled Halogenation of Indole C-H Bonds · DOI · quote verified
What did variant 0S change relative to RebH?
N470S; 2.5-fold higher yield and >99% 7-chlorination.
Source: Directed Evolution of RebH for Catalyst-Controlled Halogenation of Indole C-H Bonds · DOI · quote verified
What 6-selectivity and yield jump did 8F show versus 7M?
85% 6-chlorination and 11-fold more 6-chlorotryptamine.
Source: Directed Evolution of RebH for Catalyst-Controlled Halogenation of Indole C-H Bonds · DOI · quote verified
Do 8F and 10S need tight substrate binding for high selectivity?
No—they bind tryptamine poorly yet almost exclusively chlorinate C6 and C5.
Source: Directed Evolution of RebH for Catalyst-Controlled Halogenation of Indole C-H Bonds · DOI · quote verified
Which site is innately most reactive by HalA, and do the enzymes use it?
C2 is least reactive to chlorinate (highest HalA 177 kcal mol−1); enzymes avoid it.
Source: Directed Evolution of RebH for Catalyst-Controlled Halogenation of Indole C-H Bonds · DOI · quote verified
How large was the first epPCR library screen of 0S?
Lysates from 1000 clones assayed by automated MALDI-MS on probe 1.
Source: Directed Evolution of RebH for Catalyst-Controlled Halogenation of Indole C-H Bonds · DOI · quote verified
What 5-chlorination selectivity did 8C reach?
86%, up from 39% with 6TL, via F465C.
Source: Directed Evolution of RebH for Catalyst-Controlled Halogenation of Indole C-H Bonds · DOI · quote verified
Did 1P (S448P) already shift 5/6-selectivity?
Yes—4.5-fold increase in 5/6-chlorination versus parent.
Source: Directed Evolution of RebH for Catalyst-Controlled Halogenation of Indole C-H Bonds · DOI · quote verified
Are homologous halogenases a unique sequence solution?
No—PyrH and Thal differ from RebH by 205 and 335 residues yet solve similar 5/6 problems.
Source: Directed Evolution of RebH for Catalyst-Controlled Halogenation of Indole C-H Bonds · DOI · quote verified
What yields/selectivities were obtained on related substrates with 8F/10S?
73–98% yields and 90–100% selectivities.
Source: Directed Evolution of RebH for Catalyst-Controlled Halogenation of Indole C-H Bonds · DOI · quote verified