Angewandte
Chemie
tryptophol, and 2-methyltryptamine was also observed in
good yields (3, 4, 5, and 6). Interestingly, 6-halogenation of 5-
methyltryptamine was observed in 58% yield (7), indicating
that additional sites of the indole benzene ring can be
halogenated despite the presence of the more electron-rich
tions with enzyme loadings below those required for maximal
substrate conversion (see Supporting Information for repre-
sentative kinetic data). As expected, decreased total turnover
numbers (TTN) were observed for nonnatural substrates
(Table 3). The increasing Km values for tryptamine and
[
12]
pyrrole ring. At longer reaction times, dihalogenation of
[
24]
these substrates occurred,
so reaction monitoring was
[
a]
Table 3: Catalytic parameters for halogenation of select substrates.
required to ensure isolation of high yields of mono-halogen-
ated products. Low conversion (9–12%) of 1-methyl-l-
tryptophan and gramine to mono-halogenated products was
also observed, although the site of halogenation for these
substrates was not determined.
kcat[b]
Km[b]
Substrate
RebH
mol%]
Conv.
[%]
TTN
À1
[
[min
]
[mm]
[
c]
l-tryptophan
tryptamine
tryptoline
0.5
1
5
75
15
30
32
165
19
3.6
26
1.1
7.3
9.0
216
14
0.023
0.027
0.59
Halogenation of a number of bicyclic and tricyclic carbo-
cycles and heterocycles lacking pendent functionality was also
examined. The tricyclic 2,3-disubstituted indole tryptoline
was converted to a nearly 1:1 mixture of 5- and 6-chloro-
tryptoline (8a/b; Table 2), providing a promising starting
point for evolving halogenases with activity on more complex
2
-aminonaphthalene
1
[a] 75 mL reactions were conducted as described in Table 2 using the
indicated RebH loading and 0.5 mm phenol as an internal standard,
quenched with an equal volume of methanol, and analyzed by HPLC.
[b] Initial rate data were used to construct Hanes–Woolf plots to
determine kcat and K . [c] Reported values for the k and Km of RebH on
m
cat
[
25]
heterocyclic compounds. Comparing this result with those
for 2-methyltryptamine (7-halogenation) and 5-methyltrypt-
amine (6-halogenation) also demonstrates that different sites
of the benzene ring of nonnatural indole substrates can be
À1
[13]
l-tryptophan are 1.4 min and 2.0 mm, respectively.
tryptoline are consistent with their increasing structural
variation from tryptophan. The large structural differences
between these substrates and 2-aminonaphthalene make
direct comparisons difficult, but the relative efficacy of this
substrate presumably results from its strong electronic
activation. These data clearly show that significant improve-
ments to catalyst efficiency or stability are desirable and
provide a benchmark for further RebH optimization.
[
12]
halogenated by RebH. Again contrasting with reports for
[
11]
PrnA,
RebH provided high conversion of indole to
halogenated products. This substrate was particularly suscep-
tible to dihalogenation, and the reported 53% yield of 3-
chloroindole (9) is the maximum obtained before this process
became significant. Substituted naphthalenes were also viable
substrates, and monochlorinated compounds 10 and 11 were
isolated in high yields. While these latter substrates were
halogenated at their most activated sites, they nonetheless
illustrate the ability of RebH to accept substrates significantly
different from tryptophan.
Several notable differences between the reactivity of
RebH and that reported for PrnA deserve comment given the
significant homology of both the complete sequences (55%)
and the active sites (see below) of these two enzymes. First,
PrnA was reported to have no activity on indole, gramine, or
To further demonstrate the synthetic utility of RebH, we
[13]
[11]
confirmed that the bromination capabilities of this enzyme
1-methyl-l-tryptophan, whereas RebH halogenated each
could be translated to the preparative scale, and addition of
NaBr to the reaction medium provided 7-bromotryptophan
of these substrates. The simplest explanation for this differ-
ence is that low conversion using PrnA precluded identifica-
tion of the halogenated products. RebH has higher catalytic
(12) in 85% isolated yield. The reaction scale was also
increased to enable chlorination of 100 mg of tryptophan
using crude cell lysate, rather than purified enzyme, as
a catalyst (Scheme 4). While a significant decrease in
conversion was observed relative to the 10 mg reaction
conducted using purified enzyme, a 69% product yield was
still obtained. A strong dependence of yield on the reaction
surface area/volume ratio was also observed and suggests that
improvements may be achieved by controlling dissolved
efficiency than PrnA (reported k values on tryptophan are
cat
À1
À1 [27]
1.4 min versus 0.1 min ),
and our improved reaction
conditions would have further improved this advantage.
Second, PrnA was reported to catalyze 2-halogenation of
[11]
both 5- and N-W-methyltryptamine, while RebH catalyzes
6- and 7-halogenation of these substrates, respectively. Crystal
structures for both of these enzymes bound to FAD, chloride,
[
16,9d]
and tryptophan are available.
Analysis of residues (24
[26]
oxygen concentration.
Finally, the differing catalytic efficiency of RebH on
representative substrates was examined by conducting reac-
total) within 5 ꢂ of the substrate tryptophan shows only one
pair of residues (N467 and L456) that differ in identity and
only one additional pair of residues (N464 and N453)
displaying a notable conformational difference (Figure 2).
In RebH, the side chain of N467 forms a water-mediated
hydrogen bond to the substrate tryptophan. Similar hydrogen
bonding could help position other substrates for halogenation
at electronically disfavored sites during catalysis. The struc-
turally analogous residue in PrnA is L456, which cannot form
a hydrogen bond, and this difference may explain the
apparent differences in selectivity between PrnA and RebH.
In summary, co-expression of the halogenase RebH with
GroEL/ES and fusion of the flavin reductase RebF to MBP
Scheme 4. Preparative halogenation of tryptophan on 100 mg scale
(
cofactor regeneration system described in Table 2).
Angew. Chem. Int. Ed. 2013, 52, 5271 –5274
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
5273