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
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Evolved RebH chlorinates indoles at C5, C6 or C7
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Variant 0S gave 2.5-fold higher chlorination yield than RebH with >99% 7-selectivity.
Introducing this mutation into RebH gave variant 0S, which provided the highest chlorination yields of all variants evaluated (2.5-fold higher than RebH, >99% 7-chlorination, ).
8F showed 85% 6-selectivity and an 11-fold yield increase over 7M.
Remarkably, the variant with the highest selectivity for 6-chlorination (85%) as well as the highest yield of 6-chlorotryptamine (11-fold increase over 7M) from this library, 8F (7M-L111F), resulted from reversion of the F111L mutation that originally led to a significant change in 7-selectivity of 3W.
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.
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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.
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