Enzyme Promiscuity as a Remedy for the Common Problems with Knoevenagel Condensation

Article abstract of DOI:10.1002/chem.201901491

A new protocol based on lipase-catalyzed tandem reaction toward α,β-enones/enoesters is presented. For the synthesis of the desired products the tandem process based on enzyme-catalyzed hydrolysis and Knoevenagel reaction starting from enol acetates and aldehyde is developed. The relevant impact of the reaction conditions including organic solvent, enzyme type, and temperature on the course of the reaction was revealed. It was shown that controllable release of the active methylene compound from the corresponding enol carboxylate ensured by enzymatic reaction diminishes significantly the formation of the unwanted co-products. Furthermore, this protocol was extended by including a second tandem chemoenzymatic transformation engaging various aldehyde precursors. After a careful optimization of the reaction conditions, the target products were obtained with yields up to 86 % and with excellent E/Z-selectivity.

Full text of DOI:10.1002/chem.201901491

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Accepted Article
Title: Enzyme promiscuity as a remedy for the common problems with
Knoevenagel condensation
Authors: Dominik Koszelewski and Ryszard Ostaszewski
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Enzyme promiscuity as a remedy for the common problems with
Knoevenagel condensation
Dominik Koszelewski,* and Ryszard Ostaszewski
We would like to dedicate this paper to Professor Piotr Kiełbasiński on occasion of his 71st birthday
Abstract: A new protocol based on lipase-catalyzed tandem reaction
toward α,β-enones/enoesters is presented. For the synthesis of
desired products the tandem process based on the enzymatic
hydrolysis and Knoevenagel reaction starting from enol acetates and
aldehyde is developed. The relevant impact of the reaction conditions
including organic solvent, enzyme type, and temperature on the
reaction course was revealed. It was shown that controllable release
of the active methylene compound from the corresponding enol
carboxylate ensured by enzymatic reaction diminishes significantly
the formation of the unwanted co-products. Further this protocol was
extended by second tandem chemoenzymatic transformations
engaging various aldehyde’s precursors. After a careful optimization
of the reaction condition, target products were obtained with yields up
to 86% and excellent E/Z-selectivity.
As a part of our research to explore the enzymes application in
this still evolving area of enzyme-promoted tandem processes,[7]
we are focusing on the enzyme-mediated Knoevenagel
condensation, which is an environmental friendly alternative of
producing α,β-enones/enoesters in organic synthesis.[8] Still as
the years past from the moment Emil Knoevenagel reported in
1898 his reaction, this typically base-catalyzed process remains
a live subject in organic synthesis.[9] In 2010, Lai et al. reported
lipase-catalyzed tandem Knoevenagel condensation and
esterification with alcohol co-solvents.[10] Later, Yang et al. have
shown chemoenzymatic synthesis of α-cyano epoxides based on
simultaneous
tandem
Knoevenagel-epoxidation
reaction
catalyzed by single enzyme, lipase B from Candida antarctica.[11]
Alternative route to α,β-unsaturated carboxylic esters, an
important intermediates in natural product synthesis,[12] is
carbonyl olefination[13] in which among others Wittig reaction is
frequently employed.[14] However, isolation of the desired product
from the co-products in this reaction is often difficult and an
equimolar amount of bases such as n-BuLi or NaH is required.
Unfortunately, majority of the mentioned classical methods
require very low temperatures and anhydrous conditions.[15-17] In
addition to this, the used catalysts, especially metal complexes
are toxic or expensive and the products require complex
purification procedures, so they cannot be used in the
pharmaceutical and cosmetic industry.[18] Therefore, development
of a catalyst system that do not contain harmful components like
transition metals, strong acids or bases seems highly desirable.
The development of an environmentally sustainable protocols for
the α,β-enones/enoesters synthesis remains a challenge.[19] Our
previous work on an enzyme-mediated Knoevenagel
condensation[20] prompted us to explore the application of the
developed methodology in a consecutive way, using enol
acetates as the substrates. In the current study under application
of the biocatalytic promiscuity in organic synthesis, we
investigated the enzyme-catalyzed tandem reaction leading to
α,β-unsaturated carbonyl compounds using structurally
diversified enol esters as alternative of an active C-H methylene
compounds e.g. acetyloacetone[21] or β-keto esters.[22]
Introduction
Tandem reactions are of great interest for chemists since they
provide efficient protocols for the preparation of desired organic
compounds in an one-pot procedures.[1] Those processes refer to
arrangement of catalytic reactions into one synthetic operation
with a minimum workup or changes in the reaction conditions.[2] It
is important to notice, that in recent years, a new frontier, known
as an environmental sustainability, has emerged. Given the
drawbacks of existing catalysts (e.g., environmental pollution,
toxicity, strongly acidic reaction conditions, and complicated
operations), the search for novel, easily available, green catalysts
is being actively pursued. For this reasons, but not only, tandem
processes involving enzymes such as lipases has gained
significant attention.[3] The best example to demonstrate the
versatility of the chemoenzymatic tandem catalysis is dynamic
kinetic resolution (DKR) of secondary chiral alcohols and
amines.[4] In 2014, Xie et al. successfully employed trypsin in
tandem synthesis of 3,4-dihydropyrimidin-2(1H)-ones via
multicomponent Biginelli reaction with in situ formed
acetaldehyde.[5] Analogous approach engaging vinyl acetate as
an acetaldehyde vendor in Novozyme 435 catalyzed tandem
condensation reaction was reported by Gupta et al.[6]
Results and Discussion
Dr. D. Koszelewski, Prof. R. Ostaszewski
Institute of Organic Chemistry
Initial studies were dedicated to α,β-enones synthesis
Polish Academy of Sciences
under conditions for classical Knoevenagel reaction engaging
Kasprzaka 44/52, 01-224, Warsaw, Poland
E-mail: dominik.koszelewski@icho.edu.pl
https://ww2.icho.edu.pl/z20/
aldehydes
and
active
methylene
compounds
e.g.
acetylacetone.[9] Based on our previous results regarding
enzyme catalyzed Knoevenagel condensation[20] the model
reaction of 4-nitrobenzaldehyde (R1= 4-NO2C6H4) (1 mmol) and
acetylacetone (1 mmol) was conducted in sealed glass vials at
Supporting information available: Synthetic procedures; 1H NMR,
13C NMR spectra.
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20 °C for 96 hours in tert-butyl alcohol (Scheme 1). After
completion, the solvent was evaporated. The crude product was
purified by column chromatography (ethyl acetate/hexanes). The
obtained results are summarized in Table 1.
Table 1. The influence of the enzyme type on the Knoevenagel reaction
between 4-nitrobenzaldehyde (R1= 4-NO2C6H4) and acetylacetone.[a]
Entry
Catalyst
Yield of 3a [%][b]
1
2
3
4
5
6
7
8
9
-
7
BSA
14
11
23
18
29
28
37
9
Novozyme 435
Candida cylindracea lipase (CCL)
Candida rugosa lipase (CRL)
Pseudomonas cepacia lipase (PCL)
Pseudomonas fluorescence lipase (PFL)
Porcine pancreas lipase (PPL)
Porcine pancreas lipase (PPL)[c]
[a] Reaction conditions: 4-nitrobenzaldehyde (1 mmol) and acetylacetone (1
mmol), tert-butyl alcohol (2 mL), 20 °C for 96 hours, 200 rpm, catalyst
(protein content: 100 mg). [b] Yield of the isolated product 3a. [c] Thermally
deactivated enzyme via boiling in toluene for 24 h.
Scheme 1. Classical approach to enzyme catalyzed Knoevenagel reaction
emphasizing subsequent Michael addition step as a side-reaction.
Among tested commercially available lipases, six of them were
found to be active in the studied reaction resulted in desired 4-
acetoxy-3-penten-2-one (3a, R1= 4-NO2C6H4) (Table 1, entries 3-
8). Native lipase from porcine pancreas revealed to be the most
active providing product 3a with 37% yield (Table 1, entry 8). In
the absence of biocatalyst product 3a was isolated with 7% (Table
1, entry 1). To verify the promiscuity of the active center pocket of
the PPL in Knoevenagel reaction, bovine serum albumin (BSA)[23]
as well as thermally denatured PPL were also applied as catalysts.
Both, BSA and denatured PPL gave product 3a in low yields
(Table 1, entries 2 and 9). Obtained results which remain in an
agreement with literature data clearly show that the active site of
PPL is responsible for the Knoevenagel reaction.[24] In addition to
the product 3a the formation of the unwanted bis-adduct 4 (R1= 4-
NO2C6H4) was observed ((M-H)-: m/z = 332).[25]
The number of reports have shown that organic media is
one of the most important factors influencing the enzyme catalytic
performance.[26] Based on the literature data regarding an enzyme
catalyzed Knoevenagel reaction two more organic solvents;
DMSO and tert-amyl alcohol were investigated (Table
2).[8b,8c,24b,24c]. In both used media target product 3a was obtained
with reduced yield comparing to tert-butyl alcohol (Table 2, entries
11 and 12). Moreover, the formation of postulated by Majumder
and Gupta aldol product 1 has not been observed.[27]
It is known that water content strongly affects the catalytic
behavior of an enzyme in non-aqueous media.[28] Thus, we
investigated the effect of water content on the yield of the
Knoevenagel condensation catalyzed by PPL (Table 2, entries 1
and 2). Previous reports demonstrated that water was essential
in the enzymatic reactions of carbon–carbon bond formation.[8c]
The enzyme showed the highest catalytic activity at 5% water
content, resulting in the Knoevenagel adduct in a yield of 41%
(Table 2, entry 1). With the increased amount of water the yield of
the target product 3a decreased progressively (Table 2, entries 2
and 3). Thus, we chose 5% water content as an optimal for the
Knoevenagel reaction. Since the temperature plays an important
role in the enzyme-catalyzed Knoevenagel reactions,
a
temperature studies were performed.[24b] The yield of 3a was
increased from 41% to 43% and declined after 30 °C, which may
be due to the conformational change of the active site of PPL
enzyme caused by elevated temperature (Table 2, entries 1, 4-6).
Thus 30 °C was selected as the most favorable temperature for
further studies.
To improve the reaction yields, enzyme loading was
further investigated (Table 2, entries 4, 7 and 8). The results
showed that the target product 3a was obtained with the
highest yield using 100 mg of PPL. Increased amount of
enzyme up 120 mg resulted in slight yield reduction (Table 2,
entry 8) what may be caused by protein agglomeration what
leads to diffusion problems. Recently, we have shown pivotal
role of the acetic anhydride (Ac2O) on the Knoevenagel
reaction performance which arises from dehydration step
facilitation via intermediate product 2 (Scheme 1).[20a]
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undergoes further elimination to desired products 3 or 6 without
creating unwanted co-product 4.
Table 2. Optimization of the Knoevenagel reaction between 4-
nitrobenzaldehyde and acetylacetone catalyzed by porcine pancreas
lipase.[a]
Entry
Conditions
T [°C]
Yield of 3a [%][b]
1
tert-butyl alcohol / 5% H2O v/v
tert-butyl alcohol / 10% H2O v/v
tert-butyl alcohol / 15% H2O v/v
tert-butyl alcohol / 5% H2O v/v
tert-butyl alcohol / 5% H2O v/v
tert-butyl alcohol / 5% H2O v/v
tert-butyl alcohol / 5% H2O v/v[c]
tert-butyl alcohol / 5% H2O v/v[d]
tert-butyl alcohol / 5% H2O v/v[e]
tert-butyl alcohol / Ac2O[f]
20
20
20
30
40
50
30
30
30
30
30
30
30
30
41
36
32
43
35
24
32
37
45
42
24
32
28
<1
2
3
4
5
6
7
8
9
10
11
12
13
14
DMSO
tert-amyl alcohol
tert-butyl alcohol / 1,1-diacetate[g]
tert-butyl alcohol / 1,1-diacetate[h]
[a] Reaction conditions: 4-nitrobenzaldehyde (1 mmol) and acetylacetone (1
mmol), 200 rpm, porcine pancrease lipase (100 mg). [b] Yield of the isolated
product 3a. [c] PPL (80 mg). [d] PPL (120 mg). [e] Reaction time 112 h. [f]
Ac2O (1 equiv.). [g] 4-nitrobenzylidene diacetate (1 equiv.). [h] Without
enzyme.
Scheme 2. Enzyme catalyzed tandem Knoevenagel reaction on enol
carboxylates.
In the model reactions corresponding 4-acetoxy-3-penten-
2-one (5a, R1=R2=R3=Me) was synthesized from acetylacetone
using modified literature procedure.[30] The control reaction
between 4-nitroaldehyde and 4-acetoxy-3-penten-2-one in the
absence of PPL under previously optimized conditions did not
occur and the substrates were recovered (Table 3, entry 1). To
our delight the application of PPL resulted in target product 3a
with 51% yield (Table 3, entry 2). Further increase of water
content led to the product with substantially reduced yield (Table
3, entry 3). The model reaction proceeded also in neat tert-butyl
alcohol resulted in target product with only 18% yield (Table 3,
entry 4). When the water content was low, the conformation of
PPL was excessively rigid, which made the ‘‘induced-fit’’ process
of PPL difficult and led to the low enzyme activity. However, the
enzyme conformation in organic media with high water content
may be too flexible, which may also decrease the activity of the
enzyme.[31] Obtained results revealed that certain amount of water
is obligatory and the molecular water present in the enzyme
structure is insufficient for the studied tandem enzymatic
transformation. Interestingly, the yield of the product 3a
decreased with the temperature elevation reaching 46% at 40 °C
(Table 3, entry 5). Due this observation reaction was carried out
at 20 °C what resulted in enhanced reaction rate providing product
3a with 57% after 96 hours (Table 3, entry 6). Additional studies
on the substrates ratio used to the reaction revealed that the
application of enol carboxylate 5a in a 3:1 ratio to aldehyde
Although, the addition of the acetic anhydride to the reaction
medium subtly affects the yield of the target product 3a (Table 2,
entry 10), its presence completely suppresses formation of
undesired bis-adduct 4. Recently, Ogiwara et al. have shown that
an acetic anhydride used together with indium(III)-catalyst acts as
a promoter of the Knoevenagel reaction. The driving force of the
presented method was 1,1-diacetate, which was in-situ-
generated from an aldehyde. Under studied reaction conditions
with acetic anhydride we have not observed formation of such
intermediate. However, application of 4-nitrobenzylidene
diacetate resulted in target product 3a with 28% yield (Table 2,
entry 13). Reaction without biocatalyst did not occur (Table 2,
entry 14). Due to the best of our knowledge such promiscuous
enzymatic activity regarding usage of 1,1-diacetate as an
aldehyde vendor in Knoevenagel reaction has no precedent in the
literature.
Obtained results encouraged us to undertake additional
studies involving enol carboxylates 5 as an alternative of activated
methylene derivatives in enzyme catalyzed tandem process
(Scheme 2). According to propose mechanism we envision that
carboxyl group from the used enol 5 is transferred by the enzyme
on the aldol product 2. Finally, the formed intermediate 1
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resulted in the target product with significantly enhanced yield
82% (Table 3, entries 7-9). Such phenomena has not been
observed in opposite way for the excess of aldehyde (data not
shown).
Table 3. Tandem Knoevenagel reaction between 4-nitrobenzaldehyde and
enolate ester 5 catalyzed by porcine pancreas lipase.[a]
Entry
5 (equiv.)
Additives/conditions
T [°C]
Yield of 3a [%][b]
1
5a (1.0)
5a (1.0)
5a (1.0)
5a (1.0)
5a (1.0)
5a (1.0)
5a (2.0)
5a (3.0)
5a (4.0)
5a (3.0)
5b (3.0)
5c (3.0)
5a
without enzyme
30
30
30
30
40
20
20
20
20
20
20
20
20
20
<1
51
34
18
46
57
66
82
76
79
51
13
<1
<1
2
-
3
-
4
without H2O
Figure 1. Time profile of 4-acetoxy-3-penten-2-one (3a) formation in the
reaction catalyzed by PPL. Reaction conditions: 4-nitrobenzaldehyde (1 mmol)
and 4-acetoxy-3-penten-2-one (5a, R1=R2=R3=Me) (3 mmol), and PPL (100 mg)
in tert-butyl alcohol / 5% H2O v/v (2 mL) at 20 °C, 200 rpm.
5
-
6
-
7
-
Based on the above results, the Knoevenagel condensation
reactions of various aldehydes and 4-acetoxy-3-penten-2-one (5a,
R1=R2=R3=Me) were conducted in order to investigate the
generality for the scope of this new biocatalytic tandem protocol.
The results were summarized in Figure 2. All the products were
characterized by NMR and were compared with the literate data
(supplementary materials). It was found that a wide range of
aromatic aldehydes could effectively participate in PPL catalyzed
tandem Knoevenagel reaction to give the corresponding products
3b-k. It was observed that aromatic aldehydes bearing either
electron-withdrawing group or electron-donating group reacted
successfully with the yields up to 75%. Meanwhile, acetaldehyde
was also investigated, however, to corresponding product 3g was
obtained only with 14% yield. Due to the high chemical activity of
acetaldehyde its was recognized as an enzyme inhibitor.[32] As an
alternative to this low boiling reagent vinyl acetate (VA) was
applied resulting in product 3g with 31% yield. It was proved that
acetaldehyde can be provided in situ from VA enzymatic
transformation.[3,6,20a] Application of phenylacetic aldehyde
resulted in product 3h with 61%. Analogously to vinyl acetate,
ethyl β-styrylcarbonate and styryl acetate were used as the
phenylacetic aldehyde surrogates. However, corresponding
product 3h was obtained with lower yields, 44% and 47%,
respectively. The application of cinnamyl aldehyde provided
product 3i with high yield (86%). The promiscuous activity of
hydrolases toward Knoevenagel condensation of α,β-unsaturated
aldehydes with acetylacetone performed in the same reaction
medium, tert-butyl alcohol was recently reported by Wang et al.
but the reported yield of the corresponding product 3i was
lower.[24d] In addition, hetero-aromatic aldehydes 2-furaldehyde
and 2-thienylaldehyde could react with acetylacetone and ethyl
acetoacetate to provide products 3j and 3k in good yields, 77%
and 69%, respectively. Moreover, in none of the reactions
conducted under developed conditions and leading to target
compounds 3a-k unwanted bis-adduct 4 was observed. It is
noteworthy that obtained products constitute an important class
8
-
9
-
10
11
12
13
14
114 hours
-
-
deactivated enzyme[c]
BSA
5a
[a] Reaction conditions: 4-nitrobenzaldehyde (1 mmol) and enol ester 5, tert-
butyl alcohol / 5% H2O v/v (2 mL), 200 rpm, porcine pancreas lipase (100
mg). 96 hours. [b] Yield of the isolated product 3a. [c] Thermally deactivated
enzyme via boiling in toluene for 24 h.
Further the reaction time of the enzyme catalytic promiscuity
reaction was investigated. The time elongation to 114 h did not
affect the reaction yield (Table 3, entry 10). Detailed studies
shown that in 24 hours, the desired product 3a was obtained in
moderate yield (29%) while the yield did not increase substantially
after 72 hour (80%) (Figure 1). Next, we subsequently studied the
effect of the acyl group in enol carboxylates 5 on the rate of
enzyme catalyzed tandem reaction. Thus, it was observed that
both 1-methyl-3-oxo-1-enyl propionate (5b, R1=R2=Me, R3=C2H5)
and 1-methyl-3-oxo-1-enyl benzoate (5c, R1=R2=Me, R3=C6H5)
were less suitable partners under studied conditions providing
product 3a with 51% and 13%, respectively (Table 3, entries 11
and 12). Furthermore, the denatured PPL (denatured by heating)
or BSA (Table 3, entries 13 and 14) was used as catalyst in this
reaction and the result was similar to the control (Table 3, entry
1).
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of building block for the synthesis of biologically and
pharmaceutically relevant compounds.[33]
Figure 2. Unsaturated 2,4-pentanediones obtained via enzyme catalyzed
tandem Knoevenagel reaction. Reaction conditions: aldehyde (1 mmol), 4-
acetoxy-3-penten-2-one (5a, R1=R2=R3=Me) (3 mmol), and PPL (100 mg) in
tert-butyl alcohol / 5% H2O v/v (2 mL) for 72 h at 20 °C, 200 rpm. [a] With vinyl
acetate. [b] With ethyl β-styrylcarbonate. [c] With styryl acetate.
For other enol carboxylates such as ethyl 3-acetyloxy-3-
(phenyl)prop-2-enoate (5d, R1= C6H5, R2=OEt, R3=Me) and 3-
acetyloxy-3-(4-mehoxyphenyl)prop-2-enoate
(5e,
R1=
4-
MeOC6H4, R2=OEt, R3=Me), the reaction also proceed smoothly
providing products 6a-f with the yields up to 72% (Figure 3).
Electron-rich aldehydes gave relatively greater yields than their
electron-deficient counterparts. Besides, in most cases only one
isomer was obtained under developed conditions. In case of using
ethyl 3-acetyloxy-3-(4-nitrophenyl)prop-2-enoate (5f, R1=4-
NO2C6H4, R2=OEt, R3=Me) and 3-acetoxy-crotonic acid ethyl
ester (5g, R1= Me, R2=OEt, R3=Me) corresponding products 6g-
m were obtained with moderate yields (up to 36%) but still with
excellent regio-selectivity, exceeded 99%. The extraordinary
selectivity can be explain by the fact that enzyme catalyzed
formation of the acylated aldol product 2 what significantly
facilitates the formation of desired α,β-enoesters. Again the
application of vinyl acetate as a vendor of the acetaldehyde
results in product 6j with enhanced yield.
Figure 3. α,β-Enoesters obtained via enzyme catalyzed tandem Knoevenagel
reaction. Reaction conditions: aldehyde (1 mmol), enol carboxyalte 5 (3 mmol),
and PPL (100 mg) in tert-butyl alcohol / 5% H2O v/v (2 mL) for 72 h at 20 °C,
200 rpm. [a] With vinyl acetate.
The reason of the comparatively lower yields of the product
provided from ethyl 3-acetyloxy-3-(4-nitrophenyl)prop-2-enoate
(5f, R1= 4-NO2C6H4, R2=OEt, R3=Me) and 3-acetoxy-crotonic acid
ethyl ester (5g, R1= Me, R2=OEt, R3=Me) can be explain by the
fact that upon enzymatic hydrolysis of alkoxy group (R2=OEt)
spontaneous
decarboxylation
takes
place.[20,34]
4-
Nitroacetophenone was isolated as the side product under
developed conditions what clearly constitutes this assumption.
Due to this fact additional experiments were performed with tert-
butyl 3-acetoxy-2-butenoate (5h, R1= Me, R2=O-t-Bu, R3=Me).
Generally, tert-butyl esters are prone to undergo cleavage under
enzymatic conditions what should prevent accompanying
spontaneous decarboxyation. Indeed, reactions performed with
sterically bulk butyl 3-acetoxy-2-butenoate resulted in target
products 6n-q with relatively higher yields and excellent region-
selectivity.
Conclusions
In summary, we designed a highly selective protocol for the
synthesis of α,β-enones/enoesters using hydrolysis-Knoevenagel
cascade reaction of enol carboxylates with aldehydes. Under
developed method the formation of unwanted co-products e.g.
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General Procedure for Knoevenagel reaction with acetylacetone.
Porcine pancrease lipase (100 mg) was added to a 10 mL vial containing
aldehyde (1 mmol), acetylacetone (100 mg, 1 mmol), tert-butyl alcohol (4
mL with 5% v/v deionized water). The resulting mixture was shaken at 200
r/min at 30 °C for 96 hours, and monitored by thin layer chromatography
(TLC). The enzyme was filtered. The filtrate was diluted with TBME (15
mL) and washed with water (3x10 mL). The aqueous phase was extracted
with TBME (10 mL). Combined organic phase was washed with water,
dried over anhydrous Na2SO4 and concentrated in vacuum. The crude
product was purified by flash column chromatography on silica gel using
ethyl acetate/n-hexanes as an eluent.
bis-adducts 4, which are common for the classical Knoevenagel
condensation was significantly constricted. High selectivity target
unsaturated products was ensured by the controllable release of
the active methylene compound from the corresponding enol
carboxylate provided by the used biocatalyst. After a careful
optimization of the reaction condition, target products were
isolated with yields from 11% to 86% and excellent selectivity,
while chemical methods lead to the mixture of (E/Z)-isomers. The
plausible mechanism of the elementary reaction which provides
the single isomer of the target product via selective dehydration is
provided in the SI material (Figure S79). We disclosed a novel
protocol for the synthesis of desired products through a tandem
process based on the enzymatic hydrolysis of acetaldehyde
precursor vinyl acetate with subsequent Knoevenagel reaction.
All described enzymatic transformations were sequentially
catalyzed by one enzyme porcine pancreas lipase which revealed
its outstanding catalytic promiscuity. Using developed procedure
eight new (Z)-α,β-enoesters 6e-i and 6n-p have been prepared.
Moreover, application of vinyl acetate as a precursors of highly
volatile acetaldehyde simplifies the process providing products
which cannot be obtained through classical chemical approach.
This novel protocol incorporates several green chemistry
principles, biocatalyst, environmental friendliness, and simple
operational process. Moreover obtained product are not
contaminated with catalyst what meets the rigorous requirements
of pharmaceutical industry. We believe that presented work
significantly expands the utility of biocatalysts for important
carbon-carbon bond-forming reactions. Simple experimental
procedure is relevant with regard to economic and sustainable
consideration and allows us to believe that developed method
may represent a valuable alternative to the present reagents
reported in the literature.
General procedure for 4-acetoxy-3-penten-2-one (5a, R1=R2=R3=Me)
preparation. Anhydrous pyridine (6 mL, 74 mmol) was added with ice-
cooling to a mixture of 2,4-pentadione (4 g, 40 mmol) and acetyl chloride
(0.35 mL, 49 mmol). Then the reaction mixture was heated to 120 °C, hold
for 15 min., cooled to -18 °C, and poured into water (50 mL). Crude mixture
was extracted with ethyl ether (3 x 10 mL) and dried over anhydrous
Na2SO4. The solvent was evaporated, and the residue was distilled.
Product was obtained with 95% yield as a colorless liquid; b.p. 80 °C 7
Torr, [Lit. 78-80 °C 6.5 Torr [36]; LRMS (ESI) calcd. for C7H11O3 (M+H)+
143.1, found 143.1; 1H NMR (400 MHz, CDCl3) δ 6.04 (s, 1H), 2.27 (s,
3H), 2.17 (s, 3H), 2.13 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 197.2, 168.0,
162.4, 116.3, 32.0, 21.1, 18.4. 1H- and 13CNMR data were in accordance
with those reported in the literature.[37]
1-Methyl-3-oxo-1-enyl propionate (5b, R1=R2=Me, R3=C2H5). Product
was obtained according to General procedure with 74% yield as a
colorless liquid as an inseparable mixture of E/Z isomers; HRMS (ESI)
1
calcd. for C8H12O3Na (M+Na)+ 179.0684, found 179.0679; H NMR (400
MHz, CDCl3) δ 6.08 (s, 2H), 2.42 (q, J = 7.5 Hz, 2H), 2.38 (q, J = 7.5 Hz,
2H), 2.31 (s, 6H), 2.21 (s, 6H), 1.18 (t, J = 7.5 Hz, 6H); 13C NMR (100 MHz,
CDCl3) δ 197.3, 169.3, 162.6, 116.3, 30.9, 27.4, 18.4, 8.9.
Ethyl 3-acetyloxy-3-(4-methoxyphenyl)prop-2-enoate (5e, R1= 4-
MeOC6H4, R2=OEt, R3=Me). Product was obtained according to General
procedure with 82% yield as a colorless semi-solid. HRMS (ESI) calcd. for
C14H16NO5Na (M+Na)+ 287.0895, found 287.0893; 1H NMR (400 MHz,
CDCl3) δ 7.50 (d, J = 9.0 Hz, 2H), 6.86 (d, J = 9.0 Hz, 2H), 6.13 (s, 1H),
4.14 (q, J = 7.2 Hz, 2H), 3.78 (s, 3H), 2.35 (s, 3H), 1.25 (t, J = 7.2 Hz, 3H);
13C NMR (100 MHz, CDCl3) δ 190.9, 168.0, 164.3, 161.9, 157.9, 130.8,
127.5, 125.6, 114.2, 103.8, 60.0, 55.3, 20.9, 14.2.
Experimental Section
General methods. All the chemicals were obtained from commercial
sources and the solvents were of analytical grade. 1H- and 13C-NMR
spectra were recorded in CDCl3 solution using Varian Gemini 400 NMR
spectrometer (400 MHz). Chemical shifts are expressed in parts per million
using TMS as an internal standard. Mass spectra were recorded on API
365 mass spectrometer (SCIEX). TLC analyses were done on Kieselgel
60 F254 aluminum sheets. Lipases from porcine pancreas, Type II (PPL)
(catalogue number L-3126, Lot. number 108H1379), Pseudomonas
cepacia (PCL) (catalogue number 534641, Lot. number 03223JC),
Pseudomonas fluorescens (PFL) (catalogue number 534730, Lot. number
MKBH1198V), Candida rugosa (CRL) (catalogue number 90860, Lot.
number BCBH7102V), and Candida cylindracea (CCL) (catalogue number
62316, Lot. number 1336707) were purchased from Sigma-Aldrich.
Immobilized lipase from Candida antarctica B (Novozym 435) (catalogue
number LC200223) was purchased from Novo Nordisk. Bovine serum
albumin were purchased from Sigma-Aldrich. 4-Nitrobenzylidene diacetate
(catalog no. S353981) (acylal) was provided by Merck Column
chromatography was performed on Merck silica gel 60/230-400 mesh.
Enzymatic reactions were performed in a vortex (Heidolph Promax 1020)
equipped with incubator (Heidolph Inkubator 1000). To prove the ability of
the established protocol each reaction was repeated at least three times.
Ethyl β-styrylcarbonate and styryl acetate were obtained according to the
literature procedures.[35]
Ethyl 3-acetyloxy-3-(4-nitrophenyl)prop-2-enoate (5f, R1= 4-NO2C6H4,
R2=OEt, R3=Me). Product was obtained according to General procedure
with 85% yield as a colourless liquid b.p. 128 °C at 0.03 Torr; HRMS (ESI)
calcd. for C13H13NO6Na (M+Na)+ 302.0641, found 302.0640; 1H NMR (400
MHz, CDCl3) δ 8.25 (d, J = 8.9 Hz, 2H), 7.74 (d, J = 8.9 Hz, 2H), 6.35 (s,
1H), 4.22 (q, J = 7.1 Hz, 2H), 2.40 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H); 13C
NMR (100 MHz, CDCl3) δ 167.8, 163.4, 155.4, 139.5, 126.8, 124.0, 109.6,
60.7, 20.8, 14.1.
General procedure for the enzyme-catalysed Knoevenagel reaction
with enol carboxylates. Porcine pancrease lipase (100 mg) was added
to a 10 mL vial containing aldehyde (1 mmol), enol carboxylate (3 equiv.),
tert-butyl alcohol (4 mL with 5% v/v deionized water). The resulting mixture
was shaken at 200 r/min at 30 °C for 96 hours, and monitored by thin layer
chromatography (TLC). The enzyme was filtered. The filtrate was diluted
with TBME (15 mL) and washed with water (3x10 mL). The aqueous phase
was extracted with TBME (10 mL). Combined organic phase was washed
with water, dried over anhydrous Na2SO4 and concentrated in vacuum.
The crude product was purified by flash column chromatography on silica
gel using ethyl acetate/n-hexanes as an eluent.
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10.1002/chem.201901491
Chemistry - A European Journal
FULL PAPER
Ethyl (2E)-2-(4-methoxybenzoyl)-3-(4-methoxyphenyl)acrylate (6e).
Product was obtained according to General procedure with 45% yield as
a colorless semi-solid. HRMS (ESI) calcd. for C20H20O5Na (M+Na)+
363.1208, found 363.1205; Elemental Analysis calcd. for C20H20O5 C:
70.58%, H: 5.92%, found C: 70.42%, H: 5.73% 1H NMR (400 MHz, CDCl3)
δ 7.92 (d, J = 8.9 Hz, 2H), 7.84 (s, 1H), 7.30 (d, J = 8.8 Hz, 2H), 6.88 (d, J
= 9.0 Hz, 2H), 6.72 (d, J = 8.9 Hz, 2H), 4.19 (q, J = 7.1 Hz, 2H), 3.81 (s,
3H), 3.72 (s, 3H), 1.16 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ
194.5, 165.4, 164.1, 161.2, 141.7, 132.2, 131.5, 129.5, 128.8, 125.6, 114.2,
114.0, 61.2, 55.4, 55.2, 26.8, 14.0.
Tert-butyl 2-(4-nitrobenzylidene)-3-oxobutanoate (6o). Product was
obtained according to General procedure with 55% yield as a colorless
semi-solid. HRMS (ESI) calcd. for C15H17NO5Na (M+Na)+ 314.1004, found
314.1002; Elemental Analysis calcd. for C15H17NO5 C: 61.85%, H: 5.88%,
N: 4.81%, found C: 61.78%, H: 5.82%, N: 4.79%; 1H NMR (400 MHz,
CDCl3) δ 8.22 (d, J = 8.8 Hz, 2H), 7.65 (d, J = 8.7 Hz, 2H), 7.49 (s, 1H),
2.42 (s, 3H), 1.49 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 194.0, 166.0,
148.3, 139.7, 138.4, 136.6, 130.0, 123.7, 83.7, 27.8, 26.9.
Tert-butyl 2-(1-phenyl-1-propylene)-3-oxobutanoate (2p). Product
(ketone-enol form) was obtained according to General procedure with 21%
yield as a colorless semi-solid. HRMS (ESI) calcd. for C16H21O3 (M+H)+
261.1491, found 261.1487; Elemental Analysis calcd. for C16H20O3 C:
73.82%, H: 7.74%, found C: 73.77%, H: 7.68%; 1H NMR (400 MHz, CDCl3)
δ 13.60 (s, 1H), 7.42 – 7.34 (m, 5H), 6.75 (d, J = 16.3 Hz, 1H), 6.64 (d, J
= 16.3 Hz, 1H), 2.24 (s, 3H), 1.60 (s, 9H); 13C NMR (100 MHz, CDCl3) δ
174.3, 172.4, 138.4, 129.8, 128.5, 126.8, 125.8, 122.1, 102.2, 82.2, 28.3,
20.3.Experimental Details.
Ethyl
(2E)-2-(4-methoxybenzoyl)-3-(4-nitrophenyl)acrylate
(6f).
Product was obtained according to General procedure with 36% yield as
a colorless semi-solid. HRMS (ESI) calcd. for C19H17NO6Na (M+Na)+
378.0954, found 378.0952; Elemental Analysis calcd. for C19H17NO6 C:
64.22%, H: 4.82%, N: 3.94, found C: 64.05%, H: 4.75%, N: 3.88; 1H NMR
(400 MHz, CDCl3) δ 8.06 (d, J = 8.9 Hz, 2H), 7.91 (s, 1H), 7.87 (d, J = 9.0
Hz, 2H), 7.50 (d, J = 8.6 Hz, 2H), 6.89 (d, J = 9.0 Hz, 2H), 4.24 (q, J = 7.1
Hz, 2H), 3.83 (s, 3H), 1.20 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3)
δ 192.7, 164.5, 139.2, 138.8, 135.6, 131.5, 130.5, 128.8, 123.8, 114.2,
61.9, 55.5, 14.0.
Acknowledgements
Ethyl (2E)-2-(4-nitrobenzoyl)-3-(4-nitrophenyl)acrylate (6g). Product
was obtained according to General procedure with 18% yield as a
colorless semi-solid. HRMS (ESI) calcd. for C18H15N2O7 (M+H)+ 371.0879,
found 371.0875; Elemental Analysis calcd. for C18H14N2O7 C: 58.38%, H:
3.81%, N: 7.56, found C: 58.29%, H: 3.72%, N: 7.48; 1H NMR (400 MHz,
CDCl3) δ 8.29 (d, J = 8.7 Hz, 2H), 8.12 (d, J = 8.7 Hz, 2H), 8.06 (d, J = 8.8
Hz, 2H), 8.04 (s, 1H), 7.49 (d, J = 8.8 Hz, 2H), 4.26 (q, J = 7.1 Hz, 2H),
1.20 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 192.8, 163.6, 140.7,
139.9, 138.5, 134.2, 130.4, 129.9, 124.2, 124.1, 124.0, 62.3, 13.9.
We gratefully acknowledge the financial support from the National
Science Center, Poland project OPUS No. 2016/B/ST5/03307.
Keywords: Knoevenagel condensation • tandem reaction •
promiscuity • enol carboxylates • biocatalysis
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10.1002/chem.201901491
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A new protocol based on lipase-
catalyzed tandem reaction toward α,β-
enones/enoesters is presented. After
a careful optimization of the reaction
condition, target products were
obtained with yields up to 86% and
excellent E/Z-selectivity.
Dominik Koszelewski* and Ryszard
Ostaszewski
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Enzyme promiscuity as a remedy for
the common problems with
Knoevenagel condensation
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