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.
Source
Directed Evolution of RebH for Catalyst-Controlled Halogenation of Indole C-H Bonds
What they did
Authors screened error-prone and site-saturation libraries of RebH on deuterium-labeled tryptamines by MALDI/LC-MS, then characterised variants 0S, 8F and 10S for 7-, 6- and 5-chlorination including docking versus native tryptophan binding.
What they found
0S (N470S) raised 7-chlorination yield 2.5-fold at >99% selectivity. 8F reached 85% 6-selectivity and 11-fold more 6-chlorotryptamine than 7M. 8F/10S still select C6/C5 despite weak binding, not the innately reactive C2.
The limits
What it doesn't show
Docking poses are models; crystal structures of the evolved variants with tryptamine were not solved, so how mutations retune binding remains unproven.
Key terms
- RebH
- Flavin-dependent tryptophan 7-halogenase used as the evolution parent.
- Catalyst-controlled C–H halogenation
- Site selectivity set by the enzyme, not by a substrate directing group.
- 8F / 10S
- Evolved variants selective for 6- and 5-chlorotryptamine, respectively.
- MALDI-MS screen
- High-throughput mass assay using deuterated probes to score isomer ratios.
- HalA
- Computed C–H chlorination barrier used to rank innate site reactivity.
Flashcards
Research intelligence for this paper
See its role on concept claims, tensions it is part of, placement history, and related discoveries.
Quiz yourself
Native RebH prefers chlorination at:
Common questions
What native RebH site is preferred?
The 7-position of the indole.
What selectivities did 8F and 10S achieve?
Almost exclusive 6- and 5-chlorination, respectively.
Did they need a directing group on the substrate?
No—selectivity is catalyst-controlled.
How was 5 vs 6 distinguished?
Deuterium-substituted probes plus MS.
More on Catalysis