Enzymatic Method for N-Acyl Homoserine Lactones Synthesis Using Immobilized Candida antarctica Lipase

Article abstract of DOI:10.1007/s10562-017-2261-8

Abstract: An enzymatic method to produce N-acyl homoserine lactones (AHLs) is described. This report represents the first example of the synthesis of bioactive AHLs using immobilized Candida antarctica lipase as the catalyst. The reaction yields, evaluate

Full text of DOI:10.1007/s10562-017-2261-8

Catalysis Letters
https://doi.org/10.1007/s10562-017-2261-8
Enzymatic Method for N-Acyl Homoserine Lactones Synthesis Using
Immobilized Candida antarctica Lipase
Juan Vázquez‑Martínez1 · Edgar Nieto‑Álvarez1 · Enrique Ramírez‑Chávez1 · Jorge Molina‑Torres1
Received: 26 July 2017 / Accepted: 24 September 2017
© Springer Science+Business Media, LLC, part of Springer Nature 2017
Abstract
An enzymatic method to produce N-acyl homoserine lactones (AHLs) is described. This report represents the first example
of the synthesis of bioactive AHLs using immobilized Candida antarctica lipase as the catalyst. The reaction yields, evalu-
ated by GC-EIMS, ranged from 6 to 99%.
Graphical Abstract
Keywords Enzymatic synthesis · Biocatalysis · Quorum sensing · Autoinducers · N-Acyl homoserine lactones
1 Introduction
bacteria sense their cell density through the synthesis,
detection, and response to AHLs and regulate target
genes. In turn, these genes activate or deactivate diverse
cellular processes such as bioluminescence [3], biofilm
development [4], and antibiotics production [5], among
others. In nature, N-acyl homoserine lactones are biosyn-
thesized by the condensation of an acyl chain, derived
from a fatty acid, and a homoserine lactone derived from
the S-adenosyl-l-methionine. This reaction is catalyzed
by an AHL synthase as LuxI. AHLs vary in the acyl chain
length, where an oxo or a hydroxyl substituent at beta
position may be present (Fig. 1). AHL structure may dif-
fer according to the bacterial strain and the cellular pro-
cess regulated by QS [6]. Many studies are focused on
the AHL-based QS due to its role in the stimulation of
cellular processes in certain Gram-negative bacteria [7].
For example, the opportunistic human pathogenic bac-
terium Pseudomonas aeruginosa produces biofilms and
pyocyanin (a virulence factor) in response to QS. This
bacterium has considerable medical importance given
that some strains are multidrug-resistant pathogens. A
target to combat such antimicrobial resistant bacteria is
the QS system since theory and laboratory data of the
infection model suggest that social conflict reduces the
N-Acyl homoserine lactones (AHLs) are a group of bioac-
tive acylated amides produced by Gram-negative bacteria.
These compounds, called autoinducers, act as chemical
signals of a microbial communication system denomi-
nated quorum sensing (QS) [1, 2]. Via QS, Gram-negative
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s10562-017-2261-8) contains
supplementary material, which is available to authorized users.
* Jorge Molina-Torres
jmolina@ira.cinvestav.mx
Juan Vázquez-Martínez
jvazquez@ira.cinvestav.mx
Edgar Nieto-Álvarez
edgarnietoal@hotmail.com
Enrique Ramírez-Chávez
eramirez@ira.cinvestav.mx
1
Departamento de Biotecnología y Bioquímica, Centro
de Investigación y de Estudios Avanzados del IPN
(CINVESTAV), Unidad Irapuato, Libramiento Norte Km
9.6, 36824 Irapuato., Guanajuato, Mexico
Vol.:(0123456789)
1 3

J. Vázquez-Martínez et al.
Fig. 2 N-Acyl homoserine lactones synthesis by an amidation reac-
tion between homoserine lactone and fatty acids catalyzed by CAL
Fig. 1 General structure of N-acyl homoserine lactones
2 Experimental
Immobilized lipase acrylic resin from C. antarctica
(CAL), N-homoserine lactone hydrobromide, standards
of N-acyl homoserine lactones, and the fatty acids were
purchased from Sigma-Aldrich. Other reagents were all of
analytical grade and purchased from Sigma-Aldrich. All
solvents were HPLC grade.
possibility for QS resistance to evolve [8, 9]. Under this
approach, and with the objective to study the AHL-based
QS, laboratory procedures for the synthesis of natural and
non-conventional AHLs have been developed [10, 11].
The currently employed AHLs production method
is typically done using chemical synthesis as follows:
l-homoserine lactone hydrochloride diluted in water is
added with trimethylamine, an appropriate fatty acid,
and with a carbodiimide type compound. This solution
is stirred at room temperature overnight and then the sol-
vent is evaporated. Next, the residue is dissolved in ethyl
acetate. The product is sequentially washed twice with
sodium bicarbonate solution, then with potassium hydro-
gen sulphate solution, and finally with brine. Then, ethyl
acetate is dried over anhydrous NaSO4 and solvent freed.
Finally, the crude product is purified [10].
Enzymatic synthesis procedure was as follows:
N-homoserine lactone hydrobromide (1 mmol) was acti-
vated with N,N-diisopropylethylamine (molar ratio 18:1),
in 3 mL of tert-amyl alcohol. The mixture was stirred at
70 °C for 30 min. Then, was added the respective fatty
acid (1 mmol), 50 mg of CAL, and 20 mg of anhydrous
sodium sulphate (Fig. 2). This system was allowed to react
for 24 h at 70 °C, and the reaction was monitored by GC/
EIMS taking aliquots of 50 µL at 0, 1, 2, 3, 4, 5, 6 and
24 h. This methodology was applied for the synthesis of
non-conventional signals differing only in the used amine
substrates: free isobutylamine and free phenylethylamine.
For the assays of stereospecificity, dl-homoserine lactone
or l-homoserine lactone were used as substrates. After the
reaction, CAL was separated by centrifugation. Then the
system was added with 3 mL of deionized water and 3 mL
of ethyl acetate. The ethyl acetate fraction was recovered
and the water fraction was extracted twice with 2 mL of
ethyl acetate. Ethyl acetate fractions were combined and
solvent freed by nitrogen flux. Crude product was puri-
fied using silica gel preparative plates (20 cm × 20 cm
× 0.5 mm). The mobile phase consisted on acetone/hex-
ane (1:1 v/v). Hydroxylated AHLs were purified directly
on TLC since the solvent partition procedure was not
achieved successfully.
Enzymatic catalysis bids great advantages for the syn-
thesis of bioactive compounds, such as stereo-, regio- and
chemo-selectivity, shorter synthetic routes, purer prod-
ucts, and less waste discharge [12, 13]. Lipases are a ver-
satile group of biocatalysts, which naturally catalyze the
hydrolysis of esters to the respective carboxylic acids and
alcohols. Nevertheless, the activity of these enzymes in
non-aqueous systems and anhydrous conditions is more
versatile: by adding alternative nucleophiles as alcohols,
amines, and thiols, the process gives as result a transes-
terification, amidation and thiotransesterification reac-
tion instead of hydrolysis, respectively [13, 14]. These
enzymes have been applied biotechnologically for a wide
range of amide transformations, such as amines acylation
with acids, and the amidation of benzyl esters [13, 15].
While it has been documented the use of immobilized
Candida antarctica lipase B for the synthesis of bioactive
amides, such as N-acylethanolamines [15], capsaicinoids
[16] and N-alkamides [14], there is no published data
about its use for the synthesis of AHLs signals and related
compounds. Thus, the method here-described represents
the first report of enzymatic synthesis of bioactive AHLs
by immobilized C. antarctica lipase.
The yield and the purity of each of the synthesized
products were determined by GC/EIMS using a GC 7890A
series and an EIMS 5975 series (Agilent Technologies,
Inc.). Yields were calculated on the basis of calibration
curves of C4-C12 homoserine lactone standards, a yield
of 100% was considerate assuming a theoretical conver-
sion of 100% for a 1 mM reaction. The GC/EIMS system
was equipped with a capillary column HP5-MS (30 m ×
250 µm × 0.25 µm) and helium was used as carrier gas at
constant flow rate of 1 mL min−1. One microliter in split
1 3

Enzymatic Method for N-Acyl Homoserine Lactones Synthesis Using Immobilized Candid…
injection mode was injected (25:1 split ratio) and injec-
tion chamber temperature was set to 250 °C. The GC-
oven initial temperature was set to 150 °C, held 3 min,
and then increased at 4 °C min−1 to a final temperature of
280 °C, held for 25 min. Transfer line temperature was set
at 250 °C. Ionization was done at 70 eV of electron energy.
The ion source and quadrupole temperatures were 230 °C
and 150 °C respectively, operating at 2.9 scans per second.
Data produced by the GC/EIMS system was collected with
the ChemStation Workstation software (Agilent Technolo-
gies, Inc.) The software Automated Mass Spectral Decon-
volution and Identification System “AMDIS” (http://www.
amdis.net/) was used to extract the mass spectrum for each
compound from the background. Compounds identifica-
tion was done by comparison of the mass spectra and
retention times of the synthesized compounds with the
respective standards mass spectra, and/or the NIST MS
search software version 2.0 (National Institute of Stand-
ards and Technology, USA).
ion for the hydroxylated AHLs mass spectra [11] (Table 1,
compounds 6–8, see Supplementary Data S1). The 143 m/z
ion is produced by a McLafferty rearrangement and corre-
sponds to [C6H9NO3 +H]+, and the 101 m/z ion corresponds
to the homoserine lactone ring [C4H6NO2 +H]+ [19]. The
172 m/z ion corresponds to [C7H10NO4]+.
3.2 Synthesis of Aliphatic AHLs
The synthesis product of each reaction was readily observed
during the first hour of reaction. Yields at 24 h ranged from
7 to 99%, being the best for the 6C-AHL and the C8-AHL
(> 90%). For the C4, C10 and C12 AHLs, yields did not
exceed the 15% (Table 1; Fig. 3). Fifty percent of the maxi-
mum yields, observed at 24 h, were reached around 2 h of
reaction for the C4, C6, C10 and C12 AHLs, and around
1 h for the C8-AHL. See Fig. 3. In contrast, the previously
described chemical synthesis method takes around 12 h to
obtain yields ranging from 40 to 60% [7, 9]. For the ali-
phatic C6 and C8 AHLs, the procedure herein described rep-
resents a better synthesis method, compared with previously
described methods, since higher yields were obtained, in a
lower reaction time, with less solvent waste and a shorter
synthetic route.
The QS activity of synthesized compounds was tested
using Chromobacterium violaceum mutant strain CV026.
This strain lacks the AHL synthase but expresses the AHL
receptors, then the presence of exogenous C4, C6 or C8
AHLs activates its QS system and produces a purple antibi-
otic knows as violacein [17]. The procedure was as follows:
a dose of the respective compound was applied on a 5 mm
paper disk and placed in a Petri dish containing solid LB
medium with fresh and preinduced C. violaceum CV026
cells at OD=0.1. The cultures were incubated at 28 °C for
24 h. The induction was evaluated by measuring the purple
halo around the respective disk. A concentration series of 0,
20, 40, 60, 80 and 100 µg disk−1 was tested for each com-
pound. This method is a simple and fast procedure to detect
the quorum sensing induction in C. violaceum CV026 [18],
and then the bioactivity of the C4, C6 and C8 synthesized
signals. Additionally, a gel overlay assay [18] was done to
compare, at the same time, the QS induction level of the
synthesized N-hexanoyl isobutylamide, N-hexanoyl pheny-
lethylamide and C6-AHL. This methodology is described in
detail in the Supporting Data S2.
3.3 Synthesis of Hydroxylated AHLs (OH‑AHLs)
Detection of considerable amounts of the respective hydrox-
ylated product takes longer time than for aliphatic AHLs.
At 120 h of reaction, 76.6, 56.7 and 5.6% yield was reached
for C8, C10 and C12 OH-ALHs, respectively. In a simi-
lar way than for the aliphatic C8-AHL, the best yield was
obtained using the C8 OH-fatty acid. The use of hydroxy-
lated fatty acids as substrate gives an arguable result since
the hydroxylated product was detected, but the reaction
takes 20 times longer than for the aliphatic AHLs synthesis,
using the method here described. In contrast, OH-AHLs can
be obtained in 42–65% yield between 3 and 12 h using the
previously described chemical synthesis method. However,
for the chemical synthesis of OH-AHLs, in addition to the
steps for aliphatic AHLs, it is necessary a process in which
an oxo-AHL precursor is required [11]. Thus, the method
herein described represents a simpler procedure, and for C8
and C10 OH-AHLs represents a better synthesis method.
3 Results and Discussion
3.1 Mass Spectra Analysis of Synthesized AHLs
3.4 Selectivity and Stereospecificity of Enzymatic
AHLs Synthesis by CAL
A total of ten compounds were synthesized using the
methodology here described and are listed in Table 1. The
molecular ion [M]+ of all of the synthesized compounds was
detected in the respective mass spectrum (Table 1, Supple-
mentary Data S1). The characteristic 143 and 101 m/z ions
for aliphatic AHLs mass spectra [18] (compounds 1–5) were
observed in the expected proportion, as well as the 172 m/z
It was observed, under the described conditions, that CAL
has a higher selectivity for C6 and C8 fatty acids (includ-
ing the OH-C8 fatty acid), according to the yields. Using
C6 and C8 fatty acids as precursors, aliphatic AHLs were
synthesized with 92 and 99% yield, respectively. Moreover,
1 3

J. Vázquez-Martínez et al.
Table 1 Yield and fragmentation pattern of N-acyl homoserine lactones synthesized using immobilized C. antarctica lipase
Compound
MW /
Yield
%
AHL
Fragmentation pattern obtained by EIMS m/z (%)
#
[M]+
171.20
/ 171
57(99.9), 143(75.9), 71(68.0), 56(32.6), 55(12.9),
85 (11.0), 10.1 (10.9), 83(10.4), 72(9.6), 84(7.7)
1
2
3
12.9
92.7
99.4
143(99.9), 57(71.9), 71(34.0), 99(26.5), 56(25.3),
55(21.8), 125(21.7), 101(18.6), 83(18.2),
102(14.5)
199.25
/ 199
O
O
143(99.9), 57(95.5), 55(25.7), 125(21.4),
83(18.6), 56(18.2), 101(17.0), 156(15.2),
102(15.0), 127(9.1)
227.30
/ 227
N
H
H
3
C
3
O
143(99.9), 57(50.4), 55(21.6), 125(19.1),
156(17.3), 102(17.1), 83(16.8), 101(14.8),
56(14.1), 71(12.7)
255.35
/ 255
4
5
6
7
8
9
6.8
15
143(99.9), 57(42.5), 55(19.8), 156(19.1),
102(17.6), 125(16.5), 83(15.8), 101(12.3),
56(11.1), 98(7.5)
283.41
/ 283
102(99.9), 57(96.1), 172(67.5), 55(57.0),
56(43.4), 83(33.4), 143(30.5), 125(22.3),
74(20.3), 144(19.8)
243.30
/ 243
76.6
57.7
5.6
---
102(99.9), 57(93.1), 172(81.1), 143(36.0),
55(33.6), 56(33.6), 69(33.3), 74(17.5), 144(17.1),
83(14.8)
271.35
/ 271
102(99.9), 172(93.5), 57(92.7), 55(43.8),
143(38.5), 56(32.0), 83(25.9), 69(19.1),
144(18.2), 74(17.1)
299.41
/ 299
10(99.9), 99(44.2), 105(27.0), 91(26.5), 71(24.8),
128(13.4), 219(13.2), 65(12.7), 163(12.7),
77(17.1)
219.32
/ 219
115(99.9), 99(83.9), 116(61.9), 60(52.7),
71(45.4), 57(37.4), 58(33.7), 72(28.1), 128(28.1),
55(25.5)
171.27
/ 171
10
---
CAL is less efficient using hydroxylated fatty acids, but for
C8 and C10 OH-AHLs this enzymatic method is better
than the chemical synthesis method previously described.
Using the enzymatic method here described, transesterifi-
cation products were not observed in any reaction where
hydroxyl group was present in the acyl chain.
In order to determine the retention time and mass
spectrum of the C8-AHL d and l isomers, commercial
standards (Sigma-Aldrich) of N-octanoyl-dl-homoser-
ine lactone and N-octanoyl-l-homoserine lactone were
analyzed by GC/EIMS. In the analysis of the standard
N-octanoyl-dl-homoserine lactone two peaks, Rt= 17.703
1 3

Enzymatic Method for N-Acyl Homoserine Lactones Synthesis Using Immobilized Candid…
in the d-isomer mass spectrum than in the l-isomer mass
spectrum (Fig. 4).
So as to test the stereospecificity of CAL for the l- or
d-isomer, dl-homoserine lactone or l-homoserine lactone
was used as the substrates for N-octanoyl homoserine lac-
tone synthesis. When dl-homoserine lactone was used only
one peak is observed with Rt=17.7 min, which corresponds
with the commercial N-octanoyl-l-homoserine lactone. In
the same way, when l-homoserine lactone was used as the
substrate, only one peak is observed at the same Rt (Fig. 4).
The C8-AHL d-isomer was not detected under the described
conditions. Thus, it was concluded that CAL is stereospe-
cific for the l-isomer. This specificity allows the synthesis of
the bioactive l form [20, 21] in a selective way.
3.5 Quorum Sensing Induction Assay
Fig. 3 Time course of N-acyl homoserine lactones production (yield
%) catalyzed by CAL. Lines correspond to the production kinetics for
aliphatic AHLs, according to acyl chain carbon number
The bioactivity of the synthesized aliphatic C4, C6 and C8
AHLs was assayed as QS inducers in Chromobacterium
violaceum CV026. The production of violacein by C. vio-
laceum was induced by C6 and C8 AHLs at 100%, com-
pared with the induction by their respective commercial
standard. Induction of C4-AHL was 70% compared with
commercial C6-AHL, amount of 5 µg. N-hexanoyl-l-ho-
moserine lactone was used for this comparison due to the
lack of C4-AHL standard. Additionally, two non-conven-
tional QS signals were synthesized using the method here
described: the N-hexanoyl isobutylamide and the N-hexanoyl
and 19.737 min, were observed. While in the analysis
of the standard N-octanoyl-l-homoserine lactone only
one peak, Rt = 17.7 min, was observed (Fig. 4). The Rt
for the d-isomer was 19.74 min. In addition, from the
analysis of the mass spectra of the d and l N-octanoyl
homoserine lactone isomers, a characteristic ion of 74 m/z
[C3H4O2 + 2H]+ for the d-isomer mass spectrum was
observed. This m/z ion is present at a higher proportion
Fig. 4 a Total ion chromatogram (TIC) of commercial standard
N-octanoyl-dl-homoserine lactone (blue line) showing the two peaks
of N-octanoyl-l-homoserine lactone and N-octanoyl-d-homoserine
lactone at Rt=17.707 and Rt=19.737 min, respectively. TICs of ste-
reo-specifically CAL-synthesized N-octanoyl-l-homoserine lactone
using dl-homoserine lactone as substrate (red line) and CAL-synthe-
sized N-octanoyl-l-homoserine lactone using l-homoserine lactone
as substrate (green line) showing only the peak of the N-octanoyl-
l-homoserine lactone at Rt=17.707 min. b N-octanoyl-l-homoserine
lactone (red spectrum) and N-octanoyl-d-homoserine lactone (blue
spectrum) EI spectra comparison, black arrow indicates a characteris-
tic ion at 74 m/z for the d-isomer
1 3

J. Vázquez-Martínez et al.
Acknowledgements Authors thank CONACyT for funding this
research and for the scholarship granted to Juan Vázquez-Martínez. We
woul like to thank Carolyn Smith, Peace Corps volunteer for proof
reading the manuscript.
Compliance with Ethical Standards
Conflict of interest The authors declare that they have no conflict of
interest.
References
Fig. 5 Petri dish induction assay of violacein production by CAL-
synthesized N-hexanoyl phenylethylamide (A) and the N-hexanoyl
isobutylamide (B) in C. violaceum CV026. Concentration increment
clockwise from (1) as 0, 20, 40, 60, 80 and 100 µg per paper disk
1. Parsek M, Val D, Hanzelka B et al (1999) Proc Natl Acad Sci USA
96:4360
2. Fuqua C, Greenberg EP (2002) Nat Rev Mol Cell Biol 3:685
3. Engebrecht J, Nealson K, Silverman M (1983) Cell 32:773
4. Dickschat JS (2010) Nat Prod Rep 27:343
5. Thomson NR, Crow MA, McGowan SJ et al (2000) Mol Microbiol
36:539
phenylethylamide (Table 1). These analogues showed a posi-
tive effect on the induction of violacein production (Fig. 5),
being more effective the N-hexanoyl isobutylamide since this
compound induces the same response that the N-hexanoyl
phenylethylamide at concentration three times lower. N-hex-
anoyl isobutylamide induces violacein production from
10 µg, whereas N-hexanoyl phenylethylamide induces from
40 µg. The induction by 10 µg of C6-AHL is aproxymately
equivalent to double of 80 µg of N-hexanoyl isobutylamide,
and much higher than 80 µg of N-hexanoyl phenylethylamide
(Supplementary Fig. S2.1). These results demonstrate the
potential of this method for the simple synthesis of a wide
range of AHL type QS compounds.
6. Whitehead NA, Barnard AML, Slater H et al (2001) FEMS Micro-
biol Rev 25:365
7. Defoirdt T, Brackman G, Coenye T (2013) Trends Microbiol
21:619
8. Kievit T, De Gillis R, Marx S et al (2001) Appl Environ Microbiol
67:1865
9. Sepahi E, Tarighi S, Ahmadi FS, Bagheri A (2015) J Microbiol
53:176
10. Càmara M, Daykin M, Chhabra SR (1998) In: Williams P (ed)
Methods microbiology: bacterial pathogenesis, vol 27. Academic
Press, London
11. Ruysbergh E, Stevens CV, De Kimpe N, Mangelinckx S (2016)
RSC Adv 6:73717
12. Wang R, Hou M, Zhang Y et al (2015) Catal Lett 145:995
13. Gotor-Fernández V, Busto E, Gotor V (2006) Adv Synth Catal
348:797
14. Molina-Torres J, Santos-Escobar F, González-Arnao MT et al
(2008) Lat Am J Pharm 27:852
4 Conclusions
15. Whitten KM, Makriyannis A, Vadivel SK (2012) Tetrahedron Lett
53:5753
Bioactive N-acyl-l-homoserine lactones were synthesized
using immobilized Candida antarctica lipase as the catalyst
and free fatty acids and homoserine lactone as precursors.
This method provides a fast and selective alternative for the
synthesis of AHLs, dependent on the fatty acid chain length.
The method here described is stereospecific selective for the
l-isomers when dl-homoserine lactone is used as substrate.
Additionally, the synthesized C4, C6 and C8 AHLs induced
the QS system in Chromobacterium violaceum CV026. Fur-
ther, this method has a general applicability for the synthesis
of aliphatic and hydroxylated AHLs and unconventional QS
signals.
16. Kobata K, Kawamura M, Toyoshima M et al (1998) Biotechnol
Lett 20:451
17. McClean K, Winson M, Fish L (1997) Microbiology 143:3703
18. Weihua C, Dhiraj AV, Vatsala M, et al, In: Rumbaugh KP (ed)
Quorum sensing methods and protocols (2011) Humana Press,
Hatfield
19. Cataldi TRI, Bianco G, Frommberger M, Schmitt-Kopplin P
(2004) Rapid Commun Mass Spectrom 18:1341
20. Kuo A, Callahan SM, Dunlap PV (1996) J Bacteriol 178:971
21. Ikeda T, Kajiyama K, Kita T et al (2001) Chem Lett 4:314
1 3