Catalysis
Evolved RebH chlorinates indoles at C5, C6 or C7
Open access · cc by · source: Europe PMC
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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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.
Key findings
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.
Methodology
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.
Limitations
Docking poses are models; crystal structures of the evolved variants with tryptamine were not solved, so how mutations retune binding remains unproven.
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