Halohydrin dehalogenase-catalysed transformations of epifluorohydrin

Article abstract of DOI:10.1016/j.tetlet.2017.12.054

Biocatalytic ring-opening of epifluorohydrin has been performed by using halohydrin dehalogenase. The enzyme from Mycobacterium sp. GP1 (HheB2) catalysed reaction with high regioselectivity and low enantioselectivity in the presence of different nucleophi

Full text of DOI:10.1016/j.tetlet.2017.12.054

Accepted Manuscript
Halohydrin dehalogenase-catalysed transformations of epifluorohydrin
Maja Majerić Elenkov, Mirjana Čičak, Ana Smolko, Anamarija Knežević
PII:
S0040-4039(17)31560-5
https://doi.org/10.1016/j.tetlet.2017.12.054
TETL 49556
DOI:
Reference:
Tetrahedron Letters
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Received Date:
Revised Date:
Accepted Date:
23 November 2017
15 December 2017
16 December 2017
Please cite this article as: Majerić Elenkov, M., Čičak, M., Smolko, A., Knežević, A., Halohydrin dehalogenase-
catalysed transformations of epifluorohydrin, Tetrahedron Letters (2017), doi: https://doi.org/10.1016/j.tetlet.
2017.12.054
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Halohydrin dehalogenase-catalysed
transformations of epifluorohydrin
Maja Majerić Elenkov*, Mirjana Čičak, Ana Smolko and Anamarija Knežević
OH
O
HheB2
F
+
NaNu
F
Nu
Buffer
1-3 h
Yield up to 92%
Nu = N3, NO2, CN, Cl

1
Tetrahedron Letters
journal homepage: www.els evier.com
Halohydrin dehalogenase-catalysed transformations of epifluorohydrin
Maja Majerić Elenkova*, Mirjana Čičaka, Ana Smolkob and Anamarija Kneževića
a Center of Excellence for Marine Bioprospecting, Ruđer Bošković Institute, Bijenička c. 54, Zagreb 10000, Croatia
bLaboratory for Chemical Biology, Ruđer Bošković Institute, Bijenička c. 54, Zagreb 10000, Croatia
ARTICLE INFO
ABSTRACT
Article history:
Received
Biocatalytic ring-opening of epifluorohydrin has been performed by using halohydrin
dehalogenase. The enzyme from Mycobacterium sp. GP1 (HheB2) catalysed reaction with high
regioselectivity and low enantioselectivity in the presence of different nucleophiles, producing
racemic 1-substituted 3-fluoro-2-propanols. No by-products resulting from the ring-closure
reaction have been detected, confirming that vicinal fluoro alcohols are not substrates for
HHDH. Four different 3-fluoro-2-propanols were prepared under mild reaction conditions
starting from epifluorohydrin. High conversions of 85-100% were reached within 1-3h and
depending on the nucleophile used products were isolated in 31-92% yields.
Received in revised form
Accepted
Available online
Keywords:
Biocatalysis
Halohydrin dehalogenase
Epoxide ring-opening
Epifluorohydrin
Fluoro alcohols
2009 Elsevier Ltd. All rights reserved
.
Epihalohydrins are versatile substrates for various
transformations in organic synthesis.1 Due to the reactivity of the
oxirane ring and the presence of halogenated carbon atom they
can be transformed into a variety of multifunctionalised building
blocks. Next to chemical transformations, microbial enzymes are
also known to catalyse conversions of epihalohydrins.2
Biocatalytic transformations have been reported by using epoxide
hydrolases3 and halohydrin dehalogenases (HHDHs).4 Enzymes
HheC and HheB2 catalyse the transformation of epichlorohydrin
These compounds are highly promising, versatile building blocks
endowed with functional groups that can be used for the
synthesis of a variety of other fluorine-containing derivatives.
First we performed HHDH-catalysed reactions with all four
epihalohydrins (1a-1d, X = F, Cl, Br, I) in the presence of
sodium azide as nucleophile in order to compare their reactivity.
Results are presented in Table 1. The ring-opening of 1a-1d
catalysed by the enzyme from Mycobacterium sp. GP1 (HheB2)
is directed towards the terminal position of the epoxide, yielding
the corresponding 1-azido 3-halo-2-propanol 2a-2d. In the case
of 1a, fluoro alcohol 2a is the final product. However, ring-
closure of 2b-2d catalysed by the same enzyme, yields halide
ions (Cl−, Br−, I−) and epoxide 3 that can undergo another
nucleophilic attack to form diazido product 4. Due to the
complexity of reaction systems, the presence of several substrates
and occurrence of several competitive reactions, it is not
straightforward to explain the outcomes of these reactions. We
can estimate that the ratio of products depends on the intrinsic
reactivity of halohydrin 2, its affinity to the enzyme and
inhibitory effect of any the reacting species on the enzyme..
Brominated alcohol 2c seems to be favored substrate for HheB2,
and this sequential reaction is completely directed toward 4.
Compared to 2c, reactivity of chloro (2b) and iodo (2d)
derivatives is lower and the mixture of products where formed. In
this case we cannot rule out inhibitory effect of halide ions on the
conversion of 2b and 2d to 3.
(1b) and epibromohydrin (1c) in the presence of different
nucleophiles, NaCN,4a NaN3
4b,4c
NaOCN4d,4e and NaNO2.4f
Besides, it has been shown that under optimised conditions HheC
can simultaneously catalyse kinetic resolution and racemisation
of epihalohydrins, resulting in a dynamic kinetic resolution
yielding optically active products in high yields.4d,4e
Reactions of epifluorohydrin (1a) are less studied compared to
1b and 1c derivatives.1 Biocatalytic hydrolysis of 1a has been
achieved by employing epoxide hydrolase.3e The enzyme from
Sphingophyxis alaskensis hydrolysed 1a with good activity,
however no enantioselectivity was observed. In addition, the
ring-opening of 1a with nitrogen-, carbon-, halogen-, and
oxygen-nucleophiles is highly attractive since it can offer a
selective synthesis of a range of fluorinated building blocks
useful in the synthesis of medicinally important compounds.5
Because of its strong electronegativity, fluorine is a powerful
modulator of physical and chemical properties of organic
molecules and can increase pharmaceutical effectiveness,
biological half-life and the bioabsorption of the potential drug
molecules.5 In this context, we were interested in exploring the
possibility to use HHDH to perform biocatalytic ring-opening of
1a, in order to synthesise 1-substituted-3-fluoro-2-propanols.
The results described above confirmed that vicinal fluoro
alcohols are not substrates for HHDH since the enzyme is unable
−
to cleave C F bond. Previous screening of broad nucleophile
range revealed that F− is not accepted in the ring-opening of
epoxides.6

2
Tetrahedron: Asymmetry
∗ Corresponding author. Tel.: +381-1-4560-965; fax: +385-1-4680-108; e-mail: majeric@irb.hr
Table 1. Reactions catalysed by HheB2 starting from epihalohydrins 1a-1d and sodium azide.
OH
OH
4
O
O
HheB2
HheB2
X-
HheB2
+
NaN3
X
N3
X
N3
N3
N3
-
N3
1a-1d
2a-2d
3
X = Cl, Br, I
X = F
Substrate a
X
Conversion (%)
Products / Yield (%)b
F
100
100
100
100
2a / 100
1a
1b
1c
1d
Cl
Br
I
3 : 4 / 20 : 80
4 / 100
2d : 3 : 4 / 47 : 25 : 28
a Reactions were performed in a total volume of 2.5 mL with 100 mM substrate, 300 mM NaN3, 2% DMSO and cell-free extract of HheB2
in Tris-SO4 buffer pH 7.5.
b determined by GC
We further explored the potential of HHDH to catalyse the
synthesis of 1-substituted-3-fluoro-2-propanols. The ring-
opening reaction of racemic 1a was performed with different
within 1h. However, the reaction resulted in the mixture of
products due to the ambident nature of nitrite ions,10 where nitro
alcohol 2b and nitrite ester were formed. In the case of cyanide
and chloride within 1h ca. 70% of 1a was converted. While
reaction with cyanide slowly continues, chloride in this reaction
reacts reversibly and equilibrium is reached at this point.
Therefore, for preparative transformations in order to achieve
higher conversions it was necessary to slightly modify the
reaction conditions for nitrite, cyanide and chloride.
−
nucleophiles (N3−, CN−, NO2 , OCN−, SCN−, Cl−, Br−, I−) and
different enzymes (HheA, HheB2, HheC, HheC-T134A). In
addition, hydrolytic stability and reactivity of 1a in non-catalysed
reactions were tested. We found that this substrate undergoes
spontaneous hydrolysis (ca. 3%/h). For each nucleophile, the
experiments were performed in the absence of enzyme to
determine the rate of the chemical ring-opening reaction. At
epoxide and nucleophile concentration of 100 mM and 150 mM,
respectively, the non-catalysed spontaneous formation of racemic
alcohol products was moderate in all cases and had significant
impact with prolonged reaction time. This spontaneous ring-
opening has undesirable effect on the reaction where kinetic
resolutions are considered, and hamper synthesis of the
enantiomerically pure products. By using HheC as the most
enantioselective among the tested enzymes, products could be
obtained in moderate enantiomeric purity only (ee up to 62%).
Therefore we decided to focus on the synthesis of racemic
products by using an HHDH displaying low enantioselectivity
but high regioselectivity. HheB2 turn out to be the best
biocatalyst for this transformation. HheB2 was discovered in
1999,7 however, only several biocatalytical studies were
published so far, most of them referring the low
enantioselectivity and high regioselectivity.8 HheB2 has 25%
sequence identities compared to HheC the most used HHDH in
biotransformations.9 For the biocatalytic experiments, the
enzyme was produced recombinantly in E. coli MC1061 cells.
The enzyme was expressed in high concentration and was used as
a crude extract. Enzymatic reactions were first performed on
analytical scale in Tris-SO4 buffer (pH 7.5) containing 2% of
DMSO, 100 mM of 1a and 1.5 equiv. of nucleophile (NaN3,
NaCN, NaNO2 and NaCl). Enzymatic activity in the case of
NaOCN was very low, while NaSCN, NaBr and NaI
spontaneously reacted with 1a and the addition of enzyme had
rather low impact on the reaction outcome. Enzymatic reactions
were monitored by extracting products from the aqueous phase,
followed by GC analysis.
Preparative-scale experiments using 100 mg of rac-1a were
performed (Table 2). For the synthesis of 2a it was sufficient to
use 1.5 equiv. of NaN3. Reaction was completed within 60 min
and racemic product was obtained by extraction in 92% yield and
high chemical purity. For the synthesis of other derivatives high
conversions could be achieved by running reaction for a longer
time and using larger excess of the nucleophile. In the view of
catalytic efficiency 2 equiv. of NaNO2 and NaCN and 5 equiv. of
NaCl where chosen as the optimal (Table 2). Reactions were
carried out at room temperature and stopped when slowed down
after 1-3h. After extraction, the solvent was evaporated as well as
unreacted epoxide. Without further purification 2f and 2g were
obtained in 84% and 63% yield, respectively, while 2e was
purified by column chromatography (31% yield).
100
80
60
40
NaN
3
NaNO
2
20
0
NaCN
NaCl
0
20
40
60
80
100
120
As expected, the rate of the conversion of 1a was found to be
dependent on the used nucleophile (Figure 1). The reaction was
fastest with azide where complete conversion was reached within
1h (non-catalysed conversion was ca. 10%). With nitrite the
reaction was slightly slower and conversion of 85% was reached
Figure 1. Progress curves for the HheB2-catalysed conversion of 1a in the
presence of different nucleophiles. The reaction conditions: 1a (0.25 mmol,
0.1 M), NaNu (0.375 mmol, 1.5 eq), buffer (2.3 mL, 0.5 M Tris-SO4, pH 7.5),
DMSO (50 µL) and cell-free enzyme extract (200 µL of HheB2).

3
Table 2. Conversion of 1a with different nucleophiles catalysed by HheB2.a
OH
O
HheB2
Buffer
+
NaNu
F
F
Nu
1a
2a, 2e-2g
Nu
Equiv.b
t (h)
Conversion (%)c
Product
Yield (%)d
N3
1.5
2
2
1
2
3
1
100
100
100
100
2a
2e
2f
92
31e
84
63
NO2
CN
Cl
5
2g
a The reaction conditions: 1a (100 mg, 1.31 mmol, 0.1 M), NaNu (1.5 – 5 eq), buffer (0.5 M Tris-SO4, pH 7.5, 12 mL) and HheB2 (200 µL of cell-
free enzyme extract).
b Equivalents of NaNu.
c Determined by GC.
d Isolated yield.
e Yield of 2e after column chromatography.
2. Bonollo, S.; Lanari, D.; Marrocchi, A.; Vaccaro, L. Curr.
Org. Synth. 2011, 8, 319 – 329.
The most efficient reaction was the azidolysis of 1a.
Enzymatic activity is very high and 70% of 1a was converted
after 5 min. Low azide concentrations, only 1.5 equiv., were
sufficient to run the reaction to completion within 60 min.11
Azidolysis of 1a has been reported in a mixture of water and
ethanol by using 1.6 equiv. of sodium azide and 1.8 equiv. of
ammonium chloride. Reaction was run overnight at 50 °C and 2a
was obtained in 47% yield.12 HheB2 showed to be an
advantageous catalyst for the preparation of 2a. Biocatalytic
reactions occur at the high rate under mild reaction conditions
enabling the preparation of fluorinated alcohols in good yields.
3. (a) Lee, E. Y. J. Ind. Eng. Chem. 2007, 13, 159–162.
(b) Xue, F.; Liu, Z.-Q.; Wan, N.-W.; Zhu, H.-Q.; Zheng, Y.-
G. RSC Adv. 2015, 5, 31525–31532.
(c) Wistuba, D.; Schurig, V. Chirality 1992, 4, 178–184.
(d) Weijers, C. A. G. M. Tetrahedron: Asymmetry 1997,
8639–647.
(e) Kang, J. H.; Woo, J. H.; Kang, S. G.; Hwang, Y.-O.; Kim,
S.-J. J. Microbiol. Biotechnol. 2008, 18, 1445-1452.
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Yamada, H. Tetrahedron 1994, 50, 11821–11826.
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Schallmey, A. Appl. Microbiol. Biotechnol. 2016, 100, 7827-
7839.
In conclusion, regioselective synthesis of fluoro alcohols was
performed starting from racemic epifluorohydrin. Four different
racemic 1-substituted-3-fluoro-2-propanols were prepared in
moderate to high yields starting from commercially available
epifluorohydrin by a simple and fast procedure that took place at
room temperature. In addition, fluoro alcohols were found not to
be substrates for HHDH and experiments performed here
confirmed that the enzyme is unable to cleave C−F bond. Due to
the competitive chemical ring-opening reaction, only products
with moderate ee can be prepared by kinetic resolution of
employing HheC. To our knowledge, this is the first study of the
HHDH-catalysed ring-opening reaction of epifluorohydrin.
Studies for the preparation of enantiomericaly pure fluoro
alcohols are ongoing in our laboratories.
(d) Majerić Elenkov, M.; Tang, L.; Meetsma, A.; Hauer, B.;
Janssen, D. B. Org. Lett. 2008, 10, 2417–2420.
(e) Mikleušević, A.; Hameršak, Z.; Salopek-Sondi, B.; Tang,
L.; Janssen, D. B.; Majerić Elenkov, M. Adv. Synth. Catal.
2015, 357, 1709–1714.
(f) Jin, H.-X.; Hu, Z.-C.; Liu, Z.-Q.; Zheng, Y.-G. Biotechnol.
Appl. Biochem. 2012, 59, 170–177.
5. (a) Gillis, E. P.; Eastman, K. J.; Hill, M. D.; Donnelly, D. J.;
Meanwell, N. A. J. Med. Chem. 2015, 58, 8315–8359.
(b) Zhou, Y.; Wang, J.; Gu, Z.; Wang, S.; Zhu, W.; Aceña, J.
L.; Soloshonok, V. A.; Izawa, K.; Liu, H. Chem. Rev. 2016,
116, 422–518.
(c) Hagmann, W. K. J. Med. Chem. 2008, 51, 4359–4369.
6. (a) Majerić Elenkov, M.; Szymański, W.; Janssen, D. B. In
Science of Synthesis, Biocatalysis in Organic Synthesis 2;
Faber, K.; Fessner W.-D.; Turner, N. J., Ed.; Thieme,
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Acknowledgements
Financial support by the Croatian Ministry of Science and
Education (MZO grant no. KK.01.1.1.01.0002) is gratefully
acknowledged.
(b) Hasnaoui, G.; Majerić Elenkov, M.; Lutje Spelberg, J. H.;
Hauer, B.; Janssen, D. B. ChemBioChem 2008, 9, 1048–
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Supplementary Information
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Experimental data associated with this article can be found, in
the online version, at http://
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References and Notes
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Tetrahedron: Asymmetry
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Kroutil, W. Adv. Synth. Catal. 2007, 349, 1399-1404.
9. van Hylckama Vlieg, J. E. T.; Tang, L.; Lutje Spelberg, J. H.;
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11. Racemic 1a (100 mg,1.3 mmol, 100 mM final concentration)
was dissolved in a 12 mL of Tris-SO4 buffer (0.5 M, pH 7.5)
followed by the addition of NaN3 (130 mg, 2.0 mmol) and 1
mL of cell-free extract containing HheB2 in TMEG buffer.
The reaction was performed at room temperature for 1h. The
reaction mixture was extracted with ethyl acetate. The
combined organic extracts were dried over Na2SO4, filtered
and the solvents evaporated. Without further purification
racemic 2a was obtained in 92% yield (143 mg) as a
colourless liquid; chemical purity determined by GC was
98%; IR (KBr) ν (cm-1): 3398, 2962, 2110, 1278, 1103, 1020;
1H NMR (300 MHz, CDCl3) δ (ppm): 2.59 (br s, 1H), 3.39-
3.51 (m, 2H), 3.96-4.10 (m, 1H), 4.46 (ddd, 2H, JHF = 47.0
Hz, J = 4.5 Hz, 2.3 Hz); 13C NMR (75 MHz, CDCl3) δ (ppm):
52.47 (d, JCF = 7 Hz), 69.37 (d, JCF = 21 Hz), 83.94 (d, JCF
171 Hz).
=
12. Kolb, H.; Walsh, J. C.; Gadharmath, U. B.; Karimi, F.;
Padgett, H. C.; Kasi, D.; Gao, Z.; Liang, Q.; Collier, T. L.;
Duclos, B. A.; Zhao T. Pat. WO 2008/124651, 2008.

5
Highlights:
•
•
•
First enzyme catalysed ring-opening
reaction of epiflorohydrin
HheB2 was used for regioselective synthesis
of fluorinated alcohols
HHDHs were found unable to cleave C-F
bond