Combining designer cells and click chemistry for a one-pot four-step preparation of enantiopure β-hydroxytriazoles

Article abstract of DOI:10.1002/adsc.201000502

The multistep catalytic process using designer cells, either added as freshly prepared suspensions or as stable lyophilized powder, and click reaction can be performed in one pot. The sequence of four reactions allows the production of both enantiomers of β-hydroxytriazoles with high enantiomeric excess.

Full text of DOI:10.1002/adsc.201000502

COMMUNICATIONS
DOI: 10.1002/adsc.201000502
Combining Designer Cells and Click Chemistry for a One-Pot
Four-Step Preparation of Enantiopure b-Hydroxytriazoles
Wiktor Szymanski,a,b Christiaan P. Postema,a Chiara Tarabiono,a Florian Berthiol,b
Lachlan Campbell-Verduyn,b Stefaan de Wildeman,c Johannes G. de Vries,b,c,*
Ben L. Feringa,b,* and Dick B. Janssena,*
a
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen,
Nijenborgh 4, 9747 AG, Groningen, The Netherlands
Fax : (+31)-50-363-4296; e-mail: D.B.Janssen@rug.nl, B.L.Feringa@rug.nl
Center for Systems Chemistry, Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747 AG,
b
Groningen, The Netherlands
DSM Innovative Synthesis BV, P.O. Box 18, 6160, MD Geleen, The Netherlands
c
Fax : (+31)-46-476-7604; e-mail: Hans-JG.Vries-de@dsm.com
Received: June 29, 2010; Published online: September 1, 2010
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adsc.201000502.
Abstract: The multistep catalytic process using de-
signer cells, either added as freshly prepared sus-
pensions or as stable lyophilized powder, and click
reaction can be performed in one pot. The sequence
of four reactions allows the production of both
enantiomers of b-hydroxytriazoles with high enan-
tiomeric excess.
The outstanding possibilities offered by the use of
designer cells for the one-pot catalysis of a set of sub-
sequent reactions can be further broadened through
combination with a bioorthogonal catalytic process
that takes place in the same reaction vessel. We have
recently reported the possibility of combining a bio-
catalytic process, in which an isolated enzyme is used
for the enantioselective introduction of an azide
moiety into a substrate, with a subsequent Cu(I)-cata-
lyzed [2+3]-dipolar cycloaddition (“click” reaction)
of the product with acetylenes.[6]
Keywords: azides; biocatalysis; click chemistry; de-
signer cells; halohydrin dehalogenase; reaction cas-
cade
The combination of the multienzymatic cell cascade
and the bioorthogonal click reaction might offer a val-
uable method for the one-pot transformation of
simple, prochiral substrates into enantiopure, highly
The synthetic application of designer cells, that is, en- functionalized products. The triazole group which is
gineered microbial cells which overexpress one or formed by the cycloaddition is a bioisostere of the
more synthetically useful biocatalysts, attracts special peptide bond[7] and thus an important pharmaco-
attention. Recent reports, in which two genes encod- phore. Specifically, b-hydroxytriazoles have been re-
ing enzymes involved in reductive biotransformations ported to be b-adrenergic receptors blockers.[8]
were cooverexpressed in such “tailor-made” host
We report here a new, simple, one-pot procedure
cells, describe their use in the stereoselective reduc- for the preparation of enantiopure b-hydroxytriazoles
tion of ketones,[1] a-halo ketones,[2] a-hydroxy ke- 5 from simple a-halo ketones 1 (Scheme 1), which
tones[3] and a-keto esters[4] or reductive amination of combines the use of designer cells and click reactions.
a-keto esters.[5] In all of these cases one of the cloned
The biocatalytic cascade incorporates four process-
enzymes is responsible for the biocatalytic transfor- es, which transform the prochiral a-halo ketones 1
mation of the substrate, while the other one oxidizes into enantiopure b-hydroxy azides 4. In the first reac-
a sacrificial electron donor (such as glucose, methanol tion the ketone 1 is stereoselectively reduced to b-
or formate) thus regenerating the cofactor (NADP or halo alcohol 2 by an alcohol dehydrogenase (ADH).[9]
NADPH). The main advantages of such designer cells In this process NADPH is used as a cofactor. The re-
include low cost, as there is no need for expensive iso- cycling of NADPH is provided by the same enzyme
lated enzymes and cofactors, and the higher stability in a reaction which transforms isopropyl alcohol, a
of the biocatalysts in the cell environment.
sacrificial electron donor, into acetone. Compound 2
Adv. Synth. Catal. 2010, 352, 2111 – 2115
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2111

Wiktor Szymanski et al.
COMMUNICATIONS
Scheme 1. Multicatalytic cascade leading to enantiopure b-hydroxytriazoles.
serves as a substrate for halohydrin dehalogenase
(Hhe), which catalyzes the ring-closure towards epox-
ide 3 and subsequent ring-opening of 3 by azide
anion, forming compound 4, the final product of the
biocatalytic cascade. Azido alcohol 4 is in the same
reaction vessel converted to b-hydroxytriazole 5 in a
Cu(I)-catalyzed [2+3]-dipolar cycloaddition reaction.
We have decided to use the enzyme-catalyzed highly
regioselective azidolysis of the epoxide ring
(Scheme 1, 3 to 4), reported by us earlier,[10] as the
central reaction of this system, allowing for the intro-
duction of an azide into the substrates. A similar reac-
tion was explored for the incorporation of azide using
isolated enzymes and cofactor addition by Kroutil
and co-workers[11] and by researchers from Codexis
for the incorporation of cyanide for producing a statin
intermediate.[12]
Scheme 2. Whole cell-catalyzed conversion of b-halo ke-
tones 1 to b-azido alcohols 4.
We aimed at engineering cells that overexpress
both enzymes used in the cascade, thus making the
process cheaper and applicable on a larger scale. Two
such constructs were made, using combinations of ke-
toreductases (dehydrogenases) expressing opposite
stereoselectivity. In both cases cells of E. coli strain
MC1061 were used as the host and the enzymes were
cloned on a single pBad-vector. The first construct
(CT cells) expressed AdhT, an R-selective alcohol de-
hydrogenase from Thermoanaerobacter sp.[13] and
HheC, the R-selective halohydrin dehalogenase from
Agrobacterium radiobacter AD1.[14] The second type
of cells (BL cells) expressed AdhL, the S-selective al-
Scheme 3. Compounds studied in the biocatalytic reaction
cohol dehydrogenase from Lactobacillus brevis[15] and cascade.
HheBGP1, the halohydrin dehalogenase from Myco-
bacterium sp. GP1 that displays low enantioselectivi-
ty.[14] Between the genes a linker region was added rial and the formation of the epoxide 3a. No halo al-
containing a translational enhancer and a ribosome cohol 2a was observed, indication that under these
binding site, so each transcribed gene was independ- conditions the conversion of 2a to 3a is faster than
ently translated.
the formation of 2a. After 6 h a new portion of cells
We studied the applicability of the engineered cells was added, followed by sodium azide (5 equiv.). Over-
on a model process (Scheme 2), in which CT cells night incubation resulted in full conversion of the
were used for the transformations of compound 1a starting material 1a and formation of b-azido alcohol
(Scheme 3). The reaction was carried out at room 4a. Compounds 2a and 3a were no longer observed in
temperature in a two-phase system composed of the reaction mixture.[16]
HEPES buffer, octane as the organic solvent, and iso-
Having confirmed the applicability of the CT-type
propyl alcohol for regeneration of the nicotinamide designer cells in the model reaction, we studied the
cofactor of the reductase. After 6 h of incubation with scope of the whole-cell biocatalytic cascade on a
a suspension of freshly thawed CT cells we observed larger set of compounds (Scheme 3, Table 1). Trans-
high, though not full, conversion of the starting mate- formation of compound 1a (Table 1, entry 1) led to 4a
2112
asc.wiley-vch.de
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2010, 352, 2111 – 2115

Combining Designer Cells and Click Chemistry for a One-Pot Four-Step Preparation
Table 1. Conversion of halo ketones 1a–1k to azido alcohols
with CT cells.
lyophilized cells) we envisage this new one-pot, whole
cell cascade to be a valuable tool for the stereoselec-
tive preparation of an important class of chiral build-
ing blocks.
Entry
Substrate
Conversion
Isolated
ee of
of 1
yield of 4
4
Having demonstrated the possibility to convert the
halo ketones 1 into enantiopure azido alcohols 4
using the whole-cell cascade of biocatalytic transfor-
mations, we studied the possibility of combining it
with a click reaction in a one-pot process (Scheme 4).
Therefore, the addition of sodium azide in the second
step of the cascade is followed after 30 min by the in-
troduction of a model [2+3] cycloaddition partner
(phenylacetylene), a copper(I) source (CuSO4 and
sodium ascorbate) and MonoPhos, a ligand that has
recently been shown to efficiently enhance the rate of
the click reaction.[17] We used only substrates that
were within the substrate scope of the designer cells;
chlorine-substituted compounds 1b and 1f were also
1
2
3
4
5
6
7
8
1a
1b
1c
1d
1e
1f
1g
1h
1i
full
full
full
full
full
full
full
<5%
<5%
full
42% (4a)
44% (4a)
96% (R)
96% (R)
–
–
98% (R)
99% (R)
–
–
[a]
–
–
[a]
39% (4e)
35% (4e)
[a]
–
–
–
9
10
11
–
1j
1k
49% (4j)
70% (4k)
98% (R)
99% (R)
full
[a]
The formation of the expected product was not observed.
which was isolated with 42% yield and very high not included, as they gave the same results as their
enantiomeric excess. For compound 1b, an analogue bromo analogues 1a and 1g. Furthermore, we ob-
of 1a, with chloride instead of bromide as the leaving served that the addition of octane as an organic sol-
group, the results were virtually identical (Table 1, vent results in a reduction of reactivity, probably due
entry 2). Conversion of a-haloacetophenones 1c and to the extraction of phenylacetylene into the organic
1d (Table 1, entries 3 and 4) did not result in the for- phase. Therefore octane was not used.[18] The results
mation of the expected products.
of the reactions performed under one-pot conditions,
Encouraged by the good results obtained with sub- combining the whole-cell reaction cascade and the
strates having electron-withdrawing groups in the click reaction, are presented in Table 2.
para position of the phenyl ring (1a and 1b), we
The conversion of substrate 1a provided the expect-
tested the cyano-substituted compounds 1e and 1f. ed enantiopure triazole 5a with 41% isolated yield
Nearly enantiopure product 4e was obtained with 35– after flash chromatography (Table 2, entry 1). This
39% yield (Table 1, entries 5 and 6). We investigated result corresponds perfectly to the one obtained with-
further the influence of the position of the ring-sub- out the subsequent cycloaddition, where product was
stituent on the outcome of the cascade. Compounds isolated with 42% yield (Table 1, entry 1), indicating
with nitro substituents in ortho and meta positions that in the case of compound 1a the introduction of
were either not converted (Table 1, entry 8) or the ex- the fifth transformation in the same reaction vessel
pected product was not observed (Table 1, entry 7).
does not affect the efficiency of the process. The same
Substrate 1i, with an electron-donating methoxy conclusions may be drawn for compounds 1e and 1k
substituent, was also not converted. Two additional (Table 2, entries 2 and 4). The isolated yield of the
aliphatic substrates were tested. Very high enantiose- four-reaction cascade was significantly lower when
lectivities were obtained for both the ester 1j and the compared to that of the whole-cell biocatalytic cas-
aliphatic compound 1k.
cade without click reaction for compound 1j (Table 2,
In general, the isolated yields of the azido alcohols entry 3).
(35–70%) obtained in this one-pot, three-reaction
Encouraged by these results, we decided to explore
process correspond to those obtained with isolated the possibility of obtaining the other enantiomer of b-
enzymes (38–61%).[11] In view of the high enantiomer- hydroxytriazoles 5, using the BL cells, in which the
ic excess of the products and the simple reaction set- Lactobacillus brevis ketoreductase, which has an op-
up (see also the paragraph below, covering the use of posite enantiopreference,[19] was expressed. Since the
Scheme 4. One-pot whole cell catalysis and click reaction for the conversion of a-halo ketones 1 to b-hydroxytriazoles 5.
Adv. Synth. Catal. 2010, 352, 2111 – 2115
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
2113

Wiktor Szymanski et al.
COMMUNICATIONS
Table 2. Conversion of halo ketones to triazoles in a one-pot
whole-cell biocatalytic cascade with subsequent click reac-
tion.
click reactions. The use of designer cells lowers the
cost of the catalyst and avoids problems connected
with the use of isolated enzymes, giving comparable
results in terms of the product yield and enantiomeric
excess. Due to the built-in cofactor regeneration
system and the possibility of storage and easy use of
lyophilized cells, these biocatalysts which catalyze a
cofactor-dependent redox reaction and lyase reac-
tions, can be as conveniently used as hydrolytic en-
zymes, which have found widespread application in
organic synthesis.
Entry
Substrate
Cells
Isolated yield of 5
ee of 5
1
2
3
4
5
1a
1e
1j
1k
1k
CT
CT
CT
CT
BL
41% (5a)
36% (5e)
18% (5j)
65% (5k)
53% (5k)
99% (R)
99% (R)
99% (R)
99% (R)
97% (S)
halohydrin dehalogenase HheB used in this construct
prefers aliphatic compounds,[12] we chose compound Experimental Section
1k as the substrate for this transformation. Product
(S)-5k was obtained in pure form with 53% yield and General Procedure for Preparation of b-Hydroxy-
with high enantiomeric excess (Table 2, entry 5), thus triazoles (5) from a-Halo Ketones (1)
expanding the synthetic possibilities offered by this
one-pot, four-transformations methodology.
In a 50-mL flask were placed HEPES buffer (9 mL, 0.1M,
pH 7.5, KOH, 1 mM MgSO4, 1 mM ZnSO4), i-PrOH
Finally, to fully exploit the possibilities offered by
low cost and easy preparation of the biocatalyst, we
studied the application of lyophilized cells instead of
frozen cells. Such dried, whole-cell catalysts may be
stored and transported as a powder after preparation,
without any microbiological work, which increases
their value to the synthetic community. Thus we
tested the changes in activity and selectivity of the
lyophilized CT cells in time, on a model substrate –
compound 1k (Table 3). Immediately after freeze-
(0.2 mL, 1.5M), halo ketone 1 (0.2 mmol) and freshly
thawed cells (4 mL). The mixture was stirred at room tem-
perature for 6 h. Then thawed cells (4 mL) were added to-
gether with NaN3 (1 mmol in 200 mL water). After 30 min
phenylacetylene (44 mL, 0.4 mmol) and pre-formed click cat-
alyst (10 mmol CuSO4 and 50 mmol Na-ascorbate dissolved
in 1 mL water, 11 mmol MonoPhos added, all stirred for
10 min at room temperature) were added and the reaction
mixture was stirred overnight at room temperature. After
completion of the reaction the mixture was extracted using
CHCl3 (3 times 25 mL). The combined organic layers were
dried (MgSO4) and the volatiles were evaporated under re-
duced pressure. The b-hydroxytriazoles 5 were purified
using flash chromatography (SiO2, pentane/Et2O=1/1–pen-
tane/EtOAc=4/6).
Table 3. The use of lyophilized cells in the one-pot whole-
cell biocatalytic cascade and the click reaction.
Entry CT Cells
Storage
time at 48C yield of 5k 5k
Isolated
ee of
Supporting Information
Full experimental procedures, preparation of cells and the
analytical data are presented in the Supporting Information.
1
2
3
4
freshly prepared
–
–
65%
63%
63%
50%
99% (R)
99% (R)
99% (R)
99% (R)
lyophilized
lyophilized
lyophilized
3 weeks
10 weeks
Acknowledgements
drying with phosphate buffer, lyophilized cells
(Table 3, entry 2) perform as well as the freshly pre-
pared cells (Table 3, entry 1), both in terms of isolated
yield and enantiopurity of the product. The same
result was obtained when we used lyophilized cells
that were stored for 3 weeks at 48C (Table 3, entry 3),
which is a common storage temperature for chemical
catalysts. Even after 10 weeks of storage the cells
could be used in catalysis, and the enantiopure prod-
uct could be isolated with a moderate yield of 50%
(Table 3, entry 4).
The project is financially supported by the Netherlands Min-
istry of Economic Affairs and the B-Basic partner organiza-
tions (www.b-basic.nl) through B-Basic, a public-private
NWO-ACTS program (ACTS=Advanced Chemical Technol-
ogies for Sustainability) (W.S.) and IBOS (C.T. and F.B.),
and through NWO-STW (C.P.P).
References
[1] a) H. Grçger, F. Chamouleau, N. Orologas, C. Roll-
mann, K. Drauz, W. Hummel, A. Weckbecker, O. May,
Angew. Chem. 2006, 118, 5806; Angew. Chem. Int. Ed.
2006, 45, 5677; b) T. Ema, H. Yagasaki, N. Okita, M.
Takeda, T. Sakai, Tetrahedron 2006, 62, 6143.
We thus have demonstrated the synthetic applica-
bility of a system which combines five reactions in
one container, making use of the compatibility of bio-
catalytic processes and the bioorthogonality of the
2114
asc.wiley-vch.de
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2010, 352, 2111 – 2115

Combining Designer Cells and Click Chemistry for a One-Pot Four-Step Preparation
[2] A. Berkessel, C. Rollmann, F. Chamouleau, S. Labs, O.
May, H. Grçger, Adv. Synth. Catal. 2007, 349, 2697.
[3] K. Schroer, K. P. Luef, F. S. Hartner, A. Glieder, B.
Pscheidt, Metabol. Eng. 2010, 12, 8.
[4] a) T. Ema, S. Ide, N. Okita, T. Sakai, Adv. Synth. Catal.
2008, 350, 2039; b) R. Kratzer, M. Pukl, S. Egger, B. Ni-
detzky, Microb. Cell Fact. 2008, 7, 37.
Ching, S. Tam, S. Muley, J. Grate1, J. Gruber, J. C.
Whitman, R. A. Sheldon, G. W. Huisman, Nat. Biotech-
nol. 2007, 25, 338; b) S. K. Ma, J. Gruber, C. Davis, L.
Newman, D. Gray, A. Wang, J. Grate, G. W. Huisman,
R. A. Sheldon, Green Chem. 2010, 12, 81.
[13] Z. Findrik, D. Vasic-Racki, S. Lꢂtz, T. Daußmann, C.
Wandrey, Biotechnol. Lett. 2005, 27, 1087.
[5] A. Menzel, H. Werner, J. Altenbuchner, H. Grçger,
[14] J. E. T. van Hylckama Vlieg, L. Tang, J. H. Lutje Spel-
berg, T. Smilda, G. J. Poelarends, T. Bosma, A. E. J. van
Merode, M. W. Fraaije, D. B. Janssen, J. Bacteriol. 2001,
183, 5058.
[15] N. H. Schlieben, K. Niefind, J. Mꢂller, B. Riebel, W.
Hummel, D. Schomburg, J. Mol. Biol. 2005, 349, 801.
[16] The reaction is conducted in two stages because of the
inhibition of ketoreductase by high concentrations of
sodium azide. We observed that azide addition at an
earlier stage, when the conversion of 1a is still low, re-
sults in inhibition of the cascade.
Eng. Life Sci. 2004, 4, 573.
[6] L. S. Campbell-Verduyn, W. Szymanski, C. P. Postema,
R. A. Dierckx, P. H. Elsinga, D. B. Janssen, B. L. Ferin-
ga, Chem. Commun. 2010, 898.
[7] Y. Zhou, Y. Zhao, K. M. OꢁBoyle, P. V. Murphy,
Bioorg. Med. Chem. Lett. 2008, 18, 954.
[8] S. Su, J. R. Giguere, S. E. Schaus, J. A. Porco Jr., Tetra-
hedron 2004, 60, 8645.
[9] G. W Huisman, J. Liang, A. Krebber, Curr. Op. Chem.
Biol. 2010, 14, 122.
[10] a) J. H. Lutje Spelberg, J. E. T. van Hylckama Vlieg, L.
Tang, D. B. Janssen, R. M. Kellogg, Org. Lett. 2001, 3,
41; b) G. Hasnaoui-Dijoux, M. Majeric Elenkov, J. H.
Lutje Spelberg, B. Hauer, D. B. Janssen, ChemBioChem
2008, 9, 1048.
[11] J. H. Schrittwieser, I. Lavandera, B. Seisser, B. Maut-
ner, W. Kroutil, Eur. J. Org. Chem. 2009, 2293.
[12] a) R. J. Fox, S. C. Davis, E. C. Mundorff, L. M.
Newman, V. Gavrilovic, S. K. Ma, L. M. Chung, C.
[17] L. S. Campbell-Verduyn, L. Mirfeizi, R. A. Dierckx,
P. H. Elsinga, B. L. Feringa, Chem. Commun. 2009,
2139.
[18] We observed that the biocatalytic cascade without
octane proceeds slowly, but the rate was still sufficient
to result in full conversion of model substrate 1a during
overnight incubation.
[19] M. Wolberg, W. Hummel, M. Muller, Chem. Eur. J.
2001, 7, 4562–4571.
Adv. Synth. Catal. 2010, 352, 2111 – 2115
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
2115