Amide Synthesis via Aminolysis of Ester or Acid with an Intracellular Lipase

Article abstract of DOI:10.1021/acscatal.8b02713

A unique lipase (SpL) from Sphingomonas sp. HXN-200 was discovered as the first intracellular enzyme for the aminolysis of ester or acid to produce amide. Reactions of a series of esters and amines with SpL gave the corresponding amides 3a-g in high yield with high activity. SpL also showed high enantioselectivity and high activity for enantioselective ester aminolysis, producing amides (R)-3h-j in high ee from the corresponding racemic ester or amine. Moreover, SpL was found to be highly active for the aminolysis of carboxylic acid, which was generally considered infeasible with the known aminolysis enzymes. The aminolysis of several carboxylic acids afforded the corresponding amides 3a, 3d, 3k, 3l, and 3n in good yield. The intracellular SpL was expressed in Escherichia coli cells to give an efficient whole-cell biocatalyst for amide synthesis. Remarkably, high catalytic activity was observed in the presence of water at 2-4% (v/v) for free enzyme and 16% (v/v) for whole cells, respectively. Accordingly, E. coli (SpL) wet cells were used as easily available and practical catalysts for the aminolysis of ester or acid, producing a group of useful and valuable amides in high concentration (up to 103 mM) and high yield. The newly discovered intracellular SpL with unique properties is a promising catalyst for green and efficient synthesis of amides.

Full text of DOI:10.1021/acscatal.8b02713

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Article
Amide Synthesis via Aminolysis of Ester or Acid with an Intracellular Lipase
Shichao Zeng, Ji LIU, Sampson Anankanbil, Ming Chen, Joseph P. Adams, Radka Snajdrova, and Zhi Li
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b02713 • Publication Date (Web): 08 Aug 2018
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Amide Synthesis via Aminolysis of Ester or Acid with an Intracellular
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Lipase
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Shichao Zeng,†a Ji Liu,†a Sampson Anankanbil,b Ming Chen,a Joseph P. Adams,c Radka
Snajdrovac and Zhi Li*a
a. Department of Chemical and Biomolecular Engineering, National University of
Singapore, 4 Engineering Drive 4, Singapore 117585
b. Department of Engineering, Faculty of Science and Technology, Aarhus University,
8000 Aarhus, Denmark
c. Chemical Sciences, GSK R&D Medicines Research Centre, Gunnelswood Road,
Stevenage, SG1 2NY, UK
†The authors contributed equally to this work.
* Corresponding author. Eꢀmail: chelz@nus.edu.sg
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ABSTRACT
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A unique lipase (SpL) from Sphingomonas sp. HXNꢀ200 was discovered as the first
intracellular enzyme for the aminolysis of ester or acid to produce amide. Reactions of a
series of esters and amines with SpL gave the corresponding amides 3a-g in high yield
with high activity. SpL also showed high enantioselectivity and high activity for
enantioselective ester aminolysis, producing amides (R)ꢀ3h-j in high ee from the
corresponding racemic ester or amine. Moreover, SpL was found to be highly active for
the aminolysis of carboxylic acid, which was generally considered infeasible with the
known aminolysis enzymes. The aminolysis of several carboxylic acids afforded the
corresponding amides 3a, 3d, 3k, 3l and 3n in good yield. The intracellular SpL was
expressed in Escherichia coli cells to give an efficient wholeꢀcell biocatalyst for amide
synthesis. Remarkably, high catalytic activity was observed in the presence of water at
2ꢀ4% (v/v) for free enzyme and 16% (v/v) for whole cells, respectively. Accordingly, E.
coli (SpL) wetꢀcells were used as easily available and practical catalysts for the
aminolysis of ester or acid, producing a group of useful and valuable amides in high
concentration (up to 103 mM) and high yield. The newly discovered intracellular SpL
with unique properties is a promising catalyst for green and efficient synthesis of amides.
KEYWORDS: Amide synthesis, aminolysis of ester, aminolysis of acid, enzyme
catalysis, intracellular lipase, whole cells
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INTRODUCTION
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Amide bonds are present in many drug molecules.1 Amide synthesis is thus an
important task in pharmaceutical industry, evidenced by a recent survey.2 However, the
commonly used methods for amide synthesis via Nꢀacylation involve the use of acid
chlorides and coupling reagents with poor atom economy.2ꢀ3 In 2005, the ACS Green
Chemistry Institute Pharmaceutical Roundtable identified amide bond formation as the
most frequently used reaction in pharmaceutical syntheses but requiring green and efficient
methods.3
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Enzymeꢀcatalyzed aminolysis of ester or carboxylic acid offers a green and direct
synthesis of amides.4ꢀ6 Compared with chemically used methods, enzymatic amide
formation has several advantages such as high selectivity, mild reaction conditions, no use
of toxic or shock sensitive reagents, and high atom economy.4 Three types of serine
hydrolases are used for aminolysis of esters ꢀ lipase, subtilisin, and penicillin acylase.5
Since subtilisin requires highly active esters such as 2, 2, 2ꢀtrifuoroethyl butyrate as acyl
donors and penicillin acylase needs phenyl acetic acid or its αꢀsubstituted derivatives as
acyl donors, these enzymes are exclusively used for the kinetic resolution of chiral amines
via the aminolysis of esters.7ꢀ11 In contrast, lipase is useful for amide synthesis in organic
solvent with no hydrolysis of amide.12
However, there are also challenges associated with lipaseꢀcatalyzed amide synthesis.
Firstly, the currently used enzymes are exclusively extracellular lipases and thus have to be
used in free or immobilized forms, which requires enzyme purification and/or
immobilization. Secondly, many enzymes, such as Candida rugosa lipase (CRL) and
Pseudomonas alcaligenes lipase (PAL), have limited substrate scope.13ꢀ14 Thirdly, even the
wellꢀknown aminolysis enzymes such as Candida antarctica lipase B (CALB)6 show
generally low activity towards relatively bulky esters and/or amines.15ꢀ16
Enzymeꢀcatalyzed aminolysis of acid offers an even more direct synthesis of amides without
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the preparation of the ester substrate. However, carboxylic acids were generally considered to be
ineffective substrates for enzymeꢀcatalyzed aminolysis reaction due to the unreactive salts formed
between carboxylic acids and amines.4 Only a few examples have been reported for using
carboxylic acids as the acyl donor for enzymatic aminolysis reaction. All these examples suffer
from incomplete reaction, long reaction time, high enzyme loading or high reaction temperature
with poor to moderate amide yield. 17ꢀ28
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We have been interested in developing a novel intracellular enzyme that catalyzes the
aminolysis of esters with unique substrate specificity for green and efficient amide synthesis. A
group of target reactions were selected (Scheme 1ꢀ2). While some of the substrates are relatively
bulky, the corresponding amides are useful and valuable products. For example, amide 3a is a
structural analogue of capsaicin with much lower pungency for the application in food industry;12
amide 3c is a new potent tyrosinase inhibitor;29 amide 3c, 3d, and 3h are a novel class of 5ꢀHT3
receptor agonists;30 the R and S enantiomer of 3h showed different anticonvulsant activity towards
mice, and (R)ꢀ3h is principally responsible for the anticonvulsant activity.31 The current syntheses
of these compounds are not efficient.15ꢀ16 For instance, Novozyme 435 was reported for the
aminolysis reactions to produce amides 3c16, 3e15, and 3j,15 respectively, but with low specific
activity.
Here we report the discovery of a novel intracellular enzyme SpL for the aminolysis of ester or
acid to prepare amide with broad substrate scope, high activity, and/or high enantioselectivity, the
engineering of recombinant E. coli expressing the intracellular SpL as active wholeꢀcells catalyst
for the aminolysis of ester or acid, and the development of the easily available wetꢀcells catalyst
for practical amide synthesis.
RESULTS AND DISCUSSION
Discovery of SpL as an intracellular lipase for amide synthesis via aminolysis of ester
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Alkanes are known to be ideal stimulators for lipase expression in many wild type
strains.32ꢀ35 Thus, alkaneꢀdegrading strains could contain lipases that might catalyze the
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aminolysis of ester. 40
nꢀoctaneꢀdegrading strains collected in our lab were thus screened
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for the aminolysis of methyl hexanoate 1a with amylamine 2d 36ꢀ40
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Sphingomonas sp.
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HXNꢀ200 was found to be the most active microorganism for the reaction, with a specific
activity of 0.055 U/g wet cells.
To identify the enzyme that catalyzes the desired aminolysis of ester, the whole genome
of Sphingomonas sp. HXNꢀ200 was sequenced, and 11 putative lipases was predicted in
the strain based on bioinformatics analysis.40 Primers were designed to amplify the 11
putative lipases from the genome by polymerase chain reaction (PCR). The amplified
fragments were digested with their respective restriction enzymes and ligated to
pRSFDuetꢀ1 vector. The resultant plasmid was transformed into chemically competent T7
E. coli. 10 out of 11 putative lipases were successfully expressed in E. coli and showed
activity for hydrolyzing
p
ꢀnitrophenyl butyrate (PNPB). But only 1 lipase (SpL) was found
ꢀpentylhexanamide.
highly active for the aminolysis of ester 1a with amine 2d to give
N
The gene of SpL consists of 948 bp, encoding a 315 amino acid protein with a
predicted molecular weight of 33.81 kDa. A BLASTP with the NCBI protein database
showed that SpL shares most similarity (86% amino acid identity) with a putative
alpha/beta hydrolase foldꢀ3 protein from Sphingopyxis alaskensis RB2256. However, there
is no functional study on this protein.
E. coli (SpL) was easily grown in TB (Terrific Broth) medium. As shown in Figure 1a, a cell
density of 6.4ꢀ7.2 g cdw/L was achieved between 16ꢀ20 h. The highest specific activity (350
U/mg cdw) for the hydrolysis of PNPB was achieved at 16ꢀ18 h in the early stationary phase.
Based on the SDSꢀPAGE of the CFE (cellꢀfree extract) of the E. coli cells taken at 16 h (Figure
1b, Lane 2), SpL is an intracellular enzyme with a molecular weight of 37 kDa. This value is very
close to the predicted molecular weight.
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E. coli strain overexpressing
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ꢀterminal hisꢀtagged SpL was then constructed to
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facilitate the purification of SpL. Cells were cultivated, harvested, and disrupted, and the
CFE was subjected to affinity chromatography with a NiꢀNTA column. Hisꢀtagged SpL
was purified to apparent homogeneity as shown in SDSꢀPAGE (Figure 1b, Lane 4). This
purified SpL was used as free enzyme for the aminolysis of ester in the following studies.
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SpL-catalyzed aminolysis of ester in the presence of water
Water was generally considered as deleterious to lipaseꢀcatalyzed aminolysis of ester
due to the undesirable hydrolysis of the acyl donor.4 Hence, lipaseꢀcatalyzed aminolysis of
ester was normally carried out in anhydrous organic solvent.4 Candida antarctica lipase B
(CALB), by far the most powerful enzyme for aminolysis of ester known thus far, is such
an enzyme. Even with only trace amount of water (0.5%; v/v), the performance of CALB
dropped significantly. To achieve anhydrous condition, strict pretreatment of the solvent,
substrate, and enzyme with activated molecular sieve is often required. Thus, it is of
advantages that an enzymatic reaction could be performed without the needs of anhydrous
condition. Other type of enzyme such as acyl transferase is able to catalyze amide
formation in aqueous condition via oneꢀstep acylation of amine by using ester as acyl
donor in the presence of coꢀsolvent.41 However, there are only three examples of
lipaseꢀcatalyzed ester aminolysis in presence of water, but all involving the use of the
strong α nucleophiles (hydroxylamine or hydrazine) as acyl acceptor.14, 42ꢀ43 To our surprise,
SpL was found to be active in presence of water for the aminolysis of ester 1a with amine
2a. As shown in Figure 1c, SpL showed no catalytic activity in totally anhydrous
n
ꢀhexane. With trace amount of water (0.5%; v/v) added, apparent activity was observed.
Increase of water content from 0.5% to 2% (v/v) increased both the specific activity and
final amide yield. 2%ꢀ4% (v/v) water content was the optimal.
E. coli (SpL) cells were then examined in the presence of water for the aminolysis. As shown
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in Figure 1d, 16% (v/v) water content was found to be the optimal for achieving the highest amide
yield while maintaining high activity.
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SpL-catalyzed aminolysis of ester for the synthesis of amides 3a-g
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Free SpL was used to catalyze a series of reactions between esters 1a-d and amines
2a-d, as shown in Scheme 1. Methyl esters were used in this study to ensure low Sheldon’s
E factor by producing methanol as byproduct.17 For comparison, parallel reactions were
carried out with free CALB under its optimal conditions. As shown in Scheme 1, SpL
accepts esters 1a-d and amines 2a-d for the amide synthesis. For the aminolysis of ester
1a-d with amine 2a, SpL showed high activities ranging from 26.7 U/g to 115.9 U/g with
91.2% to 99% yield for 3a-d. These activities and yields are much higher than those of
CALB (Table S2). It is worthwhile to note that for the aminolysis of the relatively bulky
ester 1a, 1b and 1c, SpL is 45, 76, and 26 times more active than CALB. These values are
much higher than the 3.4 fold increase observed in the aminolysis of the relatively less
bulky ester 1a. Apparently, SpL is a better enzyme for accepting relatively bulky acyl
donors.
As shown in Scheme 1, SpL showed high activities (45.1 U/g to 108.2 U/g) and yields
(84.6% to 86.2%) for the aminolysis of ester 1d with amine 2b-d to produce amide 3e-g
.
The specific activities are 16.4ꢀ155.5 times higher than those of CALB. It is interesting to
note that even for the weak nucleophile aniline 2b, SpL showed a high activity of 45.1 U/g
enzyme and a very good final amide yield of 84.6%. This indicates that SpL is a promising
enzyme for anilide derivative synthesis.
SpL-catalyzed enantioselective aminolysis of ester for the synthesis of (R)-amides 3h-j
Enzyme catalyzedꢀenantioselective aminolysis of ester is a useful reaction to prepare
enantiopure amides that are valuable intermediates in pharmaceutical manufacturing. Thus
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far, enzymatic aminolysis is exclusively used for the resolution of chiral esters as well as
chiral amines. The use of lipase for enantioselective aminolysis towards the synthesis of
enantiopure or enriched amides is rarely reported.
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SpL was found to show high enantioselectivity for ester aminolysis to produce chiral amides
(Table 1). The enantioselective aminolysis of racemic ester 1e with amine 2a gave (R)ꢀ3h in
99.1% ee and 19.3% yield, with an E of 279. For SpLꢀcatalyzed enantioselective aminolysis of
ester 1d with racemic amine 2e-f, (R)ꢀ3i and (R)ꢀ3j were obtained in 94.3ꢀ94.1% ee and
31.7ꢀ34.6% yield with E of 52ꢀ54, respectively. In comparison, CALB gave much lower low E
values for producing (R)ꢀ3h and (R)ꢀ3i, and much low activity of producing (R)ꢀ3j (Table S3).
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Preparation of amide in high concentration via ester aminolysis with wet cells of E. coli
(SpL)
The direct use of whole cells expressing intracellular enzyme provides a readily
available and lowꢀcost biocatalyst for potential industrial application.40ꢀ46 Thus far, no
wholeꢀcell biocatalyst has been used for amide synthesis via ester aminolysis, since all
enzymes reported are extracellular.
Since SpL is an intracellular enzyme, it is possible to use cells expressing SpL as
catalyst. Considering the fact that lyophilization to produce dry cells is a highly
energyꢀintensive process and E. coli (SpL) needs 16% (v/v) of water to show the best
performance, directly using wet cells is highly desirable to lower the catalyst cost. Thus,
wet cells of E.coli (SpL) were used for the synthesis of amides. Based on the measured 86%
water content of the wet cell pellet, the water content of the reaction system was controlled
to be 16% (v/v) by adjusting catalyst loading to 185 mg wet cells/mL. In a typical reaction,
927 mg of freshly prepared wet cell pellet of E. coli (SpL) was mixed with 5 mL nꢀhexane
to give 16% (v/v) water content.
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Wet cells of E. coli (SpL) were demonstrated as efficient catalyst for the synthesis of
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the useful and valuable amides 3a-g and (R)ꢀ3h at high yield and high substrate
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concentration. As shown in Table 2, the aminolysis of ester 1a-b with amine 2a gave the
corresponding amide 3a-b in >99% yield with 80ꢀ100 mM. The reaction was very fast and
finished within 0.5 h. For the aminolysis of ester 1c with amine 2a and of 1d with 2c at
high substrate concentration of 120 mM, amide 3c and 3f were obtained in 80.7% and 85.9%
yield, respectively within 2 h. 79.9% and 71.5% yields of amide 3d-e were obtained for the
aminolysis between ester 1d and amine 2a-b, respectively. In comparison, the yield of 71.5%
(71.5 mM) for 3e was much better than the 25ꢀ55% yield by using Novozyme 435 or
Pseudomonas stutzeri lipase (PSL) after 20 h at 50 °C.15 The superior ability of E. coli
(SpL) cells to accept aniline 2b as a nucleophile for the synthesis of amide 3e at high
substrate concentration offers its promising application in the synthesis of anilide
derivatives in industry. Wet cells of E. coli (SpL) also catalyzed the enantioselective aminolysis
of racemic ester 1e (50 mM) with amine 2a (60 mM) to give (R)ꢀ3h in 97.9% ee with 23.2% yield.
During the biotransformation, no side reaction was observed, indicating no undesired catalytic
activity of the cells. The water was confined inside the cells, and no aqueousꢀorganic twoꢀphase
was observed in the reaction mixture.
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Preparative biotransformations were carried out with 4.6 g wet cells of E.coli (SpL) in 25 mL
nꢀhexane at 30 °C and 500 rpm for 2 h to produce amides 3a and 3b. Extraction with chloroform
and purification by flash chromatography gave pure amide products in 90ꢀ94% isolated yield.
Mechanistic study on SpL-catalyzed aminolysis of ester
Since water is involved in SpLꢀcatalyzed aminolysis of ester, it is interesting to check
whether carboxylic acid is the intermediate (Figure 2a). The aminolysis of ester 1d with
amine 2a using SpL in partially hydrated nꢀhexane (4%, v/v) was chosen as the model
reaction. As shown in Figure 2b, in the first 30 min, carboxylic acid 4a was accumulated at
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0.91 mM, indicating the occurring of ester hydrolysis. The carboxylic acid concentration
was then gradually reduced to 0.34 mM at 2 h, suggesting the possible formation of amide
3d from carboxylic acid 4a. Thus, pathway II in Figure 2a should be involved in the amide
synthesis.
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To investigate whether pathway II is the major pathway for amide formation, the hydrolysis of
ester 1d with SpL in the same medium was investigated. As shown in Figure 2c, only small amount
of carboxylic acid 4a was produced, the rate at 10 min is 84.1 ꢁmol (minꢀ1·gꢀ1 protein). This value is
10 times lower than the measured total reaction rate for SpLꢀcatalyzed aminolysis of ester 1d with
amine 2a (849 ꢁmol (minꢀ1·gꢀ1 protein)), demonstrating that the pathway II is the minor one (10%).
SpL-catalyzed aminolysis of carboxylic acid for the synthesis of amide
The unique ability of SpL to catalyze aminolysis of carboxylic acid to give amide was
confirmed. SpLꢀcatalyzed aminolysis of carboxylic acid 4a and amine 2a in partially
hydrated nꢀhexane was shown in Figure 2d. The reaction of 10 mM of carboxylic acid 4a
with 20 mM of amine 2a finished in 1 h to give 9.9 mM amide 3d in 99% yield. This
reaction rate is even 1.76 times faster than that using the methyl ester as the substrate,
suggesting that carboxylic acid is a more suitable substrate for SpLꢀcatalyzed aminolysis.
This unique character of SpL allows for amide formation directly from carboxylic acid
without the need of the preparation of ester as the substrate. So far, only several examples
have been reported for aminolysis of carboxylic acid for the enzymatic amide synthesis,
but required long reaction time, high enzyme loading and gave poor to moderate
yields.17ꢀ28 On the other hand, many wellꢀknown enzymes such as CALꢀB require
anhydrous condition to avoid hydrolysis. This is not suitable for the aminolysis of acid,
which produces water as a byꢀproduct. In comparison, SpLꢀcatalyzed amide formation via
aminolysis of carboxylic acid benefits from the unique water tolerance of the enzyme.
Possibly, water was excluded from the catalytic site, which was more accessible to the
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amine. In our future study, we plan to investigate the protein structure of SpL and explore
the structural basis of its water tolerance.
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Aminolysis of carboxylic acid to the synthesis of amide 3a, 3d, 3k-n with wet-cells of E. coli
(SpL)
As SpL was shown to be able to catalyze amide synthesis via aminolysis of carboxylic acid, the
wet cells of E. coli (SpL) were used to explore the amide synthesis. As shown in the Table 3, the
aminolysis of acid 4a (80 mM) with amine 2a (90 mM) in nꢀhexane containing wet cells (185 mg
cells/mL) gave amide 3d in 99% yield in 1 h, with a specific activity of 99.8 U/g cdw. The method
was further examined with 4 aliphatic fatty acids 4b-e (40 mM) with amine 2a (45 mM). For the
aminolysis of the acids with in medium chain 4b-d, E. coli (SpL) showed similar specific activity
(55.1ꢀ57.0 U/g cdw) and afforded amides 3a, 3k and 3l in 55ꢀ75% yield within 8 h (Scheme 3).
For the aminolysis of oleic acid 4e of long fatty acid chain (C18), E. coli (SpL) cells showed
lower activity (3.4 U/g cdw). Nevertheless, amide 3m was obtained in 19% yield after 24 h. It is
interesting to note that the cells could also catalyze the aminolysis of cyclic carboxylic acid 4f
with amine 2a to afford amide 3n in 51% yield within 8 h. The results suggested a broad substrate
scope of SpLꢀcatalyzed aminolysis of acid, including aromatic, aliphatic and cyclic carboxylic
acids.
The practical potential of amide synthesis from acid by using the wet cells of E. coli (SpL) was
demonstrated by the preparation of amides 3d, 3a and 3k-n in 25 mL nꢀhexane with 4.6 g wet
cells of E.coli (SpL) at 30 °C and 500 rpm. After 24 h reaction, the amide 3d, 3a, 3k and 3n were
extracted with chloroform and purified by simple washing with base and acid, giving amide
products in 53%, 60%, 55% and 39% yield, respectively. For the preparation of amide 3m, the
amide product was isolated through flash chromatography in 11% isolated yield. The structures of
the prepared amides were confirmed with NMR (Fig. S6ꢀS10) and high resolution MS (S14ꢀS18).
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CONCLUSIONS
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The first intracellular enzyme that catalyzes the aminolysis of ester or acid to produce
amide has been developed. Intracellular SpL from Sphingomonas sp. HXNꢀ200 showed
high activity for the aminolysis of a series of esters with different amines, accepting
relatively bulky substrates and broadening the substrate scope of enzymatic ester
aminolysis. SpL catalyzed also the highly enantioselective reactions with either racemic
ester or racemic amine, providing a new method for the synthesis of chiral amides in high
ee. Moreover, SpL is able to catalyze the aminolysis of carboxylic acid, giving rise to the
highꢀyielding enzymatic synthesis of amide directly from acid without the need of
preparing ester as the substrate. The amide synthesis with SpL could be performed in the
presence of water, without the need of anhydrous reaction medium. While cells of E. coli
expressing intracellular SpL was proven to be efficient catalyst for amide synthesis, the
use of easily available wetꢀcells of E. coli (SpL) allows for even more practical synthesis
of amide in high concentration and yield. The newly discovered intracellular SpL is a
potentially useful enzyme for green synthesis of amides in pharmaceutical manufacturing.
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EXPERIMENTAL SECTION
Screening of microorganisms for the aminolysis of ester 1a with amine 2d to produce
N-pentylhexanamide
40 octane degrading strains stored in our lab were first inoculated into 10 mL LB
medium and grown at 30 °C, and 250 rpm for 24 h. 10 mL of the seed culture was then
inoculated into 100 mL M9 medium and grown on octane vapor as the sole carbon source
as described previously.35 After growth for 3 days, the cells were harvested by
centrifugation, washed with deionized water, and then subjected to activity test.
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The activity test was carried out with 200 mg wet cell pellet, 1 mL nꢀhexane, 10 mM
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methyl hexanoate 1a and 20 mM amylamine 2d in 2 mL safeꢀlock tube on a mixing block
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at 30 °C, and 1000 rpm for 24 h. After centrifugation, the supernatant was taken for GC
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analysis to determine the formation of Nꢀpentylhexanamide.
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Construction of recombinant E. coli (SpL) and E.coli (his-tagged SpL)
The whole genomic DNA of Sphingomonas sp. HXNꢀ200 was prepared by using the
DNeasy Blood & Tissue Kit from Qiagen and used as the PCR template. The following
primers were used for amplifying SpL: Forward Primer: ACT GCA TAT GAC CGA CAG
CAC GAC CCA TTA CAC (NdeI restriction site is underlined); Reverse Primer: ACT
GAG ATC TTC ATG CCG CTC CGG TAG CCT CGG CG (BglII restriction site is
underlined).
To facilitate the purification of SpL, the following primers were used to engineer an
Nꢀterminal hisꢀtagged SpL: Forward Primer: ACT GGG ATC CGA TGA CCG ACA GCA
CGA CCC ATT ACA (BamHI restriction site is underlined); Reverse Primer: ACT GCT
CGA GTC ATG CCG CTC CGG TAG CCT CGG CG (XhoI restriction site is
underlined).
PCRs were carried out using a Biorad MycyclerTM thermo cycler with the following
program: initial denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing
at 72 °C for 30 s, extension at 72 °C for 45 s, total 33 cycles, final extension at 72 °C for
10 min. PCR products were purified on 1% agarose gel in TAE buffer and then subjected
to double digestion with appropriate restriction enzymes followed by ligation to
pRSFDuetꢀ1 vector. The ligation product was then used to transform E.coli T7 express
competent cells and the positive transformants were selected on LB agar plate containing
50 ꢁg/mL of kanamycin. DNA sequencing confirmed the successful construction of the
recombinant E.coli (SpL) and E.coli (hisꢀtagged SpL).
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Cell growth, specific activity, and enzyme expression of E. coli (SpL)
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The recombinant E. coli (SpL) was firstly grown in LB medium containing kanamycin
(50 ꢁg/mL) at 37 °C for 4ꢀ6 h and then inoculated into TB (Terrific Broth) medium
containing kanamycin (50 ꢁg/mL). When OD600 reached 0.6ꢀ0.8, IPTG (0.25 mM) was
added to induce the expression of protein. The cells continued to grow for 14−16 h at 22
°C. The cells were harvested by centrifugation (5000 g, 5 min), washed with deionized
water, and used for biotransformation. During the cell growth, the culture was taken at
different time points for measuring OD600, specific activity for hydrolyzing PNPB and
SDSꢀPAGE analysis.
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Purification and characterization of his-tagged SpL from E. coli (his-tagged SpL)
The cells of E. coli (hisꢀtagged SpL) expressing SpL with a 6×hisꢀtag at the Nꢀterminal
were cultivated and harvested at 16ꢀ18 h using the same method for E.coli (SpL). The cells
were then resuspended in KP buffer (10 mM, pH 8.0) and let pass through a cell
homogenizer (Stansted fluid power LTD) twice, followed by centrifugation (23000 g, 4
oC) for 50 min to remove the cell debris. The cellꢀfree extract (CFE) was filtered with 0.2
ꢁm Supor® membranes to remove small particles present and then subjected to FPLC for
protein purification on an AKTA purifier with a HisPrep FF 16/10 column, and UV
o
detection at 280 nm at 4 C. The untagged proteins were washed out with a wash buffer
(20 mM sodium phosphate, 500 mM NaCl, pH 7.4) containing 30 mM imidazole.
Afterwards, a wash buffer (20 mM sodium phosphate, 500 mM NaCl, pH 7.4) containing
300 mM imidazole was used to wash out the hisꢀtagged SpL. The salts and imidazole in
the collected enzyme fraction were removed by washing with deionized water in an
Amicon Ultraꢀ15 Centrifugal Filter Units (20 kD). The purity and molecular weight of
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hisꢀtagged SpL were checked by SDSꢀPAGE and MALDIꢀTOFꢀMS respectively. Free
SpL was prepared by lyophilization of purified hisꢀtagged SpL.
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Amide synthesis via aminolysis of ester with free SpL in presence of water
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A mixture consisting of 10 mM methyl hexanoate 1a, 20 mM benzylamine 2a, and 10
mg free SpL in 5 mL
n
ꢀhexane containing 0ꢀ12% (v
/v) water was shaken at 250 rpm and
o
30 C. For the case of 0% (v
/
v) water, 200 mg of activated 4A molsieve was added to
ensure totally anhydrous condition. Specific activity was measured at 30 min, and amide
yield was determined at 20 h.
General procedure for aminolysis of ester to give amide 3a-g and (R)-3h-j with free SpL
10 mg free SpL was added to a 10 mL shaking flask containing 5 mL nꢀhexane, 200 ꢁL
deionized water, and 20 mM corresponding amine (2a-f). The reaction was initiated by
adding 10 mM corresponding ester (1a-e), and the mixture was shaken at 30 °C, and 250
rpm for certain reaction time. 5 mL tertꢀbutyl methyl ether (MTBE) was added to dissolve
any possible insoluble amide, and anhydrous sodium sulfate was added to remove water. 1
mL reaction mixtures were taken for centrifugation, followed by mixing 200 ꢁL
supernatant with 200 ꢁL MTBE (2 mM benzyl alcohol as internal standard). The samples
were analyzed by HPLC for quantification of the concentration of amide 3a-g and
concentration and ee of amide 3h-j. The specific activity and amide yield were calculated
based on the product concentration.
Aminolysis and hydrolysis in the aminolysis of ester 1d with amine 2a to give amide 3d with
free SpL
5 mg/mL SpL aqueous solution was prepared by dissolving 50 mg free SpL in 10 mL
deionized water. 200 uL of the SpL aqueous solution was dispensed into a series of 10 mL
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shaking flasks, each containing 5 mL
n
ꢀhexane and 20 mM amine 2a. The reaction was
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initiated by adding 10 mM ester 1d and the mixtures were shaken at 30 °C, 250 rpm for 10
min to 2 h, respectively for monitoring the concentrations of ester 1d, amide 3d and
carboxylic acid 4a as the reaction progressed.
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Hydrolysis of ester 1d to give carboxylic acid 4a with free SpL
200 uL of 5 mg/mL SpL aqueous solution was dispensed into a series of 10 mL shaking
flasks, each containing 5 mL
30 °C and 250 rpm for 10 min to 2 h for monitoring the concentrations of ester 1d and
carboxylic acid 4a
nꢀhexane and 10 mM ester 1d. The mixtures were shaken at
.
Aminolysis of carboxylic acid 4a with amine 2a to give amide 3d with free SpL
200 uL of 5 mg/mL SpL aqueous solution was dispensed into a series of 10 mL shaking
flasks, each containing 5 mL
nꢀhexane and 20 mM amine 2a. The reaction was initiated by
adding 10 mM carboxylic acid 4a and the mixtures were shaken at 30 °C and 250 rpm for
10 min to 2 h for monitoring the concentrations of carboxylic acid 4a and amide 3d
.
Amide synthesis via aminolysis of ester with E. coli (SpL) cells in presence of water
The harvested cells of E.coli (SpL) were lyophilized to give dry cell powder. A mixture
consisting of 100 mM methyl hexanoate 1a, 120 mM benzylamine 2a and 150 mg E. coli
(SpL) dry cell powder in 5 mL
rpm and 30 oC.
nꢀhexane containing 0ꢀ20% (v/v) water was shaken at 250
General procedure for aminolysis of ester to give amide 3a-g and (R)-3h with wet cells of E.
coli (SpL)
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The wet cell pellet of E. coli (SpL) was measured to contain 86 wt% of water. 927 mg
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5
wet cell pellet was added to 5 mL nꢀhexane to give the optimal 16% (v/v) of water in the
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system. The mixture was stirred with magnetic stirrer, instead of shaking incubator, to
ensure good dispersion of the wet cell pellet. 60ꢀ120 mM of amine 2a-d was added first
and the mixture was stirred with a magnetic stirrer at 30 °C and 1400 rpm for 5 min.
50ꢀ120 mM of ester 1a-e was then added to initiate the reaction and the mixture was
stirred with a magnetic stirrer at 30 °C and 500 rpm for 30 min for measuring specific
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activity and 0.5ꢀ7 h for measuring amide yield or ee
.
General procedure for aminolysis of carboxylic acid for the synthesis of amide 3a, 3d and
3k-n with wet cells of E. coli (SpL)
927 mg freshly prepared cells (86% water content) were added to 5 mL
nꢀhexane (185
mg cells/mL) to give the optimal 16% (v v) of water in the system. Amine 2a (45ꢀ90 mM)
/
was added, and the reaction mixture was stirred with a magnetic stirrer at 30 °C and 1400
rpm for 5 min. Carboxylic acid 4a-f (40ꢀ80 mM) was then added to initiate the reaction.
The reactions were performed at 30 °C and 500 rpm for 24 h and samples were taken at 30
min (3d), 2 h (3a, 3k-l and 3n) or 8 h (3m) for measuring specific activity and at 1 h (3d),
8 h (3a 3k-l and 3n) or 24 h (3m) for measuring amide yield.
,
Preparation of amide 3a and 3b via aminolysis of ester using wet cells of E.coli (SpL)
4.6 g freshly prepared wet cells of E.coli (SpL) was added to 25 mL ꢀhexane at a
n
loading of 185 mg cells/mL in a 100 mL round bottom flask. Amine at specific
concentration (90 mM 2a for 3b synthesis; 120 mM 2a for 3a synthesis) was added, and
the reaction mixture was stirring with a mixing propeller at 30 °C and 500 rpm for 5 min.
The corresponding ester at a specific concentration (100 mM 1a for 3a synthesis; 80 mM
1b for 3b synthesis) was added, and the reaction was performed by stirring with the
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mixing propeller at 30 °C and 500 rpm for 2 h. After reaction, 25 mL chloroform was
added to the reaction mixture, and anhydrous sodium sulfate was added to remove water.
The cells were removed by filtration. Crude product was acquired by removing the solvent
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via evaporation. Column chromatography on silica gel (Rf=0.3;
nꢀhexane: ethyl
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acetate=10:1 for 3b;
nꢀhexane: ethyl acetate=5:1 for 3a) affords product 3a and 3b.
3a: obtained as a white solid; yield: 90%; H NMR (400 MHz, CDCl3, TMS) 51: δ
1
7.35ꢀ7.25 (m, 5H), 5.75 (s, br, 1H), 4.44 (d,
J
= 5.6 Hz, 2H), 2.20 (t,
J = 7.2 Hz, 2H), 1.66
(p,
J
= 7.6 Hz, 2H), 1.33ꢀ1.29 (m, 4H), 0.89 (t, J
= 6.8 Hz, 3H). 13C NMR (100 MHz,
CDCl3, TMS) 51: δ 173.0, 138.4, 128.7, 127.8, 127.5, 43.6, 36.8, 31.5, 25.4, 22.4, 13.9.
TOFMSꢀESI (m/z): [M+H]+, [M+Na]+ and [M+Na+ACN]+ calculated for C13H19NO,
206.154, 228.136, 269.163; Found, 206.153, 228.135, 269.161.
3b: obtained as a white solid; yield: 91%; H NMR (400 MHz, CDCl3, TMS) 52: δ
1
7.35ꢀ7.25 (m, 5H), 5.72 (s, br, 1H), 4.44 (d,
J = 5.6 Hz, 2H), 2.11 (tt, J = 11.8, 3.5 Hz, 1H),
1.91ꢀ1.66 (m, 5H), 1.51ꢀ1.42 (m, 2H), 1.31ꢀ1.19 (m, 3H). 13C NMR (100 MHz, CDCl3,
TMS) 52: δ 175.9, 138.5, 128.7, 127.7, 127.4, 45.6, 43.4, 29.7, 25.7. TOFMSꢀESI (m/z):
[M+H]+, [M+Na]+ and [M+Na+ACN]+ calculated for C14H19NO, 218.154, 240.136 and
281.163; Found, 218.154, 240.133 and 281.160.
Preparation of amides 3a, 3d, 3k-n via aminolysis of carboxylic acid 4a-f and amine 2a with
E. coli (SpL)
4.6 g freshly prepared wet cells of E.coli (SpL) was added to 25 mL nꢀhexane at a
loading of 185 mg cells/mL in a 100 mL round bottom flask. Amine at specific
concentration (90 mM 2a for 3d synthesis; 45 mM 2a for 3a and 3k-n synthesis) was
added, and the reaction mixture was stirring with a mixing propeller at 30 °C and 500 rpm
for 5 min. The corresponding carboxylic acid at a specific concentration (80 mM 4a for 3d
synthesis; 40 mM 4b-f for 3a and 3k-n synthesis) was added, and the reaction was
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performed by stirring with the mixing propeller at 30 °C and 500 rpm for 2 h (24 h for 3m
synthesis). After reaction, 25 mL chloroform was added for dissolving any possibly
insoluble amide, and anhydrous sodium sulfate was added to remove water. The cells were
removed by filtration. Crude product was acquired by removing the solvent via
evaporation.
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Column chromatography on silica gel was used to purify 3d
(
Rf=0.3;
n
ꢀhexane: ethyl
acetate=5:1) and 3m ꢀhexaneꢀethyl acetate, 10:1ꢀ5:1). For the amide 3a
(
n
,
3k 3l and 3n,
,
crude products were dissolved in 15 mL chloroform, washed 3 times with 1% Na2CO3
solution to remove the unreacted carboxylic acid, and then 3 times with 0.1 M HCl to
remove remaining amine. The organic phase was separated, dried with anhydrous sodium
sulfate and the solvent was removed by rotatory evaporator to give the amide products.
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53
3d: obtained as a white solid; yield: 94% H NMR (400 MHz, CDCl3, TMS) : δ
7.32ꢀ7.26 (m, 5H), 7.23ꢀ7.14 (m, 5H), 5.62 (s, br, 1H), 4.40 (d,
J = 5.7 Hz, 2H), 3.00 (t, J
13
= 7.6 Hz, 2H), 2.52 (t,
J
= 7.6 Hz, 2H). C NMR (100 MHz, CDCl3, TMS) 53: δ 171.8,
140.7, 138.1, 128.6, 128.5, 128.4, 127.7, 127.4, 126.2, 43.6, 38.5, 31.7. TOFMSꢀESI (m/z):
[M+H]+ and [M+Na]+ calculated for C16H17NO, 240.138, 262.121; Found 240.136,
262.120.
3k: obtained as a white solid; yield: 60%. 1HꢀNMR (400 MHz, CDCl3, 25 °C, TMS) 54: δ =
0.86ꢀ0.89 (m), 1.26ꢀ1.31 (m), 1.62ꢀ1.66 (m), 2.19ꢀ2.22 (m), 4.43ꢀ4.44 (m), 5.77 (m), 7.26ꢀ7.35
13
(m). CꢀNMR (400 MHz, CDCl3, 25 °C, TMS) 54: δ = 14.07, 22.60, 25.79, 29,01, 29.28, 31.69,
36.81, 43.58, 127.47, 127.81, 128.69, 138.45, 172.99. TOFMSꢀESI (m/z): [M+H]+ calculated
for C15H24NO 234.186; found 234.186.
1
3l: obtained as a white solid; yield: 55%. HꢀNMR (400 MHz, CDCl3, 25 °C, TMS) 55:
δ= 0.86ꢀ0.89 (m), 1.26ꢀ1.31 (m), 1.62ꢀ1.66 (m), 2.19ꢀ2.22 (m), 4.43ꢀ4.44 (m), 5.77 (m),
7.26ꢀ7.35 (m). 13CꢀNMR (400 MHz, CDCl3, 25 °C, TMS) 55: δ = 14.010, 22.66, 25.79,
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29.26, 29.32, 29.35, 29.45, 31.88, 36.82, 43.60, 127.49, 127.82, 128.70, 138.43, 172.99.
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TOFMSꢀESI (m/z): [M+H]+ calculated for C17H28NO 262.217; found 262.217.
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3m: obtained as a white solid; yield: 11%. HꢀNMR (400 MHz, CDCl3, 25 °C, TMS) 56:
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δ= 0.88 (m), 1.28 (m), 1.65 (m), 2.01 (m), 2.24ꢀ2.18 (t), 4.44 (d), 5.34 (m), 5.70 (s), 7.37 – 7.24
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(m). C NMR (100 MHz, CDCl3) 56
:
δ = 14.11, 22.68, 25.76, 27.18, 27.23, 29.14, 29.25, 29.29,
29.32, 29.33, 29.52, 29.70, 29.77, 31.91, 36.82, 43.63, 127.52, 127.85, 128.73, 129.75, 130.01,
138.41, 172.98. TOFMSꢀESI (m/z): [M+H]+ calculated for C25H42NO 372.326; found
372.326.
3n: obtained as a white solid; yield: 39%. HꢀNMR (400 MHz, CDCl3, 25 °C, TMS) 57:
1
δ=1.54ꢀ1.59 (m), 1.72ꢀ1.88 (m), 2.52ꢀ2.56 (m), 4.42ꢀ4.43 (d, JHz=4 MHz), 5.88 (m),
7.25ꢀ7.34 (m). 13CꢀNMR (400 MHz, CDCl3, 25 °C, TMS) 57: δ=25.93, 30.47, 43.58, 45.88,
127.42, 127.73, 128.68, 138.57, 176.17. TOFMSꢀESI (m/z): [M+H]+ calculated for
C13H18NO 204.139; found 204.138.
ASSOCIATED CONTENT
Supporting Information. The Supporting Information is available free of charge on the ACS
Publications website. Detailed experimental procedures, characterization of products, NMR
spectra and HPLC chromatograms (PDF).
AUTHOR INFORMATION
Corresponding Author
* Eꢀmail: chelz@nus.edu.sg. Phone: +65ꢀ6516 8416. Fax: +65ꢀ6779 1936.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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This research was supported by GlaxoSmithKline (GSK) and Singapore Economic
Development Board (EDB) through a Green and Sustainable Manufacturing grant (project No.
279ꢀ000ꢀ331ꢀ592).
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