Regio- and Enantioselective Sequential Dehalogenation of rac-1,3-Dibromobutane by Haloalkane Dehalogenase LinB

Article abstract of DOI:10.1002/cbic.201600227

The hydrolytic dehalogenation of rac-1,3-dibromobutane catalyzed by the haloalkane dehalogenase LinB from Sphingobium japonicum UT26 proceeds in a sequential fashion: initial formation of intermediate haloalcohols followed by a second hydrolytic step to produce the final diol. Detailed investigation of the course of the reaction revealed favored nucleophilic displacement of the sec-halogen in the first hydrolytic event with pronounced R enantioselectivity. The second hydrolysis step proceeded with a regioselectivity switch at the primary position, with preference for the S enantiomer. Because of complex competition between all eight possible reactions, intermediate haloalcohols formed with moderate to good ee ((S)-4-bromobutan-2-ol: up to 87 %). Similarly, (S)-butane-1,3-diol was formed at a maximum ee of 35 % before full hydrolysis furnished the racemic diol product.

Full text of DOI:10.1002/cbic.201600227

DOI: 10.1002/cbic.201600227
Full Papers
Regio- and Enantioselective Sequential Dehalogenation of
rac-1,3-Dibromobutane by Haloalkane Dehalogenase LinB
[a]
[b]
[c]
[a]
[a]
ˇ
Johannes Gross, Zbynek Prokop, Dick Janssen, Kurt Faber, and Mꢀlanie Hall*
The hydrolytic dehalogenation of rac-1,3-dibromobutane cata-
lyzed by the haloalkane dehalogenase LinB from Sphingobium
japonicum UT26 proceeds in a sequential fashion: initial forma-
tion of intermediate haloalcohols followed by a second hydro-
lytic step to produce the final diol. Detailed investigation of
the course of the reaction revealed favored nucleophilic dis-
placement of the sec-halogen in the first hydrolytic event with
pronounced R enantioselectivity. The second hydrolysis step
proceeded with a regioselectivity switch at the primary posi-
tion, with preference for the S enantiomer. Because of complex
competition between all eight possible reactions, intermediate
haloalcohols formed with moderate to good ee ((S)-4-bromo-
butan-2-ol: up to 87%). Similarly, (S)-butane-1,3-diol was
formed at a maximum ee of 35% before full hydrolysis fur-
nished the racemic diol product.
Introduction
Haloalkane dehalogenases (EC 3.8.1.5) are hydrolytic enzymes
that convert a broad range of natural and synthetic halogenat-
ed compounds (e.g., alkanes, alkenes, cycloalkanes, alcohols,
epoxides, carboxylic acids, esters, ethers, amides, and nitriles)
by replacing the halogen atom (chloro, bromo, or iodo) with
a hydroxyl group.[1] The first haloalkane dehalogenases were
discovered in bacteria isolated from contaminated soil, includ-
ing DhlA from Xanthobacter autotrophicus GJ10,[2] DhaA from
Rhodococcus sp. 13064,[3] and LinB from Sphingobium japoni-
cum UT26.[4] Later, haloalkane dehalogenases were identified in
diverse organisms, including the symbiotic bacteria Bradyrhizo-
enzyme–ester intermediate; 2) hydrolysis of the latter by
a water molecule activated by a His/acid pair, thereby releasing
the corresponding alcohol; 3) a pair of H-bond donor residues
(Asn/Trp, Trp/Trp or Trp/Tyr) form a halide binding site, which
stabilizes the halide released in the first step.[11]
LinB was identified as an enzyme in the degradation path-
way of g-hexachlorocyclohexane (g-HCH; “lindane”, a potent
insecticide) in S. japonicum UT26. LinB catalyzes the successive
hydrolytic dechlorination of 1,3,4,6-tetrachlorocyclohexa-1,4-
diene (1,4-TCDN) resulting in 2,4,5-trichlorocyclohexa-2,5-dien-
1-ol (2,4,5-DNOL), following initial dehydrochlorination of g-
HCH by LinA, in two consecutive steps.[4] The enzyme shows
catalytic activity with a broad range of substrates, including
halogenated alkanes, alkenes, cycloalkanes, alcohols, carboxylic
acids, esters, amides, and nitriles.[12] In some cases, LinB is able
to catalyze kinetic resolutions because of the exquisite enan-
tioselectivity, as observed for a-bromo-amides and -esters (E
value >200).[13a] Simpler secondary haloalkanes are accepted,
but stereorecognition is poor (maximum E value=16 for 2-
bromopentane).[13] Dihalogenated compounds are also con-
verted, with a general preference for a,w-dihalo-n-alkanes.[12a]
Terminally and internally halogenated dihaloalkanes are partic-
ular substrates, as hydrolysis to the corresponding diols in-
volves competitive attack between prim- and sec-halogenated
positions. In addition to this regio-divergent behavior, enantio-
selective recognition at the secondary position (in both hydrol-
ysis steps) renders the overall pathway complex. 1,2-Dibromo-
propane, for instance, was shown to yield 1-bromopropan-2-ol,
thus indicating a LinB preference for the secondary brominat-
ed atom.[12b] In contrast, DhlA was selective for the primary
position in 1,3-dihaloalkanes.[14] Over the years, enantioselec-
tive haloalkane dehalogenases have been applied in synthetic
schemes, for example, for the preparation of enantiopure
building blocks.[13b,15]
bium
japonicum USDA110
and Mesorhizobium loti
MAFF303099,[5] the plant pathogenic bacteria Agrobacterium
tumefaciens C58,[6] human pathogenic Mycobacterium tubercu-
losis H37Rv,[7] and the sea urchin Strongylocentrotus purpura-
tus.[8] Structurally similar, haloalkane dehalogenases belong to
the a/b-hydrolase fold family, and are classified phylogenetical-
ly into subfamilies.[9] Although “universal” substrates have been
identified, classification according to substrate specificity has
been proposed.[10] These enzymes share a catalytic triad (Asp-
His-Glu/Asp), and have three key mechanistic features: 1) an
Asp residue acts as nucleophile and replaces the halogen atom
by an SN2 mechanism, thus leading to formation of an
[a] J. Gross, Prof. K. Faber, Dr. M. Hall
Department of Chemistry, University of Graz
Heinrichstrasse 28, 8010 Graz (Austria)
E-mail: melanie.hall@uni-graz.at
[b] Prof. Z. Prokop
Department Experimental Biology, Faculty of Science, Masaryk University
Kamenice 5A13, 62500 Brno (Czech Republic)
[c] Prof. D. Janssen
Biomolecular Sciences and Biotechnology Institute, University of Groningen
Nijenborgh 4, 9747AG Groningen (NL)
Supporting information for this article can be found under http://
dx.doi.org/10.1002/cbic.201600227.
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Scheme 1. Sequential dehalogenation of rac-1 catalyzed by haloalkane de-
halogenase LinB. Thicker arrows indicate faster reactions.
Thus, LinB-catalyzed hydrolysis of rac-1,3-dibromobutane (1)
was studied, with a focus on the enantio- and regioselective
behavior of the enzyme (Scheme 1). Sequential dehalogenation
leads to the formation of butane-1,3-diol (3), whereas accumu-
lation of haloalcohols 2a and/or 2b as intermediates indicates
variable selectivity in both hydrolytic steps.
Results and Discussion
The enzymatic hydrolysis of rac-1,3-dibromobutane (1) to
butane-1,3-diol (3) follows a complex pathway and involves
four possible routes, depending on the regio- and enantio-
selectivity of the enzyme for the prim- or sec-halide and the R
or S enantiomer, respectively (Scheme 1). During displacement
of the prim-halogen, the configuration of the sec-halogen is
retained (i.e., (R)-1!(R)-2b and (S)-1!(S)-2b); reaction of the
sec-halogen atom would proceed with an SN2-type inversion of
the configuration (i.e., (R)-1!(S)-2a and (S)-1!(R)-2a). Analo-
gous considerations are applicable to the second step, that is,
retention for (S)-2a!(S)-3 and (R)-2a!(R)-3, and inversion for
(R)-2b!(S)-3 and (S)-2b!(R)-3. Overall, during the first step,
kinetic resolution of rac-1 takes place, along with the forma-
tion of four products, that is, enantiomeric pairs of the two
regioisomeric haloalcohols 2a and 2b. In the second step, re-
gioisomers 2a and 2b are kinetically resolved to yield one pair
of enantiomers of the final hydrolysis product 3. The enantio-
meric compositions of 1, 2a, 2b, and 3 over time depend on
the selectivity constants of each step.
Figure 1. A) Composition of reaction mixture during sequential dehalogena-
tion of rac-1 (5 mm) catalyzed by LinB (values for 3 not plotted due to in-
complete recovery; calculated at 120 min from total mass balance:
2.30 mm). B) Enantiomeric composition of 2a and 2b.
(2a) during the first 10 min, thus highlighting a strong prefer-
ence for replacement of the sec-halogen atom[12b] in the first
step of the sequential dehalogenation (Figure 1A). Gradually,
formation of 3-bromobutan-1-ol (2b) was observed in the first
60 min, but at a rate which stayed below that for 2a (Table S1
in the Supporting Information, entries 1–2), thus confirming
LinB regioselectivity for the secondary position. After 60 min,
the concentration of 2a began to decrease due to hydrolysis
at the primary halogen position, while 2b continued to accu-
mulate. A slow decline in 2b concentration due to hydrolysis
at the secondary position started to occur only after 90 min,
while the hydrolysis rate of 2a further increased (Table S1,
entry 1), thus highlighting a preference for the primary posi-
tion in the hydrolysis of these haloalcohols. It is noteworthy
that all velocity data in Table S1 are apparent values, as both
secondary and primary hydrolysis reactions occur simultane-
ously at some point (e.g., 1 to 2a or 2b and 2a or 2b to 3).
In terms of enantiomer distribution, it appeared that the
two regiocomplementary hydrolytic reactions with rac-1 have
opposite enantiopreference: (S)-2a accumulated as predomi-
nant enantiomer from sec-hydrolysis of (R)-1, whereas (S)-2b
was the major enantiomer in the prim-hydrolysis of (S)-1 (Fig-
ure 1B). The concentrations of (R)-2a and (R)-2b remained low
(<0.4 mm), and the reaction rates were lowest for these two
Detailed analysis of the course of the reaction was per-
formed by monitoring the composition of the reaction mixture
over time, including distribution of regioisomeric haloalcohols
2a and 2b. Rac-1 (5 mm) was incubated with lyophilized LinB
(6 mm) in Tris·sulfate buffer (50 mm, pH 8.2) at 218C with shak-
ing (120 rpm), and samples were withdrawn at intervals. (S)-3
and (R)-3 are commercially available, but both enantiomers of
2a and 2b had to be synthesized independently to allow iden-
tification of all reaction products (see the Experimental Sec-
tion).
Careful analysis of the reaction mixture indicated rapid con-
sumption of 1, with exclusive formation of 4-bromobutan-2-ol
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enantiomers (Table S1, entries 5–6). Surprisingly, similar reac-
tion velocities were observed for the formation of 2a and 2b
between 30 and 60 min (Table S1, entries 3 and 6: 0.028 and
0.027 mmolminꢀ1 respectively). The delayed hydrolysis of 2b is
likely responsible for the highest rate constant in the whole
pathway for the formation of (S)-2b (0.043 mmolminꢀ1 be-
tween 60 and 90 min), as 2a is already being further hydro-
lyzed.
haloalcohols 2a and 2b gave detailed insight into the complex
selectivity behavior of LinB in the replacement of primary and
secondary halogen atoms as well as enantiorecognition of all
isomers formed. Pronounced regiopreference for the sec-halo-
gen atom in the first hydrolysis step with significant enantiose-
lectivity led to initial accumulation of (S)-2a, while regioisomer-
ic (S)-2b formed more slowly. A regioselectivity switch from
sec- to prim-halogen in the second hydrolysis step was respon-
sible for faster conversion of (S)-2a compared to (S)-2b. Enan-
tiopreference in the replacement of prim-halogen atom of (S)-
1 and (S)-2a was conserved, regardless of the substitution at
the g-position.
Taken together, these data explain the ee profiles observed
over time (Figure 2): the faster reaction of (R)-1 (at the secon-
Snapshots obtained at different time points of the reaction
indicate that full hydrolysis does not occur from a lack of enan-
tioselectivity, but rather from two regiospecific pathways with
relatively high reaction rates. This clearly sets LinB apart from
DhlA. Shutting down one of the four predominant pathways
could be sufficient to obtain the diol product in a highly enan-
tiopure form, but this calls for challenging protein engineering.
Reaction engineering techniques, on the other hand, might
afford the isolation of haloalcohols with high ee values.
Experimental Section
General: All chemicals, substrates and reference materials were
from Sigma–Aldrich, Fluka, and Lancaster Synthesis and used with-
out purification. All solvents were purchased from Roth. Anhydrous
solvents were dried over a 4 ꢂ molecular sieve. Anhydrous THF
was distilled over sodium/benzophenone. LinB was prepared as re-
ported previously,[16] and was dissolved (10 mgmLꢀ1) in phosphate
buffer (50 mm, pH 7.5) and lyophilized for use in further reactions.
All biotransformations and rehydration of enzyme preparation
were performed at 218C in Eppendorf tubes (2 mL) mounted hori-
zontally on a Unitron AJ 260 shaker (120 rpm; HT Infors, Bottmin-
gen, Switzerland). Centrifugation was performed in a Heraeus Bio-
fuge pico (Thermo Fisher Scientific) at 13000 rpm at room temper-
ature.
Figure 2. Time profile of ee values of 1, 2a, and 2b during LinB catalyzed-
dehalogenation of rac-1 (values for (S)-1 calculated from total mass balance,
enantiomers not separable).
dary position in the initial step) leads to higher ee values for
(S)-1, whereas the ee value for (S)-2b increases (after a lag
phase) when its formation is accelerated by faster prim-hydrol-
ysis of (S)-1 (this indicates possible inhibition of hydrolysis of
(S)-1 by (R)-1). This pattern is most likely attributable to differ-
ences in affinity rather than rate constants, and further studies
will be conducted to elucidate the role of each step on the
overall pathway. The ee value of (S)-2b remains high because
of the overall low reactivity of (R)-1 at the primary position. In
the second step of the sequential dehalogenation, 2a is hydro-
lyzed, and highest rate of degradation is for (S)-2a, thus indi-
cating a conserved S enantiopreference in the replacement of
the prim-halogen, as seen for the hydrolysis of rac-1 to (S)-2b.
This appears to be the main reason for the transient accumula-
tion of (S)-3 (maximum 35% ee; data not shown).
Eventually, all reactions achieve completion, and rac-1 deliv-
ers 100% rac-3. Because of competition in replacement be-
tween the primary and secondary halogen atoms and the com-
plex enantioselective behavior of LinB in both steps of the se-
quential dehalogenation, the ee value of (S)-2a could be main-
tained at a reasonable level (>65%) during the major part of
the process, whereas (S)-2b reached 87% ee at high accumu-
lated concentration (~2 mm).
NMR spectra were recorded in CDCl3 with TMS (0.03%) as the stan-
dard on a Bruker NMR device at 300 MHz. Chemical shifts are re-
ported in ppm, and coupling constants are in Hz. GC-MS analysis
was on a 7890A GC-System (Agilent Technologies) coupled to
a 5975C inert XLMSD detector with an HP-5 MS column (phenylme-
thylsiloxane (5%), 30 mꢃ0.25 mmꢃ0.25 mm; J&W Scientific/Agilent
Technologies). GC-FID measurements on a chiral stationary phase
were carried out on the 7890A GC-system with a Hydrodex b-
TBDAc column (50 mꢃ0.25 mm i.d.ꢃ0.4 mm o.d., Macherey–
Nagel). Details are in the Supporting Information. TLC analysis was
on silica gel 60 F254 (Merck Millipore). Cerium ammonium molyb-
date/H2SO4 was used as the spray reagent.
General biotransformation procedure: In 2 mL Eppendorf vials,
rac-1 (0.6 mL, 5 mm) was added to Tris·sulfate (1 mL, 50 mm,
pH 8.2) followed by LinB (125 mL of a 1.5 mgmLꢀ1 stock solution;
final 6 mm), and the solution was incubated with shaking (120 rpm)
for 6 h at 218C. The reaction was stopped by saturating the solu-
tion with NaCl and adding ethyl acetate (500 mL). The preparation
was mixed and centrifuged (13000 rpm, 5 min), and the products
were extracted. After a second extraction, the combined organic
layers were dried over Na2SO4 and transferred into GC glass vials
for analysis. Product concentrations were calculated based on GC
calibration curves generated for each intermediate available as au-
Conclusion
Analysis of the sequential dehalogenation of rac-1,3-dibromo-
butane (rac-1) to butane-1,3-diol (3) by LinB via intermediate
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thentic references (see below). Analytical methods are described in
the Supporting Information.
10 mL). The organic layer was washed with water and brine, dried
over Na2SO4, and concentrated under reduced pressure. The prod-
uct was purified on silica gel with hexane/ethyl acetate (1:1) to
Synthesis of reference materials: Haloalcohol (R)-2b was ob-
tained in a three-step procedure: 1) protection of the prim-OH
group of (S)-3 with trityl chloride to yield (S)-4; 2) Appel reaction
by inversion of the configuration to furnish (R)-5; 3) final deprotec-
tion under acidic conditions to yield (R)-2b (overall yield, 51%).
Enantiomer (S)-2b was prepared by the same procedure starting
from (R)-3 (overall yield, 54%). (R)-2a was prepared in two steps:
1) activation of the prim-OH group of (R)-3 with tosyl chloride to
yield (R)-6; 2) nucleophilic substitution using LiBr to furnish (R)-2a
(overall yield, 24%). The same protocol with (S)-3 yielded (S)-2a
(overall yield, 22%). Product identity and purity were confirmed by
NMR on 2a and 2b at the end of each synthetic sequence.
1
yield (R)-2a (74 mg, 39%) or (S)-2a (82 mg, 40%). (R)-2a: H NMR:
d=4.03–3.99 (m, 1H), 3.61–3.48 (m, 2H), 2.05–1.92 (m, 2H), 1.65 (s,
1H), 1.26 (d, J=6.24 Hz, 3H); 13C NMR: d=66.1, 41.5, 30.4,
23.5 ppm. Both 1H NMR and 13C NMR spectra were in accordance
with those in ref. [22].
Assignment of absolute configuration: Co-injection of enzymatic
samples with authentic reference materials allowed unambiguous
assignment of the absolute configuration for all products. GC
methods, retention times, and chromatograms are in the Support-
ing Information.
(R)- and (S)-4-(Triphenylmethoxy)butan-2-ol ((R)- and (S)-4):[17]
(R)-3 or (S)-3 (180 mg, 2 mmol) in pyridine (350 mL) was added to
trityl chloride (631 mg, 2.2 mmol, 1.1 equiv) in pyridine (1.75 mL).
DMAP (6 mg, 0.004 mmol) was added, and the mixture was stirred
for 48 h at room temperature. After addition of water (5 mL, 08C),
the residue was extracted with dichloromethane (3ꢃ10 mL), dried
over Na2SO4, and purified by chromatography on silica gel with
hexane/ethyl acetate (2:1) to yield (R)-4 (614 mg, 92%) or (S)-4
(570 mg, 85%).
Acknowledgements
This work was in part supported by the Czech Ministry of Educa-
tion, Youth and Sports (CZ.1.05/2.1.00/19.0382 and LO1214) and
by Grant Agency of the Czech Republic (GA16–07965S).
Keywords: biocatalysis
dehalogenase · hydrolysis · LinB · regioselectivity
·
enantioselectivity
·
haloalkane
(R)- and (S)-[(3-Bromobutoxy)methanetrityl]tribenzene ((R)- and
(S)-5):[18] Triphenylphosphine (Ph3P; 493 mg, 1.88 mmol, 1.1 equiv)
was added over 30 min to ice-cooled (S)-4 (570 mg, 1.71 mmol)
and CBr4 (623 mg, 1.88 mmol, 1.1 equiv) in CH2Cl2 (750 mL) was
added. Alternatively, Ph3P (535 mg, 2.04 mmol, 1.1 equiv) was
added over 30 min to ice-cooled (R)-4 (614 mg, 1.85 mmol) and
CBr4 (670 mg, 2.02 mmol, 1.1 equiv) in CH2Cl2 (800 mL). After 3 h of
stirring at room temperature, hexane (10 mL) was added. The
white precipitate was filtered off, and the remaining solution was
concentrated under reduced pressure. The product was purified by
chromatography on silica gel with hexane/ethyl acetate (6:1) to
yield (S)-5 or (R)-5 (650 mg, 95%).
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dissolved in acetic acid (11 mL) at 508C with stirring. Water
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62%) or (R)-2b (158 mg, 63%). (S)-2b: 1H NMR: d=4.39–4.26 (m,
1H), 3.85–3.81 (m, 2H), 2.10–1.96 (m, 2H), 1.76 (d, J=6.70 Hz, 3H);
´
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1
13C NMR: d=60.87, 47.99, 43.24, 26.55 ppm. H NMR spectra were
in accordance with those in ref. [19].
(R)- and (S)-3-Hydroxybutyl 4-methylbenzenesulfonate ((R)- and
(S)-6):[20] p-Toluenesulfonyl chloride (472 mg, 2.48 mmol, 1.1 equiv)
was added to (R)-3 or (S)-3 (200 mg, 2.22 mmol) in pyridine
(1.5 mL) cooled on ice (08C). After stirring for 3 h, the reaction mix-
ture was diluted with water and extracted with diethyl ether (3ꢃ
10 mL). The organic layer was washed with saturated CuSO4 and
dried over Na2SO4. After solvent evaporation under reduced pres-
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tate (2:1) to yield (R)-6 (305 mg, 56%) or (S)-6 (327 mg, 60%).
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(R)- and (S)-4-Bromobutan-2-ol (2a):[21] Lithium bromide (163 mg,
1.87 mmol, 1.5 equiv) was added to (R)-6 (305 mg, 1.25 mmol) or
(S)-6 (327 mg, 1.34 mmol) in dry DMF (600 mL). After stirring at
room temperature for 5 h, the reaction mixture was diluted with
a small amount of water and extracted with diethyl ether (3ꢃ
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Full Papers
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Manuscript received: April 18, 2016
Accepted article published: May 25, 2016
Final article published: && &&, 0000
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These are not the final page numbers! ÞÞ

FULL PAPERS
J. Gross, Z. Prokop, D. Janssen, K. Faber,
M. Hall*
The hydrolytic dehalogenation of rac-
1,3-dibromobutane catalyzed by the
haloalkane dehalogenase LinB proceeds
in a sequential fashion via initial inter-
mediate haloalcohols followed by a
second hydrolytic step to produce the
final diol. Two simultaneous regiospecif-
ic pathways with high reaction rates
were revealed, along with pronounced
enantioselectivity in each pathway.
&& – &&
Regio- and Enantioselective
Sequential Dehalogenation of rac-1,3-
Dibromobutane by Haloalkane
Dehalogenase LinB
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ChemBioChem 2016, 17, 1 – 6
www.chembiochem.org
6
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!