Biocatalytic N-Acylation of Amines in Water Using an Acyltransferase from Mycobacterium smegmatis

Article abstract of DOI:10.1002/adsc.201801061

A straightforward one-step biocatalyzed synthesis of different N-acyl amides in water was accomplished using the versatile and chemoselective acyltransferase from Mycobacterium smegmatis (MsAcT). Acetylation of primary arylalkyl amines was achieved with a range of acetyl donors in biphasic systems within 1 hour and at room temperature. Vinyl acetate was the best donor which could be employed in the N-acetylation of a large range of primary amines in excellent yields (85–99%) after just 20 minutes. Other acyl donors (including formyl-, propionyl-, and butyryl-donors) were also efficiently employed in the biocatalytic N-acylation. Finally, the biocatalyst was tested in transamidation reactions using acetamide as acetyl donor in aqueous medium, reaching yields of 60–70%. This work expands the toolbox of preparative methods for the formation of N-acyl amides, describing a biocatalytic approach easy to accomplish under mild conditions in water. (Figure presented.).

Full text of DOI:10.1002/adsc.201801061

Title: Biocatalytic N-Acylation of Amines in Water Using an
Acyltransferase from Mycobacterium smegmatis
Authors: Martina Contente, Andrea Pinto, Francesco Molinari, and
Francesca Paradisi
This manuscript has been accepted after peer review and appears as an
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the VoR from the journal website shown below when it is published
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To be cited as: Adv. Synth. Catal. 10.1002/adsc.201801061
Link to VoR: http://dx.doi.org/10.1002/adsc.201801061

10.1002/adsc.201801061
Advanced Synthesis & Catalysis
UPDATE
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Biocatalytic N-Acylation of Amines in Water Using an
Acyltransferase from Mycobacterium smegmatis
Martina Letizia Contente [a], Andrea Pinto[b], Francesco Molinari*[b], Francesca
Paradisi*[a]
a
School of Chemistry, University of Nottingham, University Park, Nottingham, NG7 2RD, United Kingdom
Phone: +44(0)115 74 86267; e-mail: francesca.paradisi@nottingham.ac.uk
Department of Food, Environmental and Nutritional Science, DeFENS, University of Milan, via Mangiagalli 25, Milan,
b
Italy
Phone: (+39)-0250319148; e-mail: francesco.molinari@unimi.it
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######.((Please
delete if not appropriate))
Abstract. A straightforward one-step biocatalyzed synthesis of different N-acyl amides in water was
accomplished using the versatile and chemoselective acyltransferase from Mycobacterium smegmatis (MsAcT).
Acetylation of primary arylalkyl amines was achieved with a range of acetyl donors in biphasic systems within 1
hour and at room temperature. Vinyl acetate was the best donor which could be employed in the N-acetylation of
a large range of primary amines in excellent yields (85-99%) after just 20 minutes. Other acyl donors (including
formyl-, propionyl-, and butyryl- donors) were also efficiently employed in the biocatalytic N-acylation. Finally,
the biocatalyst was tested in transamidation reactions using acetamide as acetyl donor in aqueous medium,
reaching yields of 60-70%. This work expands the toolbox of preparative methods for the formation of N-acyl
amides, describing a biocatalytic approach easy to accomplish under mild conditions in water.
Keywords: Amide synthesis; Biocatalysis; Transamidation; Acyltransferase; Mycobacterium smegmatis
Amide bond formation is among the most
important reactions in organic chemistry due to the
carbodiimides), reaction of an amine with an easily
available acid derivative (e.g., an ester) or with
acylating agents (e.g., acyl halides and symmetrical
or mixed anhydrides), or from alcohols or aldehydes
along with a stoichiometric oxidant.[5] However,
available methodologies tend to be inefficient and
“amide formation avoiding poor atom economy” was
put as one of the top challenges for organic chemists
in the last decade.[6] As an example, the preparation
of 1 kg of the anti-HIV agent Fuzeon (36 aminoacids)
requires 45 kg of raw material, excluding the solvents
used in the synthesis and purification step.[7]
Furthermore, commonly employed harsh reaction
conditions, and the lack of selectivity in the presence
of additional functional groups, eliminates most
biomolecules from the range of target substrates.
Enzymatic strategies for amide bond formation have
been achieved with ATP-dependent enzymes
(generating acylphosphate intermediates), while
transacylation reactions require proteases and
carboxylesterases as biocatalysts.[8] Hydrolases such
as lipases form an acyl-enzyme intermediate, which
can yield N-acyl amides through reaction with
suitable amines, but, in aqueous environment, the
hydrolysis of the acyl-enzyme is strongly competing,
limiting these approach to organic environments.[9]
Amide synthesis in water has been reported
widespread occurrence of amides in biologically
2]
active compounds.[1,
In vivo, peptide bonds are
created predominantly during protein biosynthesis in
the ribosomes[3], whereas N-acylation mediated by
acetylCoA-dependent N-acyltransferases is a key step
in the detoxification of arylamine xenobiotics.[4]
Synthetically, amide formation is a traditional
reaction and can be carried out using different
strategies: direct reaction between an amine and an
acid mediated by coupling reagents (e.g.,
[a]
Dr. M. L. Contente and Prof. F. Paradisi
School of Chemistry
University of Nottingham
University Park, Nottingham, NG7 2RD, UK
Tel: +44 11574 86267
[b]
Dr. A. Pinto and Prof. F. Molinari
DeFENS - Dept. of Food, Environmental and Nutritional Sciences
Università degli Studi di Milano
Via Celoria, 2, 20133 Milano, IT
E-mail: francesca.paradisi@nottingham.ac.uk
francesco.molinari@unimi.it
Supporting information for this article is given via a link at the end of the
document.
1
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801061
Advanced Synthesis & Catalysis
using penicillin acylase predominantly applied to the
production of β-lactam antibiotics;[10] kinetic control
of the synthesis is achieved through an excess of an
activated donor to saturate the active site.
Nevertheless, hydrolysis of the product is often a
deleterious side-reaction, requiring critical and
careful optimization on preparative scale to maximize
yields.[11]
Table 1. N-Acetylation of (E)-cinnamylamine (50 mM) catalyzed
by MsAcT (1 mg/mL) in phosphate buffer (100 mM, pH 8.0) with
different acetyl donors at 25 °C.
A
C-acyltransferase from Pseudomonas
protegens was recently described for its promiscuous
N-acetylation activity.[12] While good anilide yields
were achieved, reaction times between 18 and 24
hours were required. In addition, the enzyme
exhibited a narrow substrate scope working only with
anilines and acetyl donors.
Entry
R[a]
Conv.[b]
(%)
Time[c]
(min)
1
2
3
4
5
6
ethyl
isopropyl
isoamyl
p-nitro phenyl
vinyl
53
62
31
48
92
82
20
30
30
60
20
30
Acetyltransferase
from
Mycobacterium
smegmatis (MsAcT) catalyzes the formation of acetyl
esters in water.[13] Unlike many acyltransferases, it is
not cofactor dependent. Its characteristic hydrophobic
tunnel leading to the active site disfavors the ingress
of water, thus promoting ester formation over
hydrolysis.[13b-d] Over prolonged times, the hydrolysis
of the product (acetyl ester) is however observed.
MsAcT, displays a secondary activity in the acylation
isopropenyl
[a] Acetyl donor 20 eq [b] Determined by HPLC. [c] Time
corresponding to maximum conversion. No background reactions
were observed under these reaction conditions.
The highest conversion (92%) was observed
with vinyl acetate (entry 5, Table 1), after 20 minutes.
No reaction was observed without enzyme under the
selected reaction conditions after prolonged times.
The same reaction performed on preparative scale (50
mL) gave 87% isolated yield of 2a.
To further establish the synthetic applicability of
the N-acetylation catalyzed by MsAcT, its substrate
scope was investigated with different primary
arylalkyl amines; the reaction was carried out using
ethyl acetate and vinyl acetate as donors (Table 2).
of
some
primary
amines
using
methyl
methoxyacetate as acyl donor;[14] this reaction was
exploited in combination with a transaminase to shift
the equilibrium in a one-pot biocatalytic cascade for
the formation of amides from aldehydes or ketones.
In this work, we demonstrate that MsAcT has in
fact a very broad substrate scope and can be used for
the preparation of N-acetyl derivatives in water
starting from different primary amines and using a
variety of acyl donors (e.g., ethyl and vinyl esters).
Firstly, the preparation of N-cinnamylacetamide (2a)
from (E)-cinnamylamine [(E)-3-phenylprop-2-en-1-
amine] (1a) was optimized using the inexpensive
ethyl acetate as acetyl donor. Different parameters
Table 2. N-Acetylation of arylalkyl amines (50 mM) catalyzed by
MsAcT (1 mg/mL) in phosphate buffer (100 mM, pH 8.0) with
different acetyl donors (20 eq.) at 25 °C.
(pH,
substrate,
temperature
and
enzyme
concentration) were optimized, while keeping the
amount of ethyl acetate fixed at 20 eq.; maximum
conversion was used as response variable (Table 1,
Supporting information).
Under optimal conditions (amine 50 mM, 25 °C,
enzyme 1 mg/mL, phosphate buffer 100 mM, pH 8.0),
maximum yields of 53-56% were observed within 20
minutes; after prolonged times (2 h), in all cases, the
reaction went under thermodynamic control with
partial hydrolysis of the product 2a.
Gedey et al. converted amines with CAL-B and
proposed that the leaving group of the acyl donor
may remain in the proximity of the active site
influencing the transesterification reaction.[15]
Therefore, a screening of different acetyl donors was
carried out; unactivated esters (isopropyl acetate,
isoamyl acetate) and activated esters (p-nitro phenyl
acetate, vinyl acetate, isopropenyl acetate) were
tested. 20 equivalents of acetyl donors were used
(Table 1) to push the equilibrium toward the desired
products. The reactions were performed under the
optimized conditions described above (entry 1, Table
1, Supporting information).
Conv.a
(%)
Timeb
(min)
Entry
1
Substrate
Acetyl donor
EtOAc
VinylOAc
51
85
60
30
1b
EtOAc
VinylOAc
80
>99
30
20
2
3
1c
EtOAc
VinylOAc
25
>99
30
20
1d
EtOAc
VinylOAc
80
98
60
20
4
1e
EtOAc
VinylOAc
97
98
30
15
5
6
1f
EtOAc
VinylOAc
96
98
30
15
1g
2
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10.1002/adsc.201801061
Advanced Synthesis & Catalysis
1
2
3
4
5
6
7
8
9
10
11
12
13
14
H
CH2CH3
CH2CH3
CH2=CH2
CH2CH3
CH2=CH2
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2=CH2
CH2CH3
CH2=CH2
CH2CH3
CH2CH3
60
15
90
11
56
15
25
66
45
97
38
74
36
52
60
30
30
60
30
60
60
30
60
30
60
30
60
60
1a
1a
1a
1a
1a
1a
1a
1h
1h
1h
1h
1h
1h
1h
EtOAc
VinylOAc
86
98
30
20
CH3CH2
CH3CH2
7
1h
CH3CH2CH2
CH3CH2CH2
(CH3)2CH
(CH3)2CHCH2
H
CH3CH2
CH3CH2
CH3CH2CH2
CH3CH2CH2
(CH3)2CH
(CH3)2CHCH2
EtOAc
VinylOAc
98
>99
30
15
1i
1j
8
9
EtOAc
VinylOAc
98
>99
30
15
EtOAc
VinylOAc
90
>99
60
20
10
11
1k
1l
EtOAc
VinylOAc
51
85
30
20
[a] Determined by HPLC. [b] Time corresponding to maximum
conversion
[a] Determined by HPLC. [b] Time corresponding to maximum
conversion.
N-Formylation occurred with high rates and
satisfactory conversions were observed (60% entry 1,
and 66% entry 8, Table 3); the same reaction on 50
mL scale gave 55% 2m (isolated yield). Bio-
preparation of N-formyl amides with such rates and
yields in water had not been previously reported in
the literature. The most consolidated chemical
methods for the preparation of these compounds rely
on formylation of amines using formylating agents
(e.g., acetic formic anhydride, formic acid, cyanide,
All the tested amines yielded N-acetyl amides
with both acetyl donors. The use of vinyl acetate
allowed for quantitative or nearly quantitative
conversions (85-99%) in all cases; notably, high
yields were obtained also with unactivated EtOAc
with most of the investigated amines resulting in a
very efficient and affordable process.
While MsAcT has been described as an O-acyl
transferase, N-acetylation of para-aminophenol and
vanillylamine (entries 3, 5, Table 2) occurred
completely chemoselectively, and no traces of O-
acetylation were detected, affording paracetamol and
N-acetyl vanillylamine with excellent yields and
without any further purification step. The selective
enzymatic acylation of amines in the presence of
other functional groups is an efficient alternative to
specific chemical reactions, such as the use of
thioesters and Cu-catalysis.[16] Both (S) and (R) β-
methylphenylethylamine (entry 10, Table 2) were
suitable substrates for MsAcT. The enzyme equally
converted both enantiomers and did not show any
stereopreference for this particular molecule.
N,N-dimethylformamide,
formylfluoride).[17]
However, these methodologies are characterized by
unsustainable conditions, expensive metal/organo-
catalysts, and tedious procedures.[18] A few examples
of enzyme-mediate N-formylation are reported
involving predominantly commercial lipases (e.g.,
CAL-B) in an anhydrous organic environment.[19]
Aqueous enzyme-cascades or chemo-enzymatic
syntheses are described exclusively for the hydrolysis
of the N-formyl group in the deracemization of DL-
aminoacids.[20] Good yields were also observed for
the formation of N-propionyl amide (90%, entry 2,
Table 3) when vinylpropionate was used as acyl
donor, whereas upon increasing the acyl chain length
(N-butyryl amide) only a maximum yield of 56% was
achieved.
We further investigated the enzyme tolerance for
the acyl substrate with two model amines; (E)-
cinnamylamine (1a) and 2-phenylethylamine (1h),
and different esters with linear or branched acyl chain
as donors (Table 3).
Finally, an even more challenging reaction, a
biocatalytic transamidation in
a
homogeneous
aqueous system, was explored. This reaction is often
troublesome, amides are highly stable and poorly
reactive,
and
transamidation
is
generally
Table 3. N-Acylation of (E)-cinnamylamine 1a and 2-
phenylethylamine 1h catalyzed by MsAcT (1 mg/mL) in phosphate thermodynamic considerations. While formally
buffer (100 mM, pH 8.0) with different acyl donors (20 eq.) at
characterized by unfavourable kinetic and
transamidation is not dissimilar from the more
common transesterifications, chemical strategies are
scarce and usually require drastic reaction conditions
making such reaction quite an atypical synthetic
methodology.[21] Recent examples of transamidation
have been reported, involving both heterogeneous and
homogeneous catalysis.[22] However, environmental
and practical drawbacks are nearly always reported
such as the use of expensive and waste-generating
reagents (e.g., hazardous acids or basis as
transamidation catalysts), high temperatures or long
reaction times, and the need for sealed reaction
vessels to achieve water and oxygen-free conditions.
25 °C. Initial concentration of amines was 50 mM.
Conv.a Timeb
Entry Amine
R
R’
(%)
(min)
3
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10.1002/adsc.201801061
Advanced Synthesis & Catalysis
Here, (E)-cinnamylamine (1a) and 2-
Experimental Section
phenylethylamine (1h) were exploited as model
substrates with 20 eq. acetamide as donor (Table 2,
Supporting information for further details). The
maximum molar conversion observed was between
60% and 70% with rapid reaction times (1 h / 30 min,
respectively). The reaction was once again performed
in water, validating the applicability of the method
(Table 4). Transamidation of (E)-cinnamylamine (1a)
on preparative scale (50 mL) gave 57% isolated yield
of 2a.
General
NMR spectra were recorded on a Varian Gemini 300
MHz spectrometer using the residual signal of the
deuterated solvent as internal standard. H chemical
shifts (δ) are expressed in ppm, and coupling
constants (J) in hertz (Hz). Merck Silica gel 60 F254
plates were used for analytical TLC; flash column
chromatography was performed on Merck Silica gel
(200–400 mesh).
1
Table 4. N-Acetylation of (E)-cinnamylamine 1a and 2-
phenylethyalmine 1h catalyzed by MsAcT (1 mg/mL) by
transamidation with acetamide (20 eq.) in phosphate buffer (100
mM, pH 8.0) at 25 °C. Initial concentration of amines was 50
mM.
Chemicals
All reagents and solvents were obtained from
commercial suppliers and were used without further
purification.
Cloning, Overexpression and Purification of
MsAcT
A synthetic gene encoding for MsAcT (GenBank
accession: ABK70783) from Mycobacterium
smegmatis str. MC2 155 was purchased from
BaseClear. The gene was cloned into vector pET26b
(Novagen) with C-terminal his-tag. Restriction sites
for NdeI and BamHI were inserted by PCR using 5’-
CCGACATATGGCTAAACGTATTCTGTGC-3’ as
Conv.
Entry
Amine
Time (min)[b]
(%)[a]
1
2
60
70
60
30
1a
1h
forward
and
5’-
CTTGGATCCAGCAGAGAACGAACTTGT-3’ as
reverse primer. The PCR product was then cloned
into pET26b expression vector and the resulting
pET26b-MsAcT transformed into chemically
competent E. coli BL21 star (DE3) for heterologous
expression. Culture of E. coli BL21 star (DE3) pETb-
MsAcT (20 mL) was grown overnight at 37 °C in LB
medium supplemented with 25 μg/mL kanamycin.
200 mL of cultivation medium (Terrific Broth: 12 g/L
bacto-tryptone, 24 g/L yeast extract, 4 g/L glycerol,
2.3 g/L KH2PO4, 9.4 g/L K2HPO4, 25 μg/mL
kanamycin, pH 7.2) were inoculated with the
previous described seed culture to an initial OD600nm
of 0.1. Cultivation was carried out at 37 °C, 150 rpm.
Cells were grown until OD600nm reached the value of
0.5. The expression of MsAcT was induced by the
addition of isopropyl-β-d-thiogalactopyranoside
(IPTG) to a final concentration of 0.1 mM. The
culture was further incubated for 16 h at 25 °C. Cells
were then harvested by centrifugation (20 min, 4500
rpm, 4 °C), washed once with 20 mM phosphate
buffer pH 8.0 and stored at -20 °C.
[a] Determined by HPLC. [b] Time corresponding to maximum
conversion
Despite the known solubility problems of many
organic substrates in water, the possibility to
synthesize amides in aqueous media seems
noteworthy. This study shows that biocatalytic N-
acylation of different amines can be realized in water
with good-to-excellent yields using the stable and
easy-to-produce acyltransferase from Mycobacterium
smegmatis (MsAcT); the applicability and
chemoselectivity of the proposed strategy was
successfully applied for the preparation of different
amides in excellent yields. Notably, also N-formyl
amides were produced in this simple and
straightforward one-step technique, which does not
require any particular formylating agent and/or
tedious subsequent purification. Finally, we have
demonstrated that this enzyme can also efficiently
catalyze challenging transamidation reactions
between primary amines and acetamide in aqueous
medium without the need for a dry reaction
2 g of pellet were resuspended in 10 mL loading
buffer (100 mM phosphate buffer pH 8.0, 6 mM
imidazole, 100 mM NaCl) and sonicated (5 cycles of
2 min each, in ice, with 1 min interval). Cell debris
were harvested by centrifugation (45 min, 15000 rpm,
4 °C). The enzyme was purified by affinity
chromatography with HIS-Select® Nickel Affinity
Gel. Briefly, the column was equilibrated with
loading buffer and the crude extract loaded; column
was then washed with loading buffer; finally, the
environment
or
oxygen-free
conditions,
demonstrating an alternative, useful, and applicable
method for the preparation of amides. This strategy
for amide bond formation sheds light on the
importance of developing catalytic methods
addressing what is recognized as an unmet synthetic
need.
4
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10.1002/adsc.201801061
Advanced Synthesis & Catalysis
Acknowledgements
adsorbed enzyme was eluted with elution buffer
(phosphate buffer 100 mM pH 8.0, 250 mM
imidazole, 100 mM NaCl).
This project was supported by the European Union’s
Horizon 2020 research and innovation programme
under the Marie Skłodowska-Curie grant agreement
N. 792804_AROMAs-FLOW (M.L.C.). A.P. was
financially supported by the “Piano di Sostegno alla
Ricerca 2015/2017–Linea 2_Azione A” of the
University of Milan.
Crude extract, pellet and pure protein were analyzed
by SDS-PAGE (See Supporting information, Fig. S1).
The fractions showing the presence of a band of the
expected size (25.6 kDa) were pooled, dialyzed
against 100 mM phosphate buffer pH 8.0 and stored
at 4 °C. Typically, starting from 2 g of wet cell paste,
it was possible to obtain 130 mg of pure protein (8.5
mg/mL).
References
[1] A. Greenberg, C. M. Breneman, J. F. Liebman. The
amide linkage: structural significant in chemistry,
biochemistry and material science Wiley-Interscience:
New York, 2000.
MsAcT activity assay
Activity
measurements
were
performed
spectrophotometrically at 400 nm by determining the
formation of p-nitrophenol at 25 °C in a half-
microcuvette (total volume 1 mL) for 2 min. One unit
(U) of activity is defined as the amount of enzyme
which catalyzes the consumption of 1 μmol of p-
nitrophenylacetate per minute under reference
conditions, namely 0.1 mg/mL p-nitrophenylacetate,
0.1% v/v EtOH, correct amount of MsAcT in 100
mM phosphate buffer, pH 8.0. Specific activity was
800 U/mg.
[2] V. R. Pattabiraman, J. W. Bode. Nature, 2011, 480,
471-479.
[3] T. M. Schmeing, V. Ramakrishnan. Nature 2009, 461,
1234-1242.
[4] S. K. Walters, S. Boukouvala. Drug Metab Rev, 2008,
40, 479-510.
[5] J. E. Taylor, S. D. Bull. N-Acylation Reactions of
Amines in Comprehensive Organic Synthesis: Second
Edition (Eds.: P. Knochel, G. A. Molander), Elsevier,
Amsterdam, 2014, pp. 427-478.
[6] D. J. C. Constable, P. J. Dunn, J. D. Hayler, G. R.
Humphrey, J. L. Leazer Jr., R. J. Linderman, K.
Lorenz, J. Manley, B. A. Pearlman, A. Wells, A. Zaksh,
T. Y. Zhang. Green Chem. 2007, 9, 411-420.
[7] V. Marx. Chem. Eng. News 2005, 83, 16–17.
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[9] a) F. van Rantwijk, R. A. Sheldon, Tetrahedron 2004,
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[10] A. Bruggink, E. C. Roos, E. de Vroom. Org. Process.
Res. Dev. 1998, 2, 128-133.
[11] a) R. C. Giordano, M. P. Ribeiro, R. L. Giordano.
Biotechnol. Adv. 2006, 24, 27-41. b) D. Romano, R.
Gandolfi, S. Guglielmetti, F. Molinari. Food Chem.
2011, 124, 1096-1098.
Small scale batch reactions
Batch reactions using MsAcT were performed in 10
mL screw cap tubes; 1 mL reaction mixture in 100
mM phosphate buffer pH 8.0, containing 50 Mm
amine, 1 mg/mL enzyme, and 20 eq. acyl donor were
left under magnetic stirring at 25 °C. 50 µL aliquots
were quenched with NaOH 1 M or NaHCO3 5% (in
the case of vanillyamine and p-aminophenol) at
different reaction times (10 min, 20 min, 30 min, 1 h,
2 h, 4 h) and extracted with 100 µL of EtOAc for
TLC (CH2Cl2/MeOH 9:1 + 0.1% TEA). Samples,
after evaporation, were resuspended in the mobile
phase for HPLC analysis.
HPLC Analysis
See supporting information.
Preparative scale batch reactions
[12] A. Zadło-Dobrowolska, N. G. Schmidt, W. Kroutil.
Chem. Commun. 2018, 54, 3387-3390.
[13] a) I. Mathews, M. Soltis, M. Saldajeno, G. Ganshaw,
R. Sala, W. Weyler, M. A. Cervin, G. Whited, R. Bott
Biochemistry 2007, 46, 8969-8979 b) N. de Leeuw, G.
Torrelo, C. Bisterfeld, V. Resch, L. Mestrom, E.
Straulino, L. van der Weel, U. Hanefeld, Adv. Synth.
Catal. 2018, 360, 242-249. c) A. Drozdz, U. Hanefeld,
K. Szymaꢀska, A. Jarzebski, A. Chrobok, Catal.
Commun. 2016, 81, 37-40. d) K. Szymaꢀska, K.
Odrozek, A. Zniszczol, G. Torrelo, V. Resch, U.
Hanefeld, A. B. Jarzebski, Catal. Sci. Technol. 2016, 6,
4882-4888.
Preparative scale reactions using MsAcT were
performed in 100 mL round bottom flasks; 50 mL
reaction mixtures (100 mM phosphate buffer pH 8.0,
50 mM amine, 1 mg/mL enzyme, and 20 eq. acyl
donor) were left under magnetic stirring at 25 °C. The
reaction mixture was monitored by TLC
(CH2Cl2/MeOH 9:1 + 0.1% TEA), quenched with
NaOH 1 M when the maximum conversion was
reached. After extraction (EtOAc: 3 X 30 mL), the
organic phase was collected, dried over Na2SO4,
filtrated and evaporated under reduced pressure. The
crude extract was purified by silica gel column
chromatography (EtOAc/n-hexane 9:1).
[14] H. Land, P. Hendil-Forssell, M. Martinelle, P.
Berglund. Catal. Sci. Technol. 2016, 6, 2897-2900.
[15] S. Gedey, A. Liljeblad, F. Fulop, L. T. Kanerva.
Tetrahedron: Asymmetry 1999, 10, 2573–2581.
5
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10.1002/adsc.201801061
Advanced Synthesis & Catalysis
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10.1002/adsc.201801061
Advanced Synthesis & Catalysis
UPDATE
Biocatalytic N-Acylation of Amines in Water
Using an Acyltransferase from Mycobacterium
smegmatis
Adv. Synth. Catal. Year, Volume, Page – Page
Martina Letizia Contente, Andrea Pinto, Francesco
Molinari*, Francesca Paradisi*
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