Combination of olefin metathesis and enzymatic ester hydrolysis in aqueous media in a one-pot synthesis

Article abstract of DOI:10.1002/adsc.201100403

A new synthetic method for the preparation of cyclic malonic acid monoesters in aqueous media was developed based on the combination of a metathesis reaction and subsequent biocatalytic hydrolysis with a pig liver esterase in a one-pot synthesis. Both reaction steps proceed smoothly under optimized conditions in aqueous media requiring only a low amount of the metal catalyst for the metathesis reaction. Notably, the applied biocatalyst turned out to be highly compatible with the metal catalyst showing no significant influence on the enzyme activity. Copyright

Full text of DOI:10.1002/adsc.201100403

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DOI: 10.1002/adsc.201100403
Combination of Olefin Metathesis and Enzymatic Ester
Hydrolysis in Aqueous Media in a One-Pot Synthesis
Katharina Tenbrink,a,b Miriam Seßler,a Jꢀrgen Schatz,a,* and Harald Grçgera,b,*
a
Department of Chemistry and Pharmacy, University of Erlangen-Nꢀrnberg, Henkestraße 42, 91054 Erlangen, Germany
Fax : (+49)-9131-85-24707; phone: (+49)-9131-85-25766; e-mail: juergen.schatz@chemie.uni-erlangen.de
Organic Chemistry I, Faculty of Chemistry, Bielefeld University, P.O. Box 100131, 33501 Bielefeld, Germany
b
Fax : (+49)-521-106-6146; phone: (+49)-521-106-2057; e-mail: harald.groeger@uni-bielefeld.de
Received: May 17, 2011; Published online: September 5, 2011
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adcs.201100403.
Abstract: A new synthetic method for the prepara-
tion of cyclic malonic acid monoesters in aqueous
media was developed based on the combination of
a metathesis reaction and subsequent biocatalytic
hydrolysis with a pig liver esterase in a one-pot syn-
thesis. Both reaction steps proceed smoothly under
optimized conditions in aqueous media requiring
only a low amount of the metal catalyst for the
metathesis reaction. Notably, the applied biocatalyst
turned out to be highly compatible with the metal
catalyst showing no significant influence on the
enzyme activity.
systems can be used.[7] Recently it was found that
metathesis in water also proceeds with high conver-
sion when using commercially available Grubbs or
Hoveyda-Grubbs catalysts under heterogeneous con-
ditions, albeit a high catalyst loading of 4 mol% is
needed.[8–10]
A further research area of current interest is the
development of multi-step one-pot processes as they
are time and cost efficient since less work-up and pu-
rification steps and therefore less solvents are
needed.[11] In this field the combination of man-made
metal catalysts and enzymes is a particular challenge
as such reactions typically require different reaction
media. Metal-catalyzed reactions often take place in
organic solvents whereas enzymes often need aqueous
media (except, e.g., lipases). So far there are only a
few examples for such one-pot syntheses.[12] Therefore
the development of efficient chemocatalytic reactions
in water, which are compatible with a biotransforma-
Keywords: aqueous media; biocatalysis; esters;
olefin metathesis; one-pot synthesis
The metathesis reaction belongs to the most efficient tion, is desirable.
and atom-economical metal-catalyzed syntheses and
In continuation with our studies on metathesis reac-
has already found important applications on the in- tions in water[10] and chemoenzymatic one-pot synthe-
dustrial scale.[1] Currently, intensive research efforts sis in aqueous media,[12d,e,13] we became interested in
concentrate on metathesis reactions in water, which combining metal-catalyzed olefin metathesis with a
serves as a cheap, environmental friendly, easy to subsequent biotransformation in aqueous media. In
handle and industrially attractive solvent.[2] For this the following we present such a one-pot synthesis as a
purpose tailor-made water-soluble Grubbs catalysts new synthetic concept for the synthesis of cyclic ma-
turned out to be suitable.[3] In addition, polymer- or lonic acid monoesters 3 (Scheme 1). This model reac-
membrane-bound,[4,5] PEGylated ligands[6] or micellar tion was chosen as the products 3 serve as possible in-
Scheme 1. General synthetic concept: metathesis and subsequent enzymatic ester hydrolysis.
Adv. Synth. Catal. 2011, 353, 2363 – 2367
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Katharina Tenbrink et al.
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termediates in the synthesis of non-natural amino lyst, 4, led to the formation of the desired product 2a
acids bearing a quaternary carbon center.[14]
with quantitative conversion after 22 h (entry 3).
In general, excellent conversions were obtained
The combination of metal- and enzyme-catalyzed
transformations in a one-pot synthesis as shown in also on preparative scale in water (5.0–7.5 mL H2O,
Scheme 1 requires compatibility of the metathesis re- work-up with dichloromethane) independent of the
action as the first step or the resulting reaction mix- substrate concentration, which was in the range of
ture thereof with the subsequent biotransformation in 33 mM to 200 mM (Table 1, entries 4 and 5). With re-
aqueous medium. Accordingly we first focused on de- spect to the mixture of the reactants in water we ob-
veloping an olefin metathesis which is highly efficient served that the substrate, which is insoluble in water,
in aqueous media (instead of using typical organic sol- forms droplets on water, in which the metal catalyst is
vents), proceeding with very low amount of catalyst dissolved. Thus, it appears to be more likely that such
(<1 mol%).
metathesis reactions proceed “on” water and not “in”
Therefore diallyl malonate 1a was chosen as a water. Although the reaction also ran in dichlorome-
model substrate. Furthermore, we were interested in thane as a solvent, at low substrate concentrations
a comparison of the efficiency of the metathesis reac- and low catalyst loading the conversion in water is
tion in water with an organic solvent like dichlorome- quantitative after six hours (entry 5) whereas the
thane (Table 1). First, we studied the efficiency of this same reaction in dichloromethane only reaches 93%
reaction in D2O on an analytical scale (1 mL of sol- conversion (entry 8). Thus, interestingly, water turned
vent, no work-up) according to a recently published out to be the preferred solvent for the metathesis re-
protocol of Varma et al.,[15] which enabled a simple action, enabling a highly efficient synthesis of 2a with
1
analysis of the reaction mixture by H NMR spectros- excellent conversion at low catalyst loading of 0.2–
copy (deuterated solvent: MeOD-d4, Table 1, en- 0.5 mol% of 4. In addition, the use of water as a sol-
tries 1–3). We were pleased to find that even a very vent also fulfils a requirement for the desired one-pot
low catalyst loading of 0.2 mol% of Grubbs II cata- synthesis.
After successfully optimizing the metathesis reac-
tion in water we next focused on the development of
Table 1. Optimization of the metathesis reaction.[a]
an efficient enzymatic hydrolysis of 2a under selective
formation of monoester 3a. As biocatalyst we chose
pig liver esterase (PLE) due to its broad applicability
and use for hydrolyses of a wide range of non-cyclic,
disubstituted dialkyl malonates.[16,17] This type of bio-
transformation (shown in Table 2) gives access to the
cyclic mono acid ester 3a bearing a quaternary carbon
center. To monitor and control the reaction and for-
mation of product 3a, the pH is kept at 7 by titration
with an NaOH solution. When using pig liver esterase
in pure water as a solvent the hydrolysis of 2a pro-
ceeded with a conversion of 92%. However, a product
mixture was isolated consisting of a 74:26 ratio of de-
sired monoester 3a and a significant amount of decar-
boxylated side product 6 (Table 2, entry 1). In order
to avoid decarboxylation as a side reaction we system-
atically explored the influence of water-soluble organ-
ic solvents on the reaction course.[18] High yields in
the range of 91–92% as well as suppression of decar-
boxylation were observed in all experiments (en-
tries 2–5).
Entry Solvent 4 [mol%] c(1a) [M] t [h] Conversion [%]
1
2
3
4
5
6
7
8
D2O
D2O
D2O
H2O
H2O
CH2Cl2 0.5
CH2Cl2 0.5
CH2Cl2 0.5
5.0
0.5
0.2
0.5
0.5
0.150
0.300
0.300
0.200
0.033
0.500
0.300
0.033
4
6
22
18
6
6
6
6
100
100
100
100
98 (100)[b]
100
99
93
[a]
Entries 1–3: these experiments were carried out on an
analytical scale and the conversion was determined from
the reaction mixture by 1H NMR spectroscopy in
MeOD-d4. Entries 4 and 5: these experiments were car-
ried out on a preparative scale and after extraction of the
reaction mixture with CH2Cl2 the conversion was deter-
However, when using the water-soluble alcohols
methanol and isopropanol transesterification was ob-
served leading to significant formation of the unde-
sired side products 5a and 5b, respectively (Table 2,
entries 2 and 3). This effect might be due to the exis-
tence of lipases in the enzyme preparation of PLE.
By engineering of the reaction medium and using an
advantageous mixture of water and tert-butyl alcohol
[3:1 (v/v)] we were pleased to find that both decar-
boxylation and transesterification were avoided and
1
mined by H NMR spectroscopy in CDCl3. Entries 6–8:
these experiments were carried out on a preparative
scale and the conversion was determined by 1H NMR
spectroscopy in CDCl3.
[b]
In parenthesis the result of a repeat test is shown.
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Combination of Olefin Metathesis and Enzymatic Ester Hydrolysis in Aqueous Media
Table 2. Development of an efficient biotransformation.[a]
Entry
Cosolvent [% (v/v)]
t [h]
Conversion [%][b]
3a [%][c]
5 [%][c]
6 [%][c]
1
2
3
4
5
–
25
12
42
23
50
92
91
91
92
92
74
13
61
97
100
0
26
3
5
3
0
MeOH (10)
i-PrOH (10)
t-BuOH (10)
t-BuOH (25)
84 (5a)
34 (5b)
0
0
[a]
All reactions were carried out in a total volume of 10 mL with 0.25 mmol of substrate 2a and 270 units of pig liver ester-
ase.
[b]
[c]
The conversion was determined via a Titrino apparatus by titration with 0.2M NaOH solution and adjusting the pH to 7.
1
The ratios of products 3a, 5 and 6 (given in %) were determined by H NMR spectroscopy from the crude product after
work-up of the reaction mixture.
an excellent reaction yield of the product 3a was ob- investigated the reaction course of the enzymatic hy-
tained (92% conversion, 100% selectivity; entry 5). drolysis of 2a under optimized conditions in the ab-
In the next step we studied the (bio-)compatibility sence and presence of metal catalyst 4 (Figure 1).
of the Grubbs II metathesis catalyst 4 with PLE used When adding the Grubbs catalyst 4, the amount of 4
as a biocatalyst. Such types of investigations on the was in the same range (0.1 or 0.5 mol%) as in the
combination of chemocatalysts and biocatalysts to- metathesis reactions described in Table 1.
wards one-pot, multi-step procedures in water are in
Interestingly, nearly the same reaction course was
general still rare up to now[12,13] but, at the same time, determined in the absence and presence of 0.1 mol%
essential for the development of such a process. We and 0.5 mol% of Grubbs II catalyst 4, respectively,
leading to high conversions and similar reaction rates
in all cases. In the presence of 0.1 mol% of 4 the bio-
transformation proceeded with a conversion of 91%,
and even at a higher loading of 0.5 mol% of metal
catalyst 4 a high conversion of 91% was obtained al-
though a slightly prolonged reaction time was re-
quired. Thus, the Grubbs II catalyst 4 seems to have a
negligible influence on enzyme activity and turned
out to be excellently compatible with the used ester-
ase PLE.
Based on these promising results in optimization of
both the metathesis and the biotransformation with
proven excellent compatibility of the enzyme with the
metal catalyst, a two-step, one-pot process in aqueous
solution combining the metathesis and enzymatic hy-
drolysis was successfully developed (Scheme 2). The
metathesis reaction was performed according to the
optimized procedure (Table 1, entry 5) using
0.5 mol% of catalyst 4. After a reaction time of 6 h
(which turned out to be sufficient for 98% conver-
sion) the enzyme, sodium chloride and tert-butyl alco-
hol were added. The desired monoester 3a, which was
formed with a high overall conversion of ꢀ95% in
the one-pot process, was obtained in excellent yield of
94% after work-up (Scheme 2). The reaction time re-
Figure 1. Compatibilty of catalyst 4 and enzyme PLE.
quired for 95% conversion was 59 h (Scheme 2), thus
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Katharina Tenbrink et al.
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Scheme 2. One-pot synthesis of monoester 3a.
Scheme 3. One-pot synthesis of monoester 3b.
being in a similar range as the analogous biotransfor- which both catalyst components (namely the metathe-
mation starting from the isolated product 2a (50 h, sis catalyst and the hydrolase) are present in the reac-
92% yield; Table 2, entry 5).
tion mixture from the beginning.[19]
This synthetic one-pot process concept was also ap-
plied to diester 1b as a further substrate (Scheme 3),
using the same conditions for metathesis and biotrans- Experimental Section
formation as has been done for substrate 1a. A quan-
titative conversion was also observed for this meta- Procedure for the One-Pot Synthesis of Malonic Acid
thesis after 6 h at room temperature in the presence Monoesters (According to Scheme 2 and Scheme 3)
of 0.5 mol% of Grubbs II catalyst 4, and the enzymat-
ic hydrolysis proceeds with a conversion of 70% in
54 h (Scheme 3). After work-up the desired product
3b was isolated in 67% yield. The enantioselectivity
of the hydrolytic process catalyzed by a commercially
available wild-type preparation of pig liver esterase,
however, was low with 7% ee.
In a 15-mL glass vial diethyl malonate 1a or 1b (0.25 mmol)
was suspended in H2O (7.5 mL) and Grubbs II catalyst (4,
0.5 mol%) was added. The reaction mixture was stirred for
6 h at room temperature before adding t-BuOH (2.5 mL)
and NaCl (0.5 mmol). The subsequent enzymatic hydrolysis,
which was performed in a Titrino apparatus (Metrohm), was
started by addition of pig liver esterase (PLE, purchased
from Sigma–Aldrich; 17 U/mg, 1080 U/mmol of 2a or 2b).
The conversion was determined by the amount of consumed
NaOH solution. The aqueous solution was extracted with
CH2Cl2 (3ꢂ30 mL) and the combined organic phases were
re-extracted with H2O (2ꢂ30 mL). The combined aqueous
phases were acidified with 2M HCl to pH 2, and again ex-
tracted with CH2Cl2 (3ꢂ30 mL). After drying the combined
organic phases over Na2SO4, the solvent was evaporated
under vacuum. The products 3a and 3b were obtained as
slightly yellowish oils; yield: 94% and 67%, respectively.
In conclusion, we have reported the first example
of a combination of a metal-catalyzed metathesis re-
action with a biotransformation in a one-pot synthesis
in aqueous media. With excellent conversion and
without formation of any side products we obtained
monoesters 3a and 3b in 94% and 67% yield, respec-
tively. This one-pot synthesis also represents a new
synthetic approach to unsaturated cyclic acids, which
serve as interesting compounds for the synthesis of
unusual amino acids. Current work focuses on the ex-
pansion of the substrate range for the chemoenzymat-
ic one-pot syntheses of malonic acid monoesters, im-
provement of enantioselectivity of hydrolysis of 2b by
using recombinant isoenzymes of the pig liver ester-
ase in isolated form[17b] as well as the synthesis of
non-natural amino acids bearing a quaternary carbon
center from the monoesters as intermediates. Further
future work will be also related to the development
Acknowledgements
K.T. and M.S. thank the Graduate School of Molecular Sci-
ence at the University of Erlangen-Nꢀrnberg for support.
M.S. thanks the “Fonds der Chemischen Industrie” for a
of a one-pot process running in a “tandem mode”, in PhD grant.
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Combination of Olefin Metathesis and Enzymatic Ester Hydrolysis in Aqueous Media
References
Angew. Chem. 2008, 120, 9693–9696; Angew. Chem.
Int. Ed. 2008, 47, 9551–9554; e) E. Burda, W. Bauer, W.
Hummel, H. Grçger, ChemCatChem 2010, 2, 67–72;
f) A. Prastaro, P. Ceci, E. Chiancone, A. Boffi, R. Ciril-
li, M. Colone, G. Fabrizi, A. Stringaro, S. Cacchi, Green
Chem. 2009, 11, 1929–1932.
[1] a) R. H. Grubbs, (Ed.), Handbook of Metathesis,
Wiley-VCH, Weinheim, 2003; b) S. Beligny, S. Blechert,
in: N-Heterocyclic Carbenes in Synthesis, (Ed.: S. P.
Nolan), Wiley-VCH, Weinheim, 2006, pp 1–22.
[2] Review: D. Burtscher, K. Grela, Angew. Chem. 2009,
121, 450–462; Angew. Chem. Int. Ed. 2009, 48, 442–454.
[3] a) B. Mohr, D. M. Lynn, R. H. Grubbs, Organometallics
1996, 15, 4317–4325; b) T. A. Kirkland, D. M. Lynn,
R. H. Grubbs, J. Org. Chem. 1998, 63, 9904–9909;
c) J. P. Jordan, R. H. Grubbs, Angew. Chem. 2007, 119,
5244–5247; Angew. Chem. Int. Ed. 2007, 46, 5152–5155.
[4] a) S. J. Connon, S. Blechert, Bioorg. Med. Chem. Lett.
2002, 12, 1873–1876; b) M. T. Zarka, O. Nuyken, R.
Weberskirch, Macromol. Rapid Commun. 2004, 25,
858–862.
[5] M. T. Mwangi, M. B. Runge, N. B. Bowden, J. Am.
Chem. Soc. 2006 128, 14434–14435.
[6] a) S. H. Hong, R. H. Grubbs, J. Am. Chem. Soc. 2006,
128, 3508–3509; b) D. Samanta, K. Kratz, X. Zhang, T.
Emrick, Macromolecules 2008, 41, 530–532.
[7] a) A.-F. Mingotaud, M. Krꢃmer, C. Mingotaud, J. Mol.
Catal. A 2007, 263, 39–47; b) S. Liu, J. Xiao, J. Mol.
Catal. A 2007, 270, 1–43; c) A.-F. Mingotaud, C. Mingo-
taud, W. Moussa, J. Polym. Sci. A 2008, 46, 2833–2844;
d) B. H. Lipshutz, G. T. Aguinaldo, S. Ghorai, K. Voig-
tritter, Org. Lett. 2008, 10, 1325–1328; e) B. H. Lip-
shutz, S. Ghorai, G. T. Aguinaldo, Adv. Synth. Catal.
2008, 350, 953–956; f) C. Airaud, V. Hꢄroguez, Y.
Gnanou, Macromolecules 2008, 41, 3015–3022; g) L.
Gulajski, P. Sledz, A. Lupa, K. Grela, Green Chem.
2008, 10, 271–274.
[8] a) M. Scholl, S. Ding, C. Woo Lee, R. H. Grubbs, Org.
Lett. 1999, 1, 953–956; b) A. K. Chatterjee, J. P.
Morgan, M. Scholl, R. H. Grubbs, J. Am. Chem. Soc.
2000, 122, 3783–3784; c) S. B. Garber, J. S. Kingsbury,
B. L. Gray, A. H. Hoveyda, J. Am. Chem. Soc. 2000,
122, 8168–8179.
[13] a) M. Kraußer, W. Hummel, H. Grçger, Eur. J. Org.
Chem. 2007, 5175–5179; b) K. Baer, M. Kraußer, E.
Burda, W. Hummel, A. Berkessel, H. Grçger, Angew.
Chem. 2009, 121, 9519–9522; Angew. Chem. Int. Ed.
2009, 48, 9355–9358.
[14] The synthetic concept for the synthesis of amino acids
starting from compound 3 and analogous consists of a
transformation into its monoamide and subsequent
Hofmann rearrangement.
[15] Varma et al. published the metathesis of 1a in water
using Grubbs II catalyst, leading to a high conversion
when using a catalyst loading of 4 mol%: V. Polshetti-
war, R. S. Varma, J. Org. Chem. 2008, 73, 7417–7419.
[16] Review article about the use of pig liver esterase in or-
ganic synthesis: P. Dominguez de Maria, C. A. Garcia-
Burgos, D. Bargeman, R. W. van Gemert, Synthesis
2007, 1439–1452.
[17] The development of a recombinant pig liver esterase is
reported in: a) A. Musidlowska, S. Lange, U. T. Born-
scheuer, Angew. Chem. 2001, 113, 2934–2936; Angew.
Chem. Int. Ed. 2001, 40, 2851–2853; b) A. Hummel, E.
Brꢀsehaber, D. Bçttcher, H. Trauthwein, K. Doderer,
U. T. Bornscheuer, Angew. Chem. 2007, 119, 8644–
8646; Angew. Chem. Int. Ed. 2007, 46, 8492–8494. In
our experiments a commercially available wild-type
preparation of the pig liver esterase was used which
contains several isoenzymes.
[18] The addition of water-soluble solvents turned out to be
suitable to improve selectivity and reactivity of PLE-
catalyzed hydrolysis reactions, see: a) M. Schneider, N.
Engel, H. Boensmann, Angew. Chem. 1984, 96, 54–55;
Angew. Chem. Int. Ed. Engl. 1984, 23, 66; b) R. Patel,
A. Banerjee, L. Chu, S. Brozozowski, V. Nanduri, L. J.
Szarka, J. Am. Oil Chem. Soc. 1998, 75, 1473–1482;
c) P. Dominguez de Maria, B. Kossmann, N. Potgrave,
S. Buchholz, H. Trauthwein, O. May, H. Grçger, Synlett
2005, 1746–1748.
[19] Notably, enzymatic hydrolysis of 1a proceeds slower
than for 2a, thus fulfilling one prerequisite for such an
envisioned tandem reaction in the future. For example,
for 1a we obtained a conversion of 4% after 1 h where-
as 27% of 2a were hydrolyzed in the same time in the
presence of PLE. A further prerequisite for a one-pot
process running in a tandem mode, however, is stability
of the metathesis catalyst under conditions of the bio-
transformation, which is also currently under investiga-
tion.
[9] V. Polshettiwar, R. S. Varma, J. Org. Chem. 2008, 73,
7417–7419.
[10] T. Brendgen, T. Fahlbusch, M. Frank, D. T. Schꢀhle, M.
Seßler, J. Schatz, Adv. Synth. Catal. 2009, 351, 303–307.
[11] a) P. Anastas, J. C. Warner, Green Chemistry: Theory
and Practice, Oxford University Press, Oxford, 1998;
b) P. Anastas, L. G. Heine, T. C. Williamson, Green
Chemical Syntheses and Processes, American Chemical
Society, Washington DC, 2000.
[12] a) M. Makkee, A. P. G. Kieboom, H. van Bekkum, J. A.
Roels, J. Chem. Soc. Chem. Commun. 1980, 930–931;
b) J. V. Allen, J. M. J. Williams, Tetrahedron Lett. 1996,
37, 1859–1862; c) C. Simons, U. Hanefeld, I. W. C. E.
Arends, T. Maschmeyer, R. A. Sheldon, Top. Catal.
2006, 40, 35–44; d) E. Burda, W. Hummel, H. Grçger,
Adv. Synth. Catal. 2011, 353, 2363 – 2367
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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