Specific chlorination of isoquinolines by a fungal flavin-dependent halogenase

Article abstract of DOI:10.1016/j.bmcl.2012.12.038

Rdc2 is the first flavin-dependent halogenase identified from fungi. Based on the reported structure of the bacterial halogenase CmlS, we have built a homology model for Rdc2. The model suggests an open substrate binding site that is capable of binding the natural substrate, monocillin II, and possibly other molecules such as 4-hydroxyisoquinoline (1) and 6-hydroxyisoquinoline (2). In vitro and in vivo halogenation experiments confirmed that 1 and 2 can be halogenated at the position ortho to the hydroxyl group, leading to the synthesis of the chlorinated isoquinolines 1a and 2a, respectively, which further expands the spectrum of identified substrates of Rdc2. This work revealed that Rdc2 is a useful biocatalyst for the synthesis of various halogenated compounds.

Full text of DOI:10.1016/j.bmcl.2012.12.038

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1001–1003
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Specific chlorination of isoquinolines by a fungal flavin-dependent
halogenase
Jia Zeng a, Anna K. Lytle b, David Gage a, Sean J. Johnson b, Jixun Zhan a,
⇑
a Department of Biological Engineering, Utah State University, 4105 Old Main Hill, Logan, UT 84322, United States
b Department of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan, UT 84322, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Rdc2 is the first flavin-dependent halogenase identified from fungi. Based on the reported structure of the
bacterial halogenase CmlS, we have built a homology model for Rdc2. The model suggests an open sub-
strate binding site that is capable of binding the natural substrate, monocillin II, and possibly other mol-
ecules such as 4-hydroxyisoquinoline (1) and 6-hydroxyisoquinoline (2). In vitro and in vivo
halogenation experiments confirmed that 1 and 2 can be halogenated at the position ortho to the hydro-
xyl group, leading to the synthesis of the chlorinated isoquinolines 1a and 2a, respectively, which further
expands the spectrum of identified substrates of Rdc2. This work revealed that Rdc2 is a useful biocata-
lyst for the synthesis of various halogenated compounds.
Received 20 September 2012
Revised 11 December 2012
Accepted 13 December 2012
Available online 21 December 2012
Keywords:
Fungal halogenase
Flavin-dependent
Homology model
Isoquinolines
Ó 2012 Elsevier Ltd. All rights reserved.
Substrate specificity
Halogenated molecules are an important group of natural prod-
ucts because of their significant biological activities. Up to now,
more than 4500 halogenated compounds have been isolated from
nature,1,2 such as rebeccamycin, chloramphenicol and vancomycin.
Recently, biological halogenation has drawn more and more atten-
tion as a highly selective and efficient substitute for classical or-
ganic chemistry approaches.3,4 Consequently, discovery of novel
halogenases with broad substrate specificity from natural product
biosynthetic pathways is critical. Our group has recently discov-
ered a promising halogenase Rdc2 that is involved in radicicol bio-
synthesis in Pochonia chlamydosporia.5 This enzyme specifically
chlorinates at C-6 of monocillins such as monocillin II, a biosyn-
thetic precursor of radicicol, as shown in Figure 1. We have found
that this enzyme can also work on other similar macrolactones
such as dihydroresorcylide. This enzyme can catalyze both
mono- and dichlorination. Additionally, it also accepts bromide
as a halogen donor to conduct both mono- and dibromination.5
Taken these unique promising properties together, this enzyme
has shown great potential as a powerful halogenating biocatalyst.
Thus, we decided to further study this enzyme to understand its
structure and test its applicability to other types of substrates. In
this work, we report a homology model of Rdc2 for substrate
docking and chlorination of two isoquinolines by this enzyme.
Several structures of bacterial flavin-dependent halogenases
have been reported, such as PrnA (AAB97504),6 RebH
(CAC93722)7 and CmlS (AAK08979).8 Both PrnA and RebH are tryp-
tophan 7-halogenase, involved in the biosynthesis of pyrrolnitrin
and rebeccamycin, respectively, while CmlS is characterized from
the biosynthetic pathway of chloramphenicol.
We constructed a homology model of Rdc2 using the LOMETS
server.9 The highest scored solution was based on the crystal struc-
ture of CmlS (pdb ID 3i3l), using MUSTER10 to perform the thread-
ing alignment and MODELLER11 to build the final structure. The
model contains 505 residues, which corresponds to 94.7% of the
Rdc2 sequence. The final model contains an N-terminal flavin
monooxygenase domain and a C-terminal ‘winged-helix’ domain
(Fig. 2A). Additional models produced by the server vary in the
C-terminal domain but show strong agreement in the N-terminal
domain.
The putative substrate binding site is located in a hydrophobic
region of the N-terminal domain adjacent to the strictly conserved
K74. The Rdc2 substrate monocillin II was modeled into the struc-
ture using a superimposed PrnA structure bound to 7-chlorotry-
ptophan (PDB ID 2AR8) as a guide (Fig. 2B). The model suggests
an open binding site that may accommodate a variety of com-
pounds, raising the possibility that Rdc2 may be able to utilize var-
ious substrates.
To further expand the substrate spectrum and the application of
Rdc2, we tested the ability of Rdc2 to chlorinate isoquinoline
substrates. Isoquinoline is a common structural component of
many bioactive natural products, such as renieramycin M from
Xestospongia sp. and jorunnamycin C from Jorunnafunebris, both
having promising anticancer activity with IC50 values in the
⇑
Corresponding author. Tel.: +1 435 797 8774; fax: +1 435 797 1248.
E-mail address: jixun.zhan@usu.edu (J. Zhan).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.038

1002
J. Zeng et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1001–1003
important type of molecule. As shown in Figure 2C and D, both
compounds can fit into the active site of Rdc2 and thus represent
potential substrates for this enzyme.
OH
6
O
1
OH
O
Rdc2
Rdc2
O
O
HO
We then conducted in vitro enzymatic assays to examine
whether they can really be chlorinated by Rdc2. Rdc2 and the part-
ner flavin reductase Fre from Escherichia coli were expressed and
purified as previously reported.5 As shown in Figure 3, LC–MS anal-
ysis revealed that both reactions13 yielded a product peak with
lower polarity. ESI-MS revealed that both 1a and 2a had two
[M+H]+ ion peaks with a 3:1 ratio in the positive mode (data not
shown), which is a characteristic of monochlorinated molecules.
Compounds 1 and 2 are isomers with a molecular weight of 145,
while the molecular weight of both 1a and 2a is 179, which is
34 Da greater than the substrates, indicating that they are the chlo-
rinated products.
HO
Cl
O
O
Monocillin II
6-Chloro-monocillin II
OH
OH
Cl
4
4a
8a
6
8
1
N
N
4-Hydroxyisoquinoline (1)
1a
3-Chloro-4-hydroxyisoquinoline (
)
Cl
Rdc2
In order to obtain sufficient amounts of 1a and 2a for structure
elucidation to characterize the chlorination positions, we used an
in vivo biocatalytic approach. A total of 30 mg of 1 and 2 were sep-
HO
HO
N
6-Hydroxyisoquinoline ( )
Figure 1. Chlorination of 1 and 2 by Rdc2.
N
arately fed into the isopropyl b-D-1-thiogalactopyranoside (IPTG)
2
2a
5-Chloro-6-hydroxyisoquinoline (
)
induced fermentation broth of E. coli BL21-CodonPlus (DE3)-RIL/
pJZ54 to yield 8.5 mg of 1a and 11.4 mg of 2a, respectively.14
The purified products were dissolved in methanol-d4 and the
NMR spectra were acquired on a JEOL instrument (300 MHz). The
1H NMR spectrum of 1a showed two doublet and two triplet sig-
nals that belong to H-5, H-6, H-7 and H-8, indicating that the halo-
genation has not occurred on the benzene ring. In contrast, only
nanomolar range.12 In this Letter, we chose two typical isoquino-
lines shown in Figure 1, 4-hydroxyisoquinoline (1) and 6-hydroxy-
isoquinoline (2), to test whether Rdc2 could chlorinate this
Figure 2. Homology model of Rdc2. (A) Overview of model colored from N (blue) to C terminus (red). The box indicates the putative active site. Modeling of monocillin II and
two unnatural substrates 1 and 2 into the active site region are indicated in (B)–(D), respectively.
1
2
2a
1a
4
5
6
7
8
9
10
11 min
8
10
12
14
16
18
20 min
Figure 3. HPLC analysis of the in vitro chlorination of 1 (left) and 2 (right) by Rdc2.

J. Zeng et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1001–1003
1003
Table 1
Acknowledgments
1H NMR data for 1a and 2a (CD3OD, 300 MHz, J in Hz)
Position
1a
2a
This work was partly supported by a Utah Science Technology
and Research (USTAR) Veterinary Diagnostics and Infectious Dis-
ease Seed Project and a National Institutes of Health Grant
(AI065357). We thank Dr. Chad Testa at Frontier Scientific, Inc.
for providing the substrates.
1
3
4
5
6
7
8
8.64 (1H, s)
9.53 (1H, s)
8.51 (1H, d, J = 6.9)
8.47 (1H, d, J = 6.9)
8.26 (1H, d, J = 8.3)
7.79 (1H, dd, J = 8.3, 6.9)
7.68 (1H, dd, J = 8.3, 6.9)
8.06 (1H, d, J = 8.3)
7.68 (1H, d, J = 9.0)
8.34 (1H, d, J = 9.0)
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
12.038. These data include MOL files and InChiKeys of the most
important compounds described in this article.
one singlet was observed for the pyridine ring, suggesting that
either C-1 or C-3 is halogenated. The chemical shift of this signal
is d 8.64, which belongs to H-1. Thus, we can deduce that the halo-
genation occurred at C-3 position, which is ortho to the hydroxyl
group. Thus, 1a was identified as 3-chloro-4-hydroxyisoquinoline.
To determine the chlorination position in 2a, both 1H and 1H–1H
COSY spectra were recorded. As expected, there were only five pro-
ton signals in the 1H NMR spectrum, confirming that one proton
has been substituted by Cl. The 1H–1H COSY correlations of H-3
to H-4 as well as H-7 to H-8 clearly revealed that these protons
are not chlorinated. Meanwhile, the signal of H-1 can be easily lo-
cated in the low field at d 9.53, indicating that C-5 of 2 has been
chlorinated, which is also ortho to the hydroxyl group. Therefore,
2a was characterized as 5-chloro-6-hydroxyisoquinoline. The 1H
NMR signals of both products are shown in Table 1.
Almost all the previously characterized flavin-dependent halog-
enases are highly substrate specific, such as those reported trypto-
phan halogenases.15–18 In order to develop a useful halogenating
biocatalyst, an enzyme with broad substrate specificity is needed.
Rdc2 is a halogenase different from those previously reported. It
is a late tailoring enzyme in radicicol biosynthesis and can haloge-
nate other macrolactones besides its natural substrates monocil-
lins.5 In this work, we report a homology model of the first
identified fungal flavin-dependent halogenase Rdc2, which pre-
dicts that this enzyme can accommodate various substrates. We
demonstrated that Rdc2 can function on another important group
of molecule, isoquinolines. This is the first report of enzymatic syn-
thesis of chlorinated isoquinolines. Structural characterization of
1a and 2a indicated that the chlorine atom has been introduced
at the position ortho to the hydroxyl group. A hydroxyl group is a
strongly activating substituent. The resonance effect of the hydro-
xyl group directs the electron toward the ring and may contribute
to the chlorination of the ortho position. Further investigation of
the X-ray structure of Rdc2 will reveal more information about
the catalytic mechanism of this enzyme, which will allow us to
use the structure model to find more potential substrates. More-
over, this research also demonstrated a convenient and effective
way to prepare halogenated molecules with Rdc2. In vitro enzy-
matic reaction allows a quick screening of the substrates and initial
analysis of the products, while in vivo biocatalysis through engi-
neered E. coli provides an economic way to obtain sufficient
amounts of halogenated products on a large scale for structure elu-
cidation and bioactivity studies.
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1 or 2, 10 mM NaCl, 16
(pH 7.0). The reaction mixtures were placed at 30 °C for 5 h and then quenched
by addition of 50 L of methanol. The mixtures were then subjected to analysis
on an Agilent 6130 LC–MS instrument using Zorbax SB-C18 (5 m,
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M Fre, and 16 M Rdc2 in100 mM phosphate buffer
l
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a
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4.6 Â 150 mm). A gradient of acetonitrile/H2O system (10–90% over 25 min)
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E. coli BL21-CodonPlus (DE3)-RIL/pJZ54 that expresses Rdc2. The culture was
maintained at 28 °C with shaking at 250 rpm for 36 h. The ethyl acetate extract
of the broth was fractionated on a Sephadex LH-20 (20 g) column eluted with
methanol to give 14 fractions, 5 mL each. Fractions 3–6 were combined and
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1a. Similarly, 30 mg of 2 was also incubated with E. coli BL21-CodonPlus (DE3)-
RIL/pJZ54 under the same conditions. The ethyl acetate extract was
fractionated on
a Diaion HP-20 (30 g) column eluted with a stepwise
gradient of isopropanol–water (0:100, 20:80, 40:60, 60:40, 80:20, 100:0,
each 250 mL) to give 6 fractions. Further separation of fraction 3 by reverse-
phase HPLC with isocratic elution of 10% acetonitrile in H2O (each containing
0.1% TFA) over 20 min at a flow rate of 1 mL/min afforded 11.4 mg of 2a in pure
form.
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