One pot 'click' reactions: Tandem enantioselective biocatalytic epoxide ring opening and [3+2] azide alkyne cycloaddition

Article abstract of DOI:10.1039/b919434g

Halohydrin dehalogenase (HheC) can perform enantioselective azidolysis of aromatic epoxides to 1,2-azido alcohols which are subsequently ligated to alkynes producing chiral hydroxy triazoles in a one-pot procedure with excellent enantiomeric excess. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/b919434g

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One pot ‘click’ reactions: tandem enantioselective biocatalytic epoxide
ring opening and [3+2] azide alkyne cycloadditionwz
a
ab
Lachlan S. Campbell-Verduyn, Wiktor Szymanski, Christiaan P. Postema,b
´
Rudi A. Dierckx,c Philip H. Elsinga,*c Dick B. Janssen*b and Ben L. Feringa*a
Received (in Cambridge, UK) 17th September 2009, Accepted 3rd December 2009
First published as an Advance Article on the web 5th January 2010
DOI: 10.1039/b919434g
Halohydrin dehalogenase (HheC) can perform enantioselective
azidolysis of aromatic epoxides to 1,2-azido alcohols which are
subsequently ligated to alkynes producing chiral hydroxy
triazoles in a one-pot procedure with excellent enantiomeric
excess.
Scheme 1 Biocatalytic azidolysis of aromatic epoxides.5
azidolysis of substituted styrene oxides to their corresponding
chiral 1,2-azido alcohols in a highly enantioselective (E 4 200)
and b-regioselective manner (Scheme 1).
The halohydrin dehalogenase has been cloned, and brought
to overexpression, lending it potential for industrial scale
production, and making it a versatile method for synthetic
organic chemists.6,7
The discovery that copper catalyzes the 1,3-dipolar cyclo-
addition of azides and alkynes to form 1,4-disubstituted
triazoles has significantly contributed to the popularization
of ‘click’ chemistry and its subsequent application to the areas
of drug discovery, polymer chemistry, medicinal and biological
sciences, amongst others.1 Recently, efforts have been pursued
to involve the copper catalyzed azide alkyne cycloaddition
(CuAAC) in one-pot multicomponent reactions.2 The
bioorthogonality of the azide-alkyne cycloaddition makes it
uniquely suited to one-pot procedures. An attractive possibility
to execute a tandem reaction is the combination of azide
induced ring opening of epoxides with the copper catalyzed
1,3-dipolar cycloaddition of azides and alkynes. Starting from
enantiomerically pure epoxides, it has been shown that sodium
azide triggered ring opening followed by the click reaction can
occur in one pot with PEG-400 as a solvent, with retention of
the enantiomeric excess of the starting material.3 Another
approach demonstrated the possibility of enzymatically reducing
a-azidoacetophenone derivatives in an enantioselective manner
to their azido alcohol counterparts. As both the aceto-
phenones and the resulting alcohols contain the azide
functionality, the alcohol must first be isolated prior to
attachment to an alkyne via copper catalysis.4
The use of isolated enzymes can be advantageous in several
respects, particularly for simplification of product separation,
and to avoid the potential for undesired byproduct formation.4
We constructed a variant of HheC with cysteine 153 mutated into
serine.8 This increases the enzyme’s stability towards oxidation
and removes the need for addition of b-mercaptoethanol.
We envisioned that this exquisite selectivity could be
combined with the copper catalyzed [3+2] cycloaddition of
azides and alkynes to produce optically pure triazoles. These
products are particularly interesting, not only due to the
presence of the 1,2,3-triazole moiety which has proven to be
a promising pharmacophore9 but also as b-adrenergic receptor
blocker analogues and potential imaging agents.10
We demonstrate herein the first example of a one-pot tandem
biocatalytic enantioselective epoxide ring opening and click
reaction to produce optically pure hydroxy triazoles. This
method uses inexpensive and readily available racemic epoxides
and allows for the subsequent click reaction to occur in one pot,
thus limiting the experimental steps and proceeding in a more
environmentally friendly fashion. Both traditional copper(I)
catalyzed and copper free click reactions give excellent results.
The investigation was initiated by exploration of the reactivity
and selectivity of the enzyme in the presence of the click
additives, namely, CuSO4ꢀ5H2O, the reducing agent sodium
ascorbate and the MonoPhos ligand used to enhance the rate
of the azide-alkyne cycloaddition as recently demonstrated.11 In
anticipation of the one-pot reaction, the buffer used to store the
enzyme, 10 mM Tris-HCl (pH 7.5, 1.0 mM EDTA, 10%
glycerol), had to be changed due to the presence of EDTA which
has the ability to chelate copper thereby inhibiting catalysis.
Potassium phosphate buffer was chosen as a substitute (pH 7.5,
50 mM). Styrene oxide 1 was used as the initial substrate.
We were pleased to find that the alteration of buffer had no
apparent impact on the enantioselectivity of the conversion of
We have previously reported on the biocatalytic azidolysis
of aromatic epoxides using HheC, the halohydrin dehalogenase
from Agrobacterium radiobacter.5 This enzyme catalyzes
a Stratingh Institute for Chemistry, University of Groningen,
Groningen, the Netherlands. E-mail: b.l.feringa@rug.nl;
Fax: +31 50 363 4278; Tel: +31 50 363 4296
b Biochemical Laboratory, Groningen Biomolecular Sciences and
Biotechnology Institute, University of Groningen, Groningen,
the Netherlands. E-mail: d.b.janssen@rug.nl
c Department of Nuclear Medicine and Molecular Imaging, University
Medical Center Groningen, University of Groningen, Groningen,
the Netherlands. E-mail: p.h.elsinga@ngmb.umcg.nl;
Fax: +31 50 3611687; Tel: +31 50 3613247
w This article is part of a ChemComm ‘Catalysis in Organic Synthesis’
web-theme issue showcasing high quality research in organic
chemistry. Please see our website (http://www.rsc.org/chemcomm/
organicwebtheme2009) to access the other papers in this issue.
z Electronic supplementary information (ESI) available: General
experimental procedures, 1H and 13C NMR data for all compounds, full
characterization for all new compounds. See DOI: 10.1039/b919434g
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Table 1 Enzymatic activity in ‘‘click’’ conditions
Table 2 Optimization of one pot enzymatic and click reactions
Concentration Cu (mol%) Time/h Conv.b ee A(%) ee B(%) E
ee A ee B
Concentrationa Solventb Additivesc Conv.d (%) (%)
1 2.0 mM
2 4.0 mM
3 4.0 mM
4 4.0 mM
5 50.0 mM
5
5
5
1
5
24
24
67
24
67
43% 75
34% 51
39% 62
n.d.a 85
24% 23
499
97
97
99
42
4200
109
124
4200
2.8
R
E
1 H
2 H
2.0
2.0
Buffer No
Buffer Yes
Buffer No
Buffer Yes
Buffer No
Water Yes
Water No
Buffer No
47% 499 89
26% 499 35
46% 499 83
4200
4200
4200
4200
4200
3 NO2 2.0
4 NO2 2.0
5 NO2 4.0
6 NO2 25.0
7 NO2 25.0
8 NO2 50.0
50%e 97
98
a
b
Azido alcohol remaining. Max. conversion 50%.
17% 499 20
o1% n.d. n.d. n.d.
o1% n.d. n.d. n.d.
o1% n.d. n.d. n.d.
with 5 mol% of copper, no azido alcohol remained in the
reaction mixture. There appears to be a slight effect on the
efficiency of the overall process, either on the part of
the phenylacetylene, or due to a process within the catalysis
of the click reaction, on the rate of the ring opening, as only
43% conversion occurs after 24 h. The same experiment at a
higher concentration showed a slight drop in the ee of 4 (97%)
and lower conversion (Table 2, entry 2).
a
b
Epoxide concentration (mM). Reactions were conducted either in
50.0 mM potassium phosphate buffer (pH = 7.5) or in distilled
c
water. Includes copper(II) sulfate pentahydrate, sodium ascorbate
d
e
and MonoPhos. Conversion at 16 h. Max. conv. 50%. Conversion
after 24 h.
epoxide to azido alcohol 3 giving the product with 99% ee
(Table 1, entry 1) albeit with slightly lower conversion
overnight (47% of the available 50% conversion for a kinetic
resolution). The same reaction was performed in the presence
of CuSO4ꢀ5H2O (5 mol%), sodium ascorbate (25 mol%) and
MonoPhos (5.5 mol%).12 Fortunately, the presence of these
additives proved to have no effect on the optical purity of 3,
but we found that the rate decreased considerably in their
presence (Table 1, entry 2).
Repetition of the experiment with a longer reaction time
gave a slight increase in conversion (Table 2, entry 3).
Reducing the amount of catalyst to 1 mol% also gave excellent
results, 99% ee for 5 and 85% ee for the remaining epoxide
(Table 2, entry 4). Comparing entries 2 and 4 it can be
ascertained that the concentration of the catalytic additives
affects the ee. With 1 mol% of copper the effective copper
complex concentration is lowered (from 0.20 mM to 0.04 mM)
and the ee rises while the starting substrate concentration is
kept constant at 4.0 mM. However, detection of azido alcohol
in the reaction mixture indicates that with 1 mol% of catalyst
the cycloaddition slows to the extent that it becomes the rate
limiting step in the cascade. Of particular interest is a reaction
performed at 50 mM concentration (Table 2, entry 5). As
aforementioned, no trace of 4 had been detected when the
enzymatic transformation was attempted at such a high substrate
concentration (Table 1, entry 8). However, in the presence of
click additives and phenylacetylene, after 24 h, 24% conversion
to triazole 5 was detected. Although the enantioselectivity was
lower (42%) in the product than usual, it is significant that the
occurrence of the second reaction appears to promote formation
of azido alcohol in the first.y
A selection of substrates was made for further investigation.
In the instance of styrene oxide as a substrate, the results were
even more satisfying. At 4.0 mM substrate concentration
triazole was detected with 499% ee, and conversion from
epoxide to product was 44%, with 78% ee for the epoxide
(Table 3, entry 1). Reducing the catalyst loading from 5 to 3%
again proved insufficient, as azido alcohol 3 remained in the
reaction mixture (Table 3, entry 2). We also tested propiolic
acid as an alkyne substrate (Table 3, entry 4). Interestingly, the
triazole product was detected with nearly racemic distribution.
We hypothesized that the presence of the acid could impact the
functionality of the enzyme. Thus the corresponding ester,
ethyl propiolate, was tested as well (Table 3, entry 5). Indeed,
the resulting triazole showed a dramatic improvement in ee to
80% but the conversion of epoxide remained low after 24 h.
The conversion of 2-(4-nitrophenyl)oxirane 2 to its corres-
ponding azido alcohol 4 in potassium phosphate buffer was
also investigated (Table 1, entry 3). Nearly full conversion was
achieved overnight with 499% and 83% ee in the azide and
the epoxide, respectively. The same reaction in the presence of
the additives (Table 1, entry 4) showed full conversion to 4
after 24 h with 97% ee and 98% ee for the azide and the
epoxide, respectively.
A higher substrate concentration
(4.0 mM) proved to have a detrimental effect upon the
conversion while retaining the perfect enantioselectivity of
the transformation (Table 1, entry 5).
When the enzymatic conversion to azido alcohol was attempted
in water, no conversion was detected (Table 1, entry 6). In the
absence of click additives, the same result was observed
(Table 1, entry 7). The poor result is thus attributed to the
inability of the enzyme to perform under these buffer-free
conditions. High substrate concentration (50 mM) also proved
too challenging for the enzyme, and no trace of 4 was detected
(Table 1, entry 8).
Having established the ability of the reaction to proceed in
potassium phosphate buffer in the presence of the necessary
additives with essentially unaltered selectivity, it was possible
to attempt the one-pot ring opening and subsequent click
reaction. In the first attempt, with 5 mol% of catalyst, after
24 h, the triazole product 5 could be detected with 99% ee, and
the remaining epoxide with 75% ee (Table 2, entry 1). Thus,
the first step of the cascade maintains its high level of
selectivity, and the click reaction proceeds at such a rate that
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Table 3 Substrate scope
This project is financially supported by the Netherlands
Ministry of Economic Affairs and the B-Basic partner
organizations,
a
public-private NWO-ACTS. Financial
support from the University Medical Center Groningen is
gratefully acknowledged.
Ra R0
Cu (mol%) Conv. (%)b ee A(%) ee B (%) E
Notes and references
1 H
2 H
3 NO2 Ph
4 NO2 COOH
Ph
Ph
5
3
5
5
44
78
78
51
n.d.
20
99
98
97
2
4200
4200
109
n.d.
10
n.d.c
34
y The possibility of enzyme inhibition by the azidoalcohol product at
higher concentrations cannot be excluded.
n.d.
20d
1 R. Huisgen, in 1,3-Dipolar Cycloaddition Chemistry, ed. A. Padwa,
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5 NO2 COOMe 5
80
a
b
4.0 mM substrate concentration. After 24 h. Max. conversion
c
d
50%. Azido alcohol remaining in the reaction mixture. Enzyme
added in two portions (at 0 h and 12 h).
Table 4 Copper free cycloaddition of cyclooctyne
2 D. B. Ramachary and C. F. Barbas III, Chem.–Eur. J., 2004, 10,
5323; K. Odlo, E. A. Høydahl and T. V. Hansen, Tetrahedron
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Conversion (%)b
ee A (%)
ee B (%)
E
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4 H. Ankati, Y. Yang, D. Zhu, E. R. Biehl and L. Hua, J. Org.
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1
2
24
48a
20
32
24
47
96
96
83
78
a
b
Enzyme added in two portions (half at t = 0 h, half at t = 24 h). Max.
conversion 50%.
5 J. H. Lutje Spelberg, J. E. T. van Hylckama Vlieg, L. Tang,
D. B. Janssen and R. M. Kellogg, Org. Lett., 2001, 3, 41.
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We can conclude from these observations that not only is the
epoxide important as the substrate undergoing enzymatic
conversion, but the choice of accompanying acetylene is
equally relevant with regards to both rate and selectivity.
We also attempted the more biologically interesting copper
free click reaction. Cyclooctyne was chosen as a model
substrate.13 After 24 h analysis by HPLC revealed the triazole
with 96% ee, and the epoxide with 24% ee, indicating 20%
conversion (Table 4, entry 1).14 We repeated the reaction over
48 h, adding the enzyme in two portions (half at the start of the
reaction, and the other half after 24 h) to ensure constant
enzymatic activity. This resulted in a significant increase in the
ee of the epoxide (47%) along with improved conversion.
The scope of the one-pot ring opening click reaction can
therefore be extended to include the variety of strained cyclic
cyclooctyne derivatives that have been developed recently.15
In conclusion, we have developed a methodology to
enzymatically catalyze azidolysis of aromatic epoxides in an
enantioselective fashion to the corresponding azido alcohols,
and in the same pot, click the resulting azides to alkynes.
The reaction conditions are very mild, proceeding in aqueous
solution with neutral pH at room temperature. The one-pot
nature of the process allows for a simpler, faster and more
environmentally friendly reaction, work-up and purification.
We have demonstrated that biocatalysis is compatible with
one-pot multicomponent reactions. This transformation can
be promoted either through copper catalysis or by ring strain,
opening the possibility for a wide variety of applications.
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azide ringopening of the epoxides could be detected.
13 J. M. Baskin, J. A. Prescher, S. T. Laughlin, N. J. Agard,
P. V. Chang, I. A. Miller, A. Lo, J. A. Codelli and
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This journal is The Royal Society of Chemistry 2010
900 | Chem. Commun., 2010, 46, 898–900