Promiscuous activity of C-acyltransferase from: Pseudomonas protegens: Synthesis of acetanilides in aqueous buffer

Article abstract of DOI:10.1039/c8cc00290h

Amide bond formation has considerable significance in synthetic chemistry. Although the C-acyltransferase from Pseudomonas protegens has been found to catalyze C-C bond formation in nature as well as in in vitro experiments with non-natural substrates, it is now shown that the enzyme is also able to catalyze amide formation using aniline derivatives as substrates with promiscuous activity. Importantly, the amide formation was enabled in aqueous buffer. Identifying phenyl acetate as the most suitable acetyl donor, the products were obtained with up to >99% conversion and up to 99% isolated yield.

Full text of DOI:10.1039/c8cc00290h

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Promiscuous activity of C-acyltransferase from
Pseudomonas protegens: synthesis of acetanilides
in aqueous buffer†
Cite this:DOI: 10.1039/c8cc00290h
Received 17th January 2018,
Accepted 8th March 2018
a
ab
Anna Z˙a˛dło-Dobrowolska,
Nina G. Schmidtab and Wolfgang Kroutil
*
DOI: 10.1039/c8cc00290h
rsc.li/chemcomm
Amide bond formation has considerable significance in synthetic However, limitations including the rather strict specificities and
chemistry. Although the C-acyltransferase from Pseudomonas protegens relative instability in anhydrous environments inspired the search
has been found to catalyze C–C bond formation in nature as well as in for non-protease catalysts. Due to the similarities in the mechanism
in vitro experiments with non-natural substrates, it is now shown that the employing nucleophilic serine at the active site, lipases and
enzyme is also able to catalyze amide formation using aniline derivatives esterases were also found to show promiscuous amidase
as substrates with promiscuous activity. Importantly, the amide activity.9 While many biocatalytic methods in low-water media
formation was enabled in aqueous buffer. Identifying phenyl acetate have been developed, few cases have been reported concerning
as the most suitable acetyl donor, the products were obtained with amide bond formation in aqueous media.10 In addition to
up to 499% conversion and up to 99% isolated yield.
hydrolases, a few other enzymes have been described to transfer
the acyl group from acyl donors to amine acceptors under
The amide moiety is the key functional group in a wide variety physiological conditions. For instance, penicillin acylase was
of biological and synthetic structures, such as peptides, polymers, applied for dipeptide synthesis11 as well as for enantioselective
pharmaceuticals and pesticides.1 Formally, an amide bond is acylation of various amines.12 Peptiligase was reported as an
formed during the condensation of an amine and carboxylic acid efficient catalyst in peptide synthesis and cyclization.13 Also an
with the release of one molecule of water. Due to the high acyltransferase originating from Mycobacterium smegmatis (MsAcT)
activation barrier, synthetic amides are mainly produced by was shown to catalyse acylation of aliphatic amines in water.14
employing activated acid derivatives, such as acid chlorides and
An acyltransferase from Pseudomonas protegens (PpATaseCH)
anhydrides.2 Although these methods tend to be highly effective, was reported to perform C–C bond formation in the biosynthesis
they suffer from several drawbacks, such as possible racemization, of the antibiotically active polyketide 2,4-diacetylphloroglucinol
poor atom economy, the possible toxic nature of organic solvents (DAPG), excreted by several plant-associated Pseudomonas sp.
and coupling reagents. As a consequence, amide bond formation and Pseudomonas fluorescens ssp.15 Unlike most other acyltrans-
has been identified as one of the most important synthetic processes ferases,16 this heterotrimeric complex acts independently of
to be improved.1,3 Furthermore, due to the increasing demands for cofactors, CoA or ATP. The multi-component ATase from
green chemistry procedures,4 a direct, efficient, environmentally Pseudomonas protegens catalyses also C–C bond formation when
friendly catalytic process is of high importance.5
transferring an acetyl moiety from a non-natural donor sub-
Various types of biocatalysts such as proteases transform strate such as isopropenyl acetate to an electron-rich phenolic
amides and catalyse in their natural physiological role amide acceptor in a Friedel–Crafts-type acetylation reaction.17 Here we
bond hydrolysis in peptide substrates.6 Due to the principle report that the enzyme shows chemical reaction promiscuity18
reversibility of catalysis and when the thermodynamic barrier catalysing also amide formation.
(e.g. using an excess of water) can be overcome then the
Since in previous studies mainly resorcinol derivatives were
equilibrium can be shifted to favour synthesis over hydrolysis. investigated as acetyl acceptors leading to C–C bond formation,17
Numerous proteases were reported to catalyse amide bond for- the substrate scope was extended by testing a substrate bearing
mation in nearly anhydrous organic solvents7 or water mimetics.8 one amino group instead of one of the hydroxyl groups in
resorcinol, a substrate undergoing C–C bond formation. Indeed,
3-aminophenol (1a) was accepted as the substrate when using 2,4-
diacetylphloroglucinol (DAPG, 4) as the acetyl donor (Scheme 1).
However, instead of the expected C–C bond formation the enzyme
a Institute of Chemistry, University of Graz, NAWI Graz, BioTechMed Graz,
Harrachgasse 21/3, Graz, Austria. E-mail: wolfgang.kroutil@uni-graz.at
b Austrian Centre of Industrial Biotechnology, acib GmbH, Graz, Austria
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8cc00290h performed N-acetylation, thus N-(3-hydroxyphenyl)acetamide (2a)
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Table 1 Biocatalytic N-acetylation of anilines
Acetyl donor
IPEA DAPG 4
R3 Conv. [%] Conv. [%] Conv. [%]
Scheme 1 ATase catalyzes N-acylation of 3-aminophenol.
PA
Entry Substr. R1
R2
was formed as the only product instead of the expected 1-(2-amino-
4-hydroxyphenyl)ethan-1-one (3, Scheme 1), which would have
been formed via C–C bond formation.17 Thus, in the observed
reaction the enzyme breaks a C–C bond in the acetyl donor and
enables subsequently C–N bond formation. Furthermore, it has to
be noted that acetylation was observed exclusively at the amino
group while the phenolic alcohol was not acetylated. The selective
acylation of amines in the presence of other functional groups
such as phenolic alcohols is rather a difficult task. For instance,
chemical acylation of such compounds leads to the formation of
1
2
3
4
5
6
7
8
1a
1b
1c
1d
1e
1f
1g
1h
1i
H
H
OH
OH
H
H
H
H
H
OH
H
H
H
H
H
H
H
Et
H
H
43
OH o1
o1
92
o1
o1
o1
84
55
92
86
81
7
499(96)
o1
H
o1
OH o1
o1
H
63
50
499(70)
499(91)
499(83)
499(99)
499(98)
18(13)
o1
Cl
i-Pr 66
Et
H
H
H
47
33
12
9
10
11
12
1j
1k
1l
Et
NO2
H
H
o1
o1
CH2OH Cl
12
85
98(91)
the corresponding amides and esters, when acid chlorides or Reaction conditions: cell-free E. coli extract containing PpATaseCH
(0.066 U) in KPi-buffer (100 mM, pH 7.5) with acceptors 1a–l at 10 mM
and the donors: DAPG (15 mM), DMSO (10 vol%) or IPEA (100 mM) or PA,
phenylacetate (15 mM), 35 1C, 18 h. Conversion was determined by HPLC
anhydrides were employed; N-selectivity could only be achieved
with thioesters and Cu-catalysis.19
To gain deeper insight into the substrate spectrum, a range according to calibration curves using authentic samples. Values in
brackets correspond to isolated product yields from semi-preparative
scale experiments transforming 0.25 mmol of the substrate: amine
(10 mM final concentration) in KPi-buffer (100 mM, pH 7.5), cell-free extract
of aromatic amines 1b–1l containing various functional groups
was studied (Table 1). For this purpose, the substrate (10 mM)
was suspended in KPi-buffer adjusted to pH 7.5, and cell-free containing PpATaseCH (2.5 mL, 1.65 U). Semi-preparative bioacetylation
(25 mL total volume) was started by adding PA (54.9 mL, 15 mM final
concentration) and was run at 35 1C and 140 rpm for 24 h.
extract containing recombinant PpATaseCH was subsequently
added, followed by addition of the acetyl donor (DAPG 4). While
aniline derivatives bearing an OH group in the para- or ortho-
position were not converted (substrates 1b–1d), aniline 1e as
Using IPEA as the acetyl donor with substrates 1a–1l,
well as anilines with various substituents in the para-position 10 equivalents of IPEA were required to obtain moderate
(Cl, i-Pr, Et) were well accepted reaching a conversion of up to conversion, for instance, reaching 66% conversion for 1k. In
92% within 18 hours (substrates 1f–1h). Furthermore, anilines contrast, just 1.5 equivalents of PA were sufficient to observe
with an ethyl group also in the meta- or ortho-position were quantitative product formation for all substrates transformed
accepted, whereby the para-substituted substrate reacted the also with DAPG (Table 1, entries 1, 5–9 and 12), except for 1j which
fastest followed by the meta-substituted ones (substrates 1h–1i). led to lower conversion.
Having a nitro-moiety in the ortho-position instead of the ethyl
Thus, comparing the natural acetyl donor DAPG with the
group did not lead to any detectable conversion. In addition to non-natural IPEA and PA revealed that PA was the most efficient
the mono-substituted anilines, also a di-substituted aniline (1l) acetyl source, requiring only a low excess of donor to enable the
was successfully transformed.
completion of the reaction for substrates 1a and 1f–1h under
Although rather useful conversion values were achieved the reaction conditions tested, thus leading in all cases to higher
when using DAPG 4 as the acetyl donor, other acetyl donors conversion than observed with DAPG. For instance, acetylation with
were evaluated. One reason being that DAPG 4 is not commercially IPEA resulted in 50% conversion for 1f (55% with DAPG) while
available and the co-product phloroglucinol generated during 499% was achieved with PA.
reaction impedes the purification. Consequently, enol esters
Testing the transformation of aliphatic amines with the
and phenyl esters such as isopropenyl acetate (IPEA) and phenyl improved protocol showed that benzylamine (1m) or 1-octylamine
acetate (PA) were evaluated as donors in further experiments. was not accepted by the enzyme. This chemoselectivity may
For these two donors the enzyme would cleave a C–O bond.
have practical application, as ATase differentiates between the
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Table 2 Chemoselectivity of ATase: N-acetylation of aniline derivative 1a
versus benzylamine 1m
Table 3 Intensification of the N-acetylation of 1a
Substrate
Donor
Entry
concentration [mM]
concentration [mM]
Conv. [%]
Conv. of
Conv. of
Chemoselectivity
Entry
Donor
1a [%]
1m [%]
2a : 2m
1
2
3
4
5
6
10
10
10
10
20
40
100
50
15
11
30
60
80
86
499
499
499
95
1
2
3
IPEA
DAPG
PA
46
96
100
n.d.
n.d.
n.d.
2a only
2a only
2a only
Reaction conditions: cell-free E. coli extract containing PpATaseCH
(0.066 U) in KPi-buffer (100 mM, pH 7.5) with 1a at 10 mM and
benzylamine 1m at 10 mM and the donors: DAPG (15 mM), DMSO
(10 vol%) or IPEA (100 mM) or PA, phenylacetate (15 mM), 35 1C, 18 h.
Conversion was determined by HPLC via calibration curves using
authentic samples. n.d. not detected.
Reaction conditions: cell-free E. coli extract containing PpATaseCH
(0.066 U) in KPi-buffer (100 mM, pH 7.5) with acceptor 1a at 10 mM,
20 mM or 40 mM and the donor PA, phenylacetate (15 mM, 30 mM,
50 mM, 60 mM and 100 mM), 35 1C, 18 h. Conversion was measured by
HPLC according to calibration curves using authentic samples.
aniline-NH2 and an aliphatic amine. To demonstrate the
chemoselectivity, the acetylation of aniline derivative 1a and
benzylamine was tested in one pot simultaneously (Table 2).
Using either IPEA, DAPG or PA perfect chemoselectivity toward
1a was observed.
Up to now mainly acetyl and propanoyl groups have been
transferred by this enzyme.17a The expansion of the scope of
acyl donors (e.g. for butanoyl, benzoyl, etc.) by applying protein
engineering is under investigation.
substrate) led to lower conversion than when using just 1.5 of
donor PA (Table 3, entries 1–3). Actually, the equivalents of PA
could be reduced even further to 1.1 equivalents (entry 4).
Keeping the donor at 1.5 equivalents, the substrate concentration
was increased at the given enzyme concentration up to 20 mM still
leading to 499% conversion before only at 40 mM a slight drop of
conv. to 95% was observed. The latter corresponds to an apparent
total turnover number of TONapp = 5360.
The promiscuous N-acetylation activity of ATase may be
explained based on sequence alignment searches. The PhlC
unit, which is responsible for the enzyme activity, can be related
to the thiolase superfamily,21 featuring a cysteine residue at
position 88 (Cys88) which aligns to the conserved active-site
cysteine of thiolases. Cysteine C88 may form a thioester inter-
mediate with the acetyl donor, which may be prone to the amine
nucleophilic attack leading to the amidation product.
In summary, the acyltransferase from Pseudomonas protegens
was found to exhibit promiscuous N-acetylation activity towards a
broad spectrum of aniline derivatives. In contrast to previous
studies in which C–C bond formation was found, the enzyme
allowed the acetylation of aniline derivatives in aqueous medium.
After identifying phenyl acetate as an ideal donor, excellent
isolated yields of up to 99% were obtained for the N-acetylated
anilides. The transformation was demonstrated with substrate
loadings above 40 mM and a low excess of donor (1.5 eq.). The
protocol involving mild reaction conditions, a simple workup
procedure leading to high yields, may serve as an attractive
alternative to the existing N-acylation methods.
To test the acyltransferase PpATaseCH under the conditions
previously described for the acetyltransferase from Mycobacterium
smegmatis,14 an experiment was performed in CHES buffer
adjusted to pH 10. However, under these conditions substrate 1a
was transformed with only 34% conversion into the corresponding
acetanilide, while in phosphate buffer at pH 7.5 the transformation
reached completion. This result is in accordance with our previous
observations for the C–C bond forming reaction, showing that
PpATaseCH displayed higher activity at pH 7.5 to 8.5, while a
more alkaline pH resulted in lower enzyme activity. Since MsAcT
has been reported to transform only aliphatic amines, PpATaseCH
complements the substrate scope by also allowing the transforma-
tion of anilines into the corresponding acetanilides.
Furthermore, lipases are also known for their amidation
activity especially in organic solvents.20 For comparison, the
N-acetylation of 1a was tested in aqueous buffer using Novozym
435 as a representative lipase due to its broad substrate
acceptance. However, the conversion was o1% when using
either IPEA or PA after 18 h, clearly showing that Novozym 435
does not catalyze this reaction in aqueous buffer. Additionally,
it was ensured by enzyme purification that no other enzyme
present in E. coli catalyzes the observed reaction, thus the
amide formation is due to the activity of PpATaseCH.
To demonstrate the applicability of the amide formation,
semi-preparative transformations were performed starting with
0.25 mmol of the substrate. Anilines 1a and 1e–j were trans-
formed into the corresponding acetanilides using PA as the
acetyl donor within 24 h and the products were isolated with
high yields (Table 1, values in brackets). The yields were in most
cases comparable to the conversion obtained in analytical-scale
experiments.
We gratefully acknowledge support from the Austrian Science
Fund (FWF) through a Lise Meitner Fellowship Grant M 2172-B21.
NGS was financed by the Austrian FFG, BMWFJ, BMVIT, SFG,
Standortagentur Tirol and ZIT through the Austrian FFG-COMET-
Funding Program.
Conflicts of interest
There are no conflicts to declare.
Notes and references
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