Acyl Donors and Additives for the Biocatalytic Friedel–Crafts Acylation

Article abstract of DOI:10.1002/ejoc.201701079

The Friedel–Crafts acylation is a broadly applied reaction that can be conducted using various types of catalyst. However, a biocatalytic alternative has only been reported recently. In this study, the scope of acetyl donors is described, showing that, in addition to vinyl acetate derivatives, phenyl esters are also suitable donors. Furthermore, it was found that various amines enhance the reaction, whereby the effect do not seem to be correlated to the pH but to the structure of the donor. For instance, 1,4-diazabicyclo[2.2.2]octane (DABCO) turned out to be a viable alternative to imidazole; however the former performed best at pH 9.85, whereas the latter performed best at pH 8.3.

Full text of DOI:10.1002/ejoc.201701079

DOI: 10.1002/ejoc.201701079
Full Paper
Biocatalytic C-Acylation
Acyl Donors and Additives for the Biocatalytic Friedel–Crafts
Acylation
Nina G. Schmidt[a,b] and Wolfgang Kroutil*[b]
Abstract: The Friedel–Crafts acylation is a broadly applied reac- various amines enhance the reaction, whereby the effect do
tion that can be conducted using various types of catalyst. not seem to be correlated to the pH but to the structure of
However, a biocatalytic alternative has only been reported re- the donor. For instance, 1,4-diazabicyclo[2.2.2]octane (DABCO)
cently. In this study, the scope of acetyl donors is described, turned out to be a viable alternative to imidazole; however the
showing that, in addition to vinyl acetate derivatives, phenyl former performed best at pH 9.85, whereas the latter performed
esters are also suitable donors. Furthermore, it was found that best at pH 8.3.
Introduction
Recently, we demonstrated the synthetic utility of a related
acyltransferase for the regioselective C-acylation of unprotected
C4- or C5-substituted resorcinols into the corresponding mono-
C-acylated 2,4-dihydroxyphenyl ketones in a Friedel–Crafts-type
acylation reaction.[15] The products constitute key scaffolds that
are valuable for the pharmaceutical industry.[16] The enzyme
employed works independent of ATP and CoA and is a multi-
component acyltransferase (ATase) from Pseudomonas prote-
gens (PpATaseCH).
Different types of acyl donors were identified, which can be
categorized according to the bond that is cleaved by the ATase
upon acyl transfer (Scheme 1). For instance, a C–C bond is
cleaved when DAPG (C-acyl donors) serves as donor. In other
cases, formally the C–N bond of N-acetylimidazole (N-acyl
donors) or the C–O bond of isopropenyl acetate (IPEA, O-acyl
donors) is broken.
Controlling chemo-, regio-, and stereoselectivity in organic syn-
thesis is crucial, especially to access and decorate multifunc-
tional complex molecules.[1] Metal-, organo-, or biocatalysts are
routinely employed to discriminate between multiple chemical
functionalities and reaction sites.[2] Controlling selectivity
through biocatalysis has gained attention[3] because of the gen-
erally outstanding results and because of the possibility to tune
the selectivity by exploiting major advances in molecular and
computational biology, which pave the way to engineered en-
zymes with defined properties.[4] Focusing on C–C bond forma-
tion,[5] many named reactions have biocatalytic analogues, in-
cluding aldol condensations,[6] Michael-(type) additions[7] Diels–
Alder reactions,[8] cyclopropanations,[9] Wittig-type olefin-
ations[10] and Pictet–Spengler reactions.[11] There are also sev-
eral examples of enzymatic Friedel–Crafts-(type) alkylations,[12]
which enable regioselective C–C bond formation at aromatic
compounds. However, little is known about enzyme-catalyzed
Friedel–Crafts acylation.
An acyltransferase has been identified in the biosynthesis
of the antibiotically active polyketide 2,4-diacetylphloroglucinol
(DAPG), which is secreted by certain fluorescent Pseudomonas
sp. to protect plants from soilborne diseases.[13] The ATase
thereby catalyzes the reversible acetylation and deacetylation
of monoacetylphloroglucinol (MAPG), leading to the formation
of DAPG and phloroglucinol (PG) in the forward reaction,
whereas DAPG is the acyl donor for PG in the reverse direction
of the reaction.[14]
[a] ACIB GmbH,
Petersgasse 14, 8010 Graz, Austria
[b] Department Institute of Chemistry, Organic and Bioorganic Chemistry
Institution, University of Graz, NAWI Graz, BioTechMed Graz,
Heinrichstraße 28, 8010 Graz, Austria
Scheme 1. Biocatalytic Friedel–Crafts and Fries reaction.
E-mail: Wolfgang.Kroutil@uni-graz.at
http://biocatalysis.uni-graz.at/
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under https://doi.org/10.1002/ejoc.201701079.
Additionally, the ATase catalyzes the transformation of O-
acetylated substrates into the C-acetyl products by a Fries reac-
tion (Scheme 1).[15]
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For the acetylation using IPEA, it was found that imidazole is
beneficial for the reaction. Here, we investigate whether other
bases may also be used and we extend the scope of activating
groups for the acyl donor.
Table 1. PpATaseCH catalyzed Friedel–Crafts bioacetylation of resorcinol (1a)
employing aliphatic and aromatic acyl sources 5a–n in the presence or ab-
sence of imidazole.[a]
Entry
Donor
Absence of imidazole
Presence of imidazole[c]
2a
3a
4a
2a
3a
[%]
[%]
[%]
[%]
[%]
1
2
3
4
5
6
7
8
9
10
11
12
13
14
5a
5b
5c
5d
5e
5f
5g
5h
5i
25
45
1
< 1
5
n.d.
n.d.
17
n.d.
n.d.
n.d.
n.d.
3
n.d.
n.d.
n.d.
n.d.
28
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
31
98
82
n.d.
n.d.
5
> 99
> 99
> 99
> 99
27
< 1
n.d.
n.d.
n.d.
28
n.d.
n.d.
n.d.
n.d.
5
Results and Discussion
Extending the Scope of Acyl Donors for the ATase
1
n.d.
> 99
> 99
> 99
> 99
97
12
65
n.d.
< 1
Given that isopropenyl acetate (5a) and vinyl acetate (5b) have
been reported as acetyl donors,[15] the saturated analogues
5c,d were tested as well as acetic anhydride 5e for the Friedel–
Craft acetylation of resorcinol 1a as a model compound
(Scheme 2). Furthermore, the enzyme-catalyzed Fries reaction
indicated that phenyl esters might also be donors; therefore, a
range of substituted phenyl esters 5f–l was selected. Alterna-
tively, acetanilides 5m–n were tested. The transformation was
studied in the absence and presence of imidazole as additive,
because imidazole was identified to improve the transformation
with IPEA.
5j[b]
5k[b]
5l[b]
5m[b]
5n[b]
< 1
3
n.d.
n.d.
43
66
n.d.
< 1
22
n.d.
n.d.
n.d.
n.d.
n.d.
[a] Reaction conditions: cell-free E. coli extract containing the recombinant
PpATaseCH (3 U ≡ 18 mg of lyophilized cells), resorcinol (1a, 10 m ), KPi-
buffer (50 m , pH 7.56), imidazole (optional: 100 m , added from a 1 stock
solution prepared in the same buffer, pH 9.55), donor 5a–n (100 m ), pH 8.30
M
M
M
M
M
(after imidazole addition), total reaction volume: 1 mL, 35 °C, 18 h, 750 rpm.
Concentrations in % were determined by HPLC using calibration curves of
authentic samples. n.d. = not detected. [b] The donor was dissolved in DMSO
to enhance solubility. The final reaction mixture (1 mL) contained 10 vol.-%
DMSO. [c] Compound 4a was not detected under these conditions.
On the other hand, the nonactivated derivatives 5c and 5d
turned out not to be useful donors (Table 1, entry 3 and 4) as
well as acetic anhydride (5e). In contrast, phenyl acetate (5f)
and its derivatives emerged as excellent acetyl donors for the C-
acetylation, especially when substituted with electron-donating
groups such as in 5g–i. Using these donors, more than 99 %
yield of product 2a was reached both in the absence and in
the presence of imidazole (entry 6–9).
para-Substituted phenyl derivatives 5j–l with electron-with-
drawing groups were less reactive (Table 1, entry 10–12). No
product was formed with amide analogues 5m–n within the
reaction time of 18 h, which was probably due to the reduced
reactivity of acetanilides over aromatic esters (entry 13–14).
Notably, none of the investigated phenyl acetyl esters under-
went Fries rearrangement into the corresponding hydroxy-
phenyl ketones by the PpATaseCH; thus, they served exclusively
as acyl donors for the irreversible bioacetylation of 1a. This may
be explained by the preference of the enzyme for resorcinol
derivatives as acceptors.
Whereas imidazole had no clear influence on the enzyme-
catalyzed C-acetylation using phenyl acetate (5f) and its elec-
tron-rich analogues 5g–i, pronounced effects were observed for
phenolic esters bearing electron-withdrawing groups, such as
p-NO2 (5j), p-CO2H (5k) or p-CN (5l). In case of 5j, product for-
mation of 2a was lower when imidazole was present (Table 1,
entry 10, 27 % over 97 %), whereas the opposite was observed
Scheme 2. Acetic acid derivatives investigated as acetyl donors for the
PpATaseCH catalyzed bioacetylation of resorcinol (1a).
C-Acetylation with IPEA (5a) and vinyl acetate (5b) led to with 5k, where imidazole led to higher amounts of 2a (entry 11,
high levels of formation of product 2a in the presence of imid- 43 % over 12 %). With 5l, the formation of the C-acetyl product
azole (98 % and 82 %), whereas in the absence of imidazole, 2a seemed to be independent of imidazole (entry 12, 65 % vs.
the reaction led only to low amounts of 2a within 18 hours 66 %), but the spontaneous O-acetylation, especially to diester
(25 % and 45 %, Table 1, entry 1 and 2).
4a, was particularly strong without imidazole (31 %).
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The amount of detected spontaneously formed resorcinol found that even at 1
M
5a (and 300 m
M
without imidazole) the
esters 3a and 4a was only significant in the case of acetic anhy- enzyme was still active, giving up to 68 % of the C-acetyl prod-
dride (Table 1, entry 5) and in the case of two para-substituted uct 2a (Figure 1). As noted earlier, reactions with donor 5a were
phenyl esters 5k and 5l (entries 11 and 12).
more efficient in the presence of imidazole, whereas in the ab-
sence of imidazole higher donor loadings improved the C-acet-
ylation, reaching thereby a maximum at 300 mM IPEA. Whereas
product formation never exceeded 70 % (filled circles) in the
absence of imidazole, bioacetylations in the presence of imid-
To analyze the spontaneous nonenzymatic formation of the
resorcinol mono ester 3a and diester 4a, the reactions were
performed in the absence of enzyme (Table 2). The amount of
3a/4a formation correlated, in principle, with the reactivity of
the investigated carboxylic acid derivatives: no O-acetylation
occurred with less reactive aliphatic esters 5c and 5d or acet-
anilides 5m–n, whereas significant amounts of monoester 3a
were detected in reactions with the enol esters 5a and 5b, an-
hydride 5e, and the phenyl acetates 5f–l. Interestingly, diester
4a was only observed in the absence of imidazole when using
the highly reactive carboxylic acid derivatives 5e, 5j, and 5l
(> 80 %; entries 5, 10, and 12). In the biotransformations,
the amounts of 3a and 4a were lower, one reason being the
PpATaseCH catalyzed Fries reaction of the esters into 2a.
azole already gave 96 % product 2a at just 75 m
eventually went to completion with donor concentrations ran-
ging from 100 m to 300 m (Figure 1, filled squares). Notably,
no residual O-acetyl by-product was detected in the bioacetyl-
ations until reaching 500 m IPEA (Figure S1). However, in the
M IPEA and
M
M
M
absence of enzyme, the spontaneous O-acetylation was greatly
enhanced by imidazole and steadily increased with increasing
IPEA concentration (up to 55 %, Figure S2), whereas without
imidazole the formation of 3a and 4a was much lower. At this
point, the pH of the reaction system has to be discussed in
more detail.
Table 2. Spontaneous O-acetylation of resorcinol 1a in the absence of enzyme
employing acyl donors 5a–n. The formation of monoester 3a and diester 4a
was evaluated in the presence and absence of imidazole.[a]
Entry
Donor
Absence of imidazole
Presence of imidazole[c]
3a [%]
4a [%]
3a [%]
1
2
3
4
5
5a
5b
5c
5d
5e
9
57
n.d.
n.d.
10
n.d.
n.d.
n.d.
n.d.
80
24
16
n.d.
n.d.
40
6
7
8
9
10
11
12
13
14
5f
5g
5h
5i
26
8
10
7
12
n.d.
10
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
80
n.d.
89
n.d.
n.d.
16
11
15
18
13
44
7
n.d.
n.d.
5j[b]
5k[b]
5l[b]
5m[b]
5n[b]
Figure 1. PpATaseCH catalyzed formation of 2a at varied IPEA (5a) concentra-
tion. The reactions were carried out in the presence of imidazole (■) using
KPi-buffer (50 m
(50 m , pH 7.56) or in the absence of imidazole () using KPi-buffer (50 m
pH 8.30). Reagents and conditions: cell-free E. coli extract containing the re-
combinant PpATaseCH (3.3 U ≡ 20 mg of lyophilized cells), 1a (10 m ), KPi-
buffer (50 m , pH 7.5 or pH 8.3), imidazole (100 m , added from a 1
M, pH 7.56), in the absence of imidazole (●) using KPi-buffer
M
M,
[a] Reaction conditions: resorcinol (1a; 10 m
imidazole (optional: 100 m , added from a 1
the same buffer, pH 9.55), donor 5a–n (100 m
addition), total reaction volume = 1 mL, 35 °C, 18 h, 750 rpm. Concentrations
in % were determined by HPLC using calibration curves of authentic samples.
n.d. = not detected. [b] The donor was dissolved in DMSO. The final reaction
mixture (1 mL) contained 10 vol.-% DMSO. [c] Compound 4a was not de-
tected under these conditions.
M
), KPi-buffer (50 m
stock solution prepared in
), pH 8.30 (after imidazole
M, pH 7.56),
M
M
M
M
M
M
M
stock solution prepared in the respective KPi-buffer, pH 9.55), IPEA (5a), total
reaction volume = 1 mL, 35 °C, 18 h, 750 rpm.
Comparing the pH before and after the reaction in the ab-
sence of enzyme revealed a shift, which depended on the addi-
tion of imidazole and IPEA. For instance, whereas substrate 1a
generally did not alter the pH of the reaction system, the addi-
Although phenolic esters turned out to be active acyl do-
nors, IPEA was considered as superior for several reasons:[17]
First and foremost the co-product acetone can be stripped eas-
ily from the reaction mixture and, unlike the corresponding
phenol co-products, does not impede the purification of the
target hydroxyphenyl ketones. Moreover, acetone is less react-
ive compared with the co-product acetaldehyde, which emer-
ges from the reaction with vinyl acetate (5b) and may inhibit
the enzyme. For these reasons, IPEA was chosen as donor for
further studies.
tion of imidazole (100 mM) from an externally prepared stock
solution (1 stock solution prepared in the respective KPi-
M
buffer, pH 9.55) caused the pH to rise from 7.56, the pre-set
buffer pH, to approximately 8.30. When starting the reaction by
adding commercial unpurified 5a (100 mM), the pH immedi-
ately decreased by 0.18 units, due to acetic acid impurities
present (Figure S4). By contrast, the use of freshly distilled IPEA
altered the apparent pH to a lesser extent (0.09 units). The pH
eventually further decreased in the course of the reaction using
either distilled or undistilled IPEA and was approximately 6.8
after 18 h (Figure S4). In comparison, the pH in the correspond-
Optimum Concentration of IPEA 5a
The concentration of donor 5a was varied at a fixed concentra- ing reaction without imidazole only decreased to 7.2 (18 h, Fig-
tion of imidazole (100 m ) in the bioacetylation of 1a. It was ure S3), which may indicate that the donor was hydrolyzed
M
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faster in the presence of imidazole. The bioacetylation in the these divergences. Nevertheless, various amine additives had
presence of imidazole went to completion, whereas the same remarkable effects on the bioacetylation with IPEA (Figure 2):
reaction without imidazole did not. This leads to the assump- besides imidazole (6a), pyridine (6i) and also several substituted
tion that the apparent effect of imidazole to improve the enzy- imidazoles enhanced the enzymatic C-acetylation with IPEA,
matic C-acetylation might be due to the increased activity of whereby it depended on the position of the substituent as well
the ATase at higher pH. To investigate whether increasing the as on the electronic nature.
pH would be enough to obtain the same amount of product as
in the presence of imidazole, the imidazole-induced change in
pH was mimicked by performing the bioacetylations at various
IPEA concentrations in buffer with preset pH 8.30 (Figure 1,
empty circle). Surprisingly, the formation of the C-acetyl prod-
uct 2a was considerably lower than in the imidazole-supple-
mented reactions (empty circles vs. squares). Furthermore,
product 2a formation was even lower than the corresponding
non-supplemented reactions performed in buffer with preset
pH 7.56 (filled vs. empty circles), indicating that imidazole plays
another role in addition to simply changing the pH of the reac-
tion system.
Figure 2. Formation of product 2a in the bioacetylation of 1a with IPEA in the
presence of additives 6a–p. The gray horizontal line represents the product
formation of 2a without amine additive. Reagents and conditions: Lyophilized
cells of E. coli containing the recombinant PpATaseCH (20 mg), 1a (10 m
KPi-buffer (50 m , pH 8.30, 100 m amine 6), IPEA (100 m ), total reaction
volume = 1 mL, 35 °C, 18 h.
M),
Alternatives Amine Additives to Imidazole
M
M
M
To elucidate whether the bioacetylation using IPEA may also be
promoted by additives other than imidazole 6a, a broad set of
aromatic (6b–i), alicyclic (6j–m) and acyclic (6n–p) amines was
evaluated (Scheme 3).
Imidazoles with electron-withdrawing groups 6e–f, but also
2-methylimidazole (6c) had no notable effect on the bioacetyl-
ation using IPEA, whereas the reaction was improved twofold
by 4-methylimidazole (6c) and even fivefold by N-methylimid-
azole (6d) compared with the reaction without additive. The
imidazole series also promoted the spontaneous O-acetylation
in the absence of enzyme (Figure S8). Pyrazole (6g), pyrrole (6h)
and the acyclic amines 6n–p had a negative impact on the
ATase catalyzed formation of 2a. Apart from aromatic amines,
the reaction was significantly enhanced by the alicyclic com-
pounds morpholine (6k) and 1,4-diazabicyclo[2.2.2]octane
(DABCO, 6l), with the latter being superior over all other investi-
gated amine additives, leading to 32 % product formation un-
der the conditions employed. Piperidine (6j) on the other hand,
which differs from 6k by an oxygen atom, did not improve
the reaction. Remarkably, the spontaneous O-acetylation was
significantly reduced with 6k–l and also the pH changed less
than with the aromatic amines 6a–i (Figure S9).
Scheme 3. Amines investigated as additives for the biocatalytic Friedel–Crafts
acylation.
Comparing in a next step the time course of the biocatalytic
Given that 100 m
conversion into 2a at 100 m
M
imidazole (6a) enabled more than 99 % C- and spontaneous O-acetylation employing IPEA/DABCO
IPEA, these concentrations were (100 m
) to the reaction with IPEA/imidazole (100 m ) using
M
M
M
also employed for testing other amines as alternatives to imid- cell-free extract preparations as in the optimized system, it
azole. To further simplify the procedure, lyophilized cell prepa- turned out that the formation of 2a in the presence of DABCO
rations of the PpATaseCH were used and the respective amine was slower than imidazole when both reactions were per-
additive was supplemented in the reaction buffer with the pH formed at the same pH (pH 8.3), which was realized by adding
adjusted to pH 8.30 to make it comparable to the reaction in DABCO from an pH-adjusted stock solution (Figure 3). Whereas
the presence of imidazole.
Interestingly, the type of enzyme preparation effected the
bioacetylation, as indicated by the reaction with imidazole (6a),
the reaction with imidazole went to completion after 22 h, the
IPEA/DABCO system reached only 33 % product formation.
However, when handling the DABCO reaction in the same
which only gave 16 % product formation with lyophilized cells fashion as the imidazole reaction, thus without adjustment of
whereas more than 99 % was obtained with cell-free extract, pH, then the pH at the onset of the acylation reaction was
despite adapting the amounts of the enzyme preparation to pH 9.85. Interestingly, under these conditions, the acetylation
make the experiments comparable (see the Supporting Infor- in the presence of DABCO was equally fast during the first four
mation). It might be speculated that limited mass-transfer of hours as the reaction with imidazole at pH 8.3, but then slowed
the substrate through the cell to the enzyme probably caused down, eventually reaching 83 % after 22 hours. The reduction
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Table 3. PpATaseCH catalyzed bioacetylation of 1b–e using IPEA in the pres-
ence of imidazole or DABCO.
Entry
Substrate
2 [%][a]
Imidazole
DABCO
1
2
3
4
1b
1c
1d
1e
98
40[b]
2
73
72
n.d.
21
29
[a] Concentrations in % were determined by HPLC using calibration curves
of authentic samples. Reagents and conditions: Cell-free E. coli extract con-
taining the recombinant PpATaseCH (3.3 U ≡ 20 mg of lyophilized cells), 1a–
e (10 m
stock solution prepared in the same buffer, pH 9.55) or DABCO (100 m
added from a 1 stock solution prepared in the same buffer, pH 11.21), IPEA
(100 m ), total reaction volume = 1 mL, 35 °C, 18 h, 750 rpm. At the onset
of the reaction the pH for the mixture with imidazole was pH 8.3 whereas it
was pH 9.85 for DABCO. n.d. = not detected. [b] In addition to C-acetyl prod-
uct 2c, remaining O-acetylated product was also detected by HPLC.
M), KPi-buffer (50 mM, pH 7.56), imidazole (100 mM, added from a 1 M
M,
Figure 3. Time course for the PpATaseCH catalyzed formation of 2a (●) and
the spontaneous formation of monoester 3a () in the presence of ATase using
IPEA/imidazole (pH 8.3, solid line), IPEA/DABCO (pH 8.3, dashed line) or IPEA/
DABCO (w/o pH-adjustment, pH 9.85, dotted line).
M
M
of activity may be explained by denaturing of the enzyme at
the rather basic pH. These observations suggest that the gen-
eral impact of the amine additives on the C- and O-acetylation
is more complex and not only an effect of pH.
The course of the spontaneous O-acetylation in the presence
of enzyme varied also depending on the amine additive: with
DABCO, the formation of monoester 3a was always less than
3 %, independent of the pH, but with imidazole, 3a reached a
maximum after 2 h followed by a steady decrease because of
ATase catalyzed transformation of 3a into 2a.
Conclusions
It was found that PpATaseCH can utilize a broad set of acetyl
derivatives as acyl donor for the biocatalytic Friedel–Crafts
acylation of resorcinol. Typical acetyl donors were activated es-
ters, such as enol esters 5a–b or para- and meta-substituted
phenyl esters 5g–j. Nonactivated aliphatic esters 5c–d or acet-
anilides 5m–n were not transformed. Notably, the investigated
phenyl esters did not undergo Fries rearrangement, as was ob-
served for an acetyl ester of resorcinol.
To test whether DABCO might be an alternative additive in
case of other resorcinol derivatives as substrates, the bioacetyl-
ation of selected C4- or C5-substituted resorcinols 1b–e was
investigated at pH 9.85, thus without pH adjustment
(Scheme 4). As already observed in the time study with 1a,
less C-acetyl product 2b was formed in the presence of DABCO
compared with the optimized IPEA/imidazole system within 18
hours reaction time (Table 3).
Imidazole had a significant effect on the Friedel–Crafts acyl-
ation: using IPEA (5a) or vinyl acetate (5c) the amine additive
improved the reactions leading to higher concentrations of
product, whereas the opposite effect was observed when using
the p-NO2-substituted phenol ester 5j as donor; in this case,
imidazole led to diminished levels of product formation. The
effect of imidazole cannot be accounted for by a shift of pH in
the reaction mixture only (pH 7.56 to ca. 8.30 after addition)
because reactions that were performed right at pH 8.30 but
without imidazole were inferior (30 % product formation in-
stead of >99 %). Additionally, it was found that various other
amine additives, including N-methylimidazole (6b), pyridine (6i)
and the alicyclic amines morpholine (6k) and DABCO (6l) im-
proved the C- and O-acetylation (Figure 2). In particular, DABCO
enhanced the PpATaseCH catalyzed C-acetylation giving 2a
while at the same time the spontaneous O-acetylation leading
to the formation of monoester 3a was suppressed. In contrast,
O-acetylation was significant in the presence of imidazole and
Scheme 4. Structures of expected products 2b–e.
Thus, the bioacetylation of 1b gave 98 % conversion into 2b other imidazole derivatives (Figure S8). Even more interesting,
with imidazole (pH 8.3) and 73 % when using DABCO at pH 9.95 when employing DABCO under the same conditions as imidaz-
(Table 3, entry 1). In contrast, substrate 1c was transformed into ole (pH 8.3), only moderate amounts of product were formed,
2c with 72 % in the presence of DABCO, whereas only 40 % was however at pH 9.85 DABCO led to a reaction initially as fast as in
obtained with IPEA/imidazole (entry 2). In this case, there was the presence of imidazole, but which leveled off after 6 hours,
still a significant amount of mono-O-acetylated product 3c de- probably because of instability of the enzyme at that high pH.
tected in the presence of imidazole,[15] while again no O-acetyl- When applying the best conditions with DABCO (pH 9.85) for
ation was observed in the reaction with DABCO. Whereas 1d the bioacetylation of other acceptor substrates, lower concen-
was transformed only to a very small extent or not at all, the trations of product were obtained compared with the corre-
C5-substituted resorcinol 1e (5-MeO) was transformed with sponding reactions with IPEA/imidazole, except for 4-methyl-
comparable amount of product formation in both cases (en- ated resorcinol 1c (Table 3). In this case, the enzymatic C-acetyl-
try 3–4).
ation could be improved from 40 % (with imidazole) to ca. 70 %
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bioacetylation. The final reaction mixture (1 mL) was manually
shaken thoroughly directly after donor addition to ensure proper
mixing. The reaction mixture (final volume = 1 mL) was horizontally
shaken for 18 h at 35 °C in an orbital shaker at 750 rpm. The reac-
tion was worked-up and analyzed as described above. To monitor
the spontaneous formation of the O-acetyl by-products 3a and 4a,
control reactions without enzyme were performed. The pH change
was surveyed by measuring the pH at the beginning and the end
of the control reactions (see the Supporting Information).
(with DABCO). Again, no O-acetylated side products were
formed with IPEA/DABCO, whereas remaining ester in the bio-
acetylation of 1c with imidazole led to reduced amounts of the
desired C-acetyl product 2c.
Considering the importance of Friedel–Crafts acylations of
phenolic compounds in synthetic chemistry and the fact that
many common approaches still rely on harsh conditions to con-
trol selectivity, the methods described herein may initiate fur-
ther research on biocatalytic alternatives.
Acknowledgments
Experimental Section
This study was financed by the Austrian Research Promotion
Agency (FFG), the Federal Ministry for Science, Research and
Technology, the Austrian Ministry for Transport, Innovation and
Technology, the Styrian Business Promotion Agency (SFG), the
General Remarks: All compounds used in this study were obtained
from commercial suppliers and used as received unless stated oth-
erwise. pH measurements were carried out with a pH meter (Hanna
Instruments, HI2211 pH/ORP Meter), equipped with a conventional Standortagentur Tirol and the Wirtschafts-/Technologieagentur
Ag/AgCl pH-electrode (SI-Analytics, BlueLine 16 pH). Conversions
were determined by HPLC (Shimadzu-prominence liquid chromato-
graph, SPD-M20A diode array detector), equipped with a Phenome-
nex Luna® 5μ C18 (2) 100A (250 × 4.6 mm) column. Gradient elution
with H2O and MeCN (+TFA, 0.1 vol.-%) was performed to separate
the reaction products as described previously.[15] Products were
identified as described previously.[15] The following gradient was
applied: 0–15 % MeCN (0–5 min), 15–60 % MeCN (5–22 min), 60–
100 % MeCN (22–25 min), 100–0 % MeCN (25–30 min), flow rate =
1 mL min–1, sample vol. = 2 μL. The reaction products were quanti-
fied at 280 nm or 230 nm (only when diester 4a was detected) from
the peak areas based on external calibration curves using authentic
samples. The ATase from Pseudomonas protegens (PpATaseCH) was
overexpressed in E. coli BL21 (DE3) as described previously[15] and
used as cell-free extract preparations or lyophilized cell prepara-
tions.
der Stadt Wien (ZIT) through the Austrian FFG-COMET Funding
Program.
Keywords: Biocatalysis · Biotransformations · Friedel–Crafts
acylation · C–C coupling · Phenol ketones
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Representative Procedure for the Bioacetylation of Resorcinol
(1a) Using Different Acyl Donors (5a–n): Resorcinol (1a, 10 m
was dissolved in KPi-buffer (50 m , pH 7.56) and the mixture was
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Received: August 2, 2017
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Biocatalytic C-Acylation
N. G. Schmidt, W. Kroutil* ............... 1–8
Acyl Donors and Additives for the
Biocatalytic Friedel–Crafts Acylation
A biocatalytic Friedel–Crafts-like acyl- well as the influence and effect of
ation is described. The scope of the ac- amines as additive on the reaction are
tivating groups of the acetyl donors as studied.
DOI: 10.1002/ejoc.201701079
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