LipG9-mediated enzymatic kinetic resolution of racemates: Expanding the substrate-scope for a metagenomic lipase

Article abstract of DOI:10.1016/j.mcat.2019.110402

Enzymes are the main biocatalysts of biological systems and nowadays they play an important role in asymmetric organic synthesis. Microorganisms are the main source for enzymes, however, just a very small portion of them are culturable at lab conditions and, as an alternative, metagenomics approaches allow new enzymes to be accessed from so-called “non-culturable” microorganisms. Several classes of metagenomic enzymes have been described in literature. Nevertheless, studies about their potential for asymmetric biotransformation are underexploited. Therefore, we present our recent efforts to establish the substrate-scope of LipG9, a metagenomic lipase, in enzymatic kinetic resolution (EKR) of chiral substances. LipG9 was previously isolated, immobilized and successfully applied in EKR of aliphatic alcohols. In this study, a series of resolvable chiral substances were assayed with LipG9, and secondary benzyl alcohols/esters were preferentially resolved in a much superior enantioselectivity (E > 200) than those described for aliphatic alcohols (E from 4 to 63). In an opposite way, Im-LipG9 did not present activity for tertiary alcohols, amines and lactones. When compared to commercial lipases, Im-LipG9 enantioselectivity was superior to Candida rugosa lipase and equivalent to Candida antarctica lipase B. Thus, the chemo and enantioselectivity of LipG9 in EKR reactions were identified and its potential for asymmetric synthetic approaches was demonstrated.

Full text of DOI:10.1016/j.mcat.2019.110402

MolecularCatalysis473(2019)110402
Contents lists available at ScienceDirect
Molecular Catalysis
journal homepage: www.elsevier.com/locate/mcat
LipG9-mediated enzymatic kinetic resolution of racemates: Expanding the
substrate-scope for a metagenomic lipase
Juliana Christina Thomasa, Robson Carlos Alnochb, Allen Carolina dos Santos Costaa,
Pamela Taisline Bandeiraa, Martha Daniela Buricha, Suelem Kluconski Camposa,
Alfredo Ricardo Marques de Oliveiraa, Emanuel Maltempi de Souzab, Fabio de Oliveira Pedrosab,
Nadia Kriegera,b, Leandro Piovana,⁎
a Universidade Federal do Paraná, Departamento de Química, Centro Politécnico, Curitiba 81.531-980, Brazil
b Universidade Federal do Paraná, Departamento de Bioquímica e Biologia Molecular, Centro Politécnico, Curitiba 81.531-980, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Lipase
Metagenomic
Kinetic resolution
Enantioselectivity
Alcohols
Enzymes are the main biocatalysts of biological systems and nowadays they play an important role in asym-
metric organic synthesis. Microorganisms are the main source for enzymes, however, just a very small portion of
them are culturable at lab conditions and, as an alternative, metagenomics approaches allow new enzymes to be
accessed from so-called “non-culturable” microorganisms. Several classes of metagenomic enzymes have been
described in literature. Nevertheless, studies about their potential for asymmetric biotransformation are un-
derexploited. Therefore, we present our recent efforts to establish the substrate-scope of LipG9, a metagenomic
lipase, in enzymatic kinetic resolution (EKR) of chiral substances. LipG9 was previously isolated, immobilized
and successfully applied in EKR of aliphatic alcohols. In this study, a series of resolvable chiral substances were
assayed with LipG9, and secondary benzyl alcohols/esters were preferentially resolved in a much superior en-
antioselectivity (E > 200) than those described for aliphatic alcohols (E from 4 to 63). In an opposite way, Im-
LipG9 did not present activity for tertiary alcohols, amines and lactones. When compared to commercial lipases,
Im-LipG9 enantioselectivity was superior to Candida rugosa lipase and equivalent to Candida antarctica lipase B.
Thus, the chemo and enantioselectivity of LipG9 in EKR reactions were identified and its potential for asym-
metric synthetic approaches was demonstrated.
Introduction
substantially. Hence, different approaches to obtain new lipases or
modifying current lipases have been proposed, highlighting molecular
Lipases (glycerol ester hydrolases EC 3.1.1.3) are prominent bio-
catalysts, specifically carboxylesterases, that act upon carboxylic esters
promoting their hydrolysis in aqueous media or synthesis in water-re-
stricted media [1,2]. Over the last few years, the use of lipases in the
synthesis of biologically active compounds has increased substantially,
being an excellent approach to produce chiral drugs. The major appli-
cation of lipases in organic synthesis is through enzymatic kinetic re-
solution (EKR) of racemates, such as acids, esters, amines and alcohols
[3–5].
biology techniques, such as metagenomics [7]. The construction of
metagenomic libraries from natural or polluted environments is parti-
cularly promising, since it enables lipases to be obtained from so-called
“non-culturable” microorganisms. Lipases and esterases have been ob-
tained and characterized from metagenomic libraries from different
environments [8–10]. However, despite the rising in the discovery of
new lipases, there are fewer studies involving the application of these
enzymes in organic media, including EKR reactions [11–21], when
compared to commercial enzymes.
Due to the high applicability of lipases in several areas, as a result of
its ability to catalyze reactions in organic or water restricted media with
a wide variety of unnatural substrates, while allowing to obtain a di-
verse range of organic compounds with high level of regio-, chemo- and
stereoselectivity [2,6], the search for new enzymes has increased
In our previous works, a new lipase, LipG9, was identified in a
metagenomic library constructed from soil samples contaminated with
animal fat [20]. LipG9 was overexpressed and studies were performed
with free and immobilized enzyme [19,22]. The immobilized form, Im-
LipG9 showed activity and stability in polar and non-polar organic
⁎ Corresponding author.
E-mail address: lpiovan@quimica.ufpr.br (L. Piovan).
https://doi.org/10.1016/j.mcat.2019.110402
Received 10 January 2019; Received in revised form 8 May 2019; Accepted 10 May 2019
2468-8231/©2019ElsevierB.V.Allrightsreserved.

J.C. Thomas, et al.
MolecularCatalysis473(2019)110402
(Table 1, entries 5 and 8). On the other hand, CALB-mediated EKR of 1
presented 50% conversion, ee > 99% for (S)-1 and (R)-1a and E > 200
in only 2 h (Table 1 – entry 3). For 1-phenylethanol congeners, the
presence of electron donor or withdrawing groups at para-position of
aromatic ring implied mainly in a significant increase of the reaction
time. CALB-mediated EKR of alcohols 2 and 3 were performed in 24 h
and 36 h, respectively, with maintenance of excellent conversion rates
and enantioselectivity parameters (Table 1 – entries 6 and 9). Re-
garding stereoselectivity and conversion rates, Im-LipG9 exhibited su-
perior behavior in comparison with CRL and performed similarly to
CALB, a lipase already known as a good biocatalyst for substrates 1-3
[24].
After that, Im-LipG9, CRL and CALB were evaluated in EKR of more
sterically hindered benzylic alcohols (4 and 5). For Im-LipG9-mediated
EKR, high stereosselectivity was observed (E > 200) with excellent
ee > 99% for both esters (R)-4a and (R)-5a (Table 1 – entries 10 and
13). However, the main implication of changing methyl to ethyl and
butyl moieties at R2 position was the decrease on conversion rates from
49% for 1 (Table 1 – entry 1) to 30 and 10% for alcohols 4 and 5,
respectively (Table 1 – entries 10 and 13). The same was observed in
CRL-mediated reaction and low values of ee and E were observed
(Table 1 – entries 11 and 14). CALB-mediated resolution of 4 and 5 also
required longer reaction times. However, ee and E remained high for
alcohol 4, which was resolved in 36 h (Table 1 – entry 12). For com-
pound 5, since the reaction was stopped after 216 h, conversion reached
43% and, due to low conversion, (S)-5 was obtained with 75% ee
(Table 1 – entry 15).
Due to exciting results observed for EKR of alcohols 1-4, Im-LipG9
was also assayed for hydrolysis reaction employing esters 1a-4a as
substrates (Table 2). Once again, Im-LipG9 presented high enantiomeric
ratio (E > 200) in hydrolytic EKR of esters 1a-4a. Consequently, the
alcohol products (S)-1-4 were obtained with high enantiomeric excess
(ee > 98%; Table 2 – entries 1, 4, 7 and 10). However, unsatisfactory
conversion rates and reaction times were observed for reactions in-
volving compounds 1a (26%, 120 h), 3a (33%, 36 h) and 4a (10%,
192 h), and no conversion was observed for ester 5a (Table 2 – entry
13). These results indicate that Im-LipG9-mediated acylation reactions
were more efficient than hydrolytic reactions.
Fig. 1. Substrate scope for lipase-mediated enzymatic kinetic resolution eva-
luation.
solvents [19]. Additionally, a preliminary assay demonstrated that Im-
LipG9 showed excellent enantioselectivity (E > 200) in transester-
ification and hydrolysis of model compound 1-phenylethanol and its
corresponding ester, respectively [19]. Im-LipG9 was also evaluated in
the EKR of aliphatic sec-alcohols, which were resolved via transester-
ification reactions with satisfactory conversions (19–59%) and en-
antiomeric excesses for alcohols (26–88%) and esters (30–96%) [23].
Due to promising results in Im-LipG9-mediated EKR of aliphatic sec-
alcohols, a substrate scope for this biocatalyst is studied in this work.
For that purpose, a series of chiral secondary benzylic alcohols (1-5),
tertiary benzylic alcohols (6-7), secondary benzylic amines (8-10) and
lactones (11-12 - Fig. 1) were evaluated as substrates in EKR reactions
mediated by Im-LipG9.
Results and discussion
Regarding commercial lipases, CRL-mediated EKR of esters 1a-4a
revealed low E (2–10) and ee values for (S)-esters (6–38%) and (R)-
alcohols (15–53%) (Table 2 – entries 2, 5, 8 and 11). Ester 5a was not
hydrolyzed by CRL (Table 2 – entry 14). Both acylation and hydrolysis
reactions mediated by CRL presented discouraging results, which
clearly demonstrate the superior performance of Im-LipG9 over this
commercial lipase.
Compounds 1-12 and their derivatives were assayed in enzymatic
kinetic resolution employing Im-LipG9 as biocatalyst. For comparative
purposes, commercially available immobilized lipases Candida rugosa
(CRL) and Candida antarctica fraction B (CALB; Novozym 435®) were
also evaluated.
Enzymatic assays were initiated by transesterification reaction of
alcohols 1-5 with vinyl acetate in hexane at 35 °C (Table 1). Im-LipG9-
mediated reactions presented exciting performance in EKR of pheny-
lethanol and derivatives. High values of enantiomeric excess, 95% for
both (S)-1 and (R)-1a, and enantiomeric ratio (E > 200) in addition to
49% of conversion were observed after 30 h (Table 1 – entry 1). On the
other hand, EKR of 4-nitro-phenylethanol (2) took 36 h to reach only
12% conversion. Despite the low conversion, the observed value of E
(> 200) indicated the maintenance of high stereoselectivity of Im-
LipG9, since ester (R)-2a was obtained with ee > 99% (Table 1 – entry
4). For Im-LipG9-mediated resolution of 4-metoxi-phenylethanol (3),
the presence of MeO- moiety did not influence the reaction as much as
NO2 group. The reaction reached 47% conversion after 24 h, with en-
antiomeric excess of 90 and 99% for (S)-3 and (R)-3a which implied in
E > 200 (Table 1 – entry 7). These results confirmed the high degree of
stereoselectivity of Im-LipG9 towards benzylic alcohols.
As expected, CALB maintained the high performance observed in
transesterification reactions. In EKR of esters 1a-3a, all species (esters
(S)-1a-3a and alcohols (R)-1-3) were obtained with ee > 99%.
Conversion rates were 50% for all compounds and reactions took an
amount of time comparable to acylation reactions (Table 2). A con-
siderable decrease in CALB performance was observed in EKR of ester
4a since a drastic increase on reaction time was observed (36 h for
acylation of 4 versus 216 h for hydrolysis of 4a) with 43% conversion,
which implied in ee = 75% to (S)-1a. CALB was also not able to hy-
drolyze ester 5a. In summary, regarding selectivity and conversion
rates, Im-LipG9 was superior to CRL in most of hydrolitic reactions,
while presenting comparable results to CALB, a well known commercial
biocatalyst. None of the tested biocatalysts were able to hydrolize the
bulkiest substrate 5a.
Other substrates
As before mentioned, Im-LipG9 performance was compared to
commercially available lipases, starting with CRL-mediated EKR of 1,
resulting in long reaction time (60 h), poor values of ee (45% for 1 and
58% for 1a) and a value of E = 6 (Table 1 – entry 2). Low values of ee
and E were again observed for CRL-mediated resolution of 2 and 3
In order to explore the substrate scope for Im-LipG9, this biocatalyst
was also evaluated in EKR reactions of tertiary alcohols (6-7), sec-
ondary benzylic amines with electron donor and electron withdrawing
groups in the aromatic ring (8-10) and aliphatic lactones (11-12;
2

J.C. Thomas, et al.
MolecularCatalysis473(2019)110402
Table 1
Lipase-mediated enzymatic kinetic resolution of alcohols 1-5 via transesterification reactions.
Enantiomeric excess (%)b)
Entry
Substrate
Lipase
time/h
c (%)a)
(S)-1-5
(R)-1a-5a
Ec)
1
2
3
Im-LipG9
CRL
CALB
30
60
2
49
43
50
95
45
> 99
95
58
> 99
> 200
6
> 200
4
5
6
Im-LipG9
CRL
CALB
36
24
24
12
51
50
13
2
> 99
> 99
32
> 99
> 200
2
> 200
7
8
9
Im-LipG9
CRL
CALB
24
48
36
47
27
50
90
9
> 99
98
23
> 99
> 200
2
> 200
10
11
12
Im-LipG9
CRL
CALB
156
48
36
30
15
50
44
57
> 99
> 99
9
> 99
> 200
4
> 200
13
14
15
Im-LipG9
CRL
CALB
192
216
216
10
13
43
10
78
75
> 99
12
> 99
> 200
9
> 200
a)
Conversion: c = 100 x (eeS/eeS + eeP).
Enantiomeric excess: (R – S) / (R + S) x 100.
Enantiomeric ratio: E = ln {[eeP (1 – eeS)] / (eeP + eeS)} / ln {[eeP (1 + eeS)] / (eeP + eeS)}. eeS = enantiomeric excess of substrate; eeP enantiomeric excess of
b)
c)
product; R = peak area in the chromatogram for R enantiomer; S = peak area in the chromatogram for S enantiomer.
Fig. 2). Unfortunately, no reaction was observed for any of these sub-
strates. Regarding tertiary alcohols, their corresponding esters were
also evaluated in Im-LipG9-mediated hydrolysis reaction, but no pro-
ducts were observed. This absence of reactivity shows that Im-LipG9
has high selectivity between secondary and tertiary alcohols, as well as
it is not active for secondary benzylic amines and lactones, which could
be useful in reactions involving more functionalized compounds.
Table 2
Lipase-mediated enzymatic kinetic resolution of alcohols 1–5 via hydrolysis reactions.
Enantiomeric excess (%)b)
Entry
Substrate
Lipase
time/h
c (%)a)
(S)-1a-5a
(R)-1-5
Ec)
1
2
3
Im-LipG9
CRL
CALB
120
60
2
26
47
50
34
26
> 99
> 99
29
> 99
> 200
3
> 200
4
5
6
Im-LipG9
CRL
CALB
24
24
24
48
60
50
90
38
> 99
> 99
25
> 99
> 200
2
> 200
7
8
9
Im-LipG9
CRL
CALB
36
36
36
33
30
50
93
6
> 99
98
15
> 99
7
2
> 200
10
11
12
Im-LipG9
CRL
CALB
192
216
216
10
32
43
10
26
75
> 99
53
> 99
> 200
10
> 200
13
14
15
Im-LipG9
CRL
CALB
216
216
216
–
–
–
–
–
–
–
–
–
–
–
–
a)
Conversion: c = 100 x (eeS/eeS + eeP).
Enantiomeric excess: (R – S) / (R + S) x 100.
Enantiomeric ratio: E = ln {[eeP (1 – eeS)] / (eeP + eeS)} / ln {[eeP (1 + eeS)] / (eeP + eeS)}. eeS = enantiomeric excess of substrate; eeP enantiomeric excess of
b)
c)
product; R = peak area in the chromatogram for R enantiomer; S = peak area in the chromatogram for S enantiomer.
3

J.C. Thomas, et al.
MolecularCatalysis473(2019)110402
temperatures were set at 220 °C. Temperature programming for the
chiral analyses of alcohols and esters is reported below.
Compounds 1 and 1a: 110 °C (1 min) to 120 °C – gradient rate:
1 °C min−1; Retention times (min): (R)-1: 4.29; (S)-1: 4.57; (R)-1a:
3.81; (S)-1a: 3.57.
Compounds 2 and 2a: 160 °C–172 °C - gradient rate: 1 °C min−1
;
Retention times (min): (R)-2: 8.10; (S)-2: 8.57; (R)-2a: 5.61; (S)-2a:
5.43.
Compounds 3a and 3b: 110 °C–138 °C - gradient rate: 1 °C min−1
;
Retention times (min): (R)-3a: 6.84; (S)-3a: 7.11; (R)-3b: 25.13; (S)-3b:
24.74
Compounds 4a and 4b: 60 °C–110 °C – gradient rate: 2 °C min−1
;
Retention times (min): (R)-4a: 19.28; (S)-4a: 18.55; (R)-4b: 30.44; (S)-
4b: 30.10.
Chiral HPLC analyses were performed on a Shimadzu model LC-6AD
chromatograph (UV–vis 215 and 267 nm) with Phenomenex-Lux
column (250 mm x 4.6 mm. Hexanes and isopropanol (99:1) were em-
ployed as mobile phase.
Compounds 5 and 5a: Retention times (min): (R)-5: 34.90; (S)-5:
37.43; (R)-5a: 6.31; (S)-5a: 6.94.
Strains E. coli TOP10 (Invitrogen, Carlsbad, CA, USA) and BL21
(DE3) (Novagen, Madison, MI, USA), vectors pET-28a (+), (Novagen,
Madison, MI, USA) and IPTG (iso- propyl β-D thiogalactopyranoside)
(Invitrogen Life Technologies, CA, USA) were used as the recombinant
protein expression system. Overexpression and purification of LipG9
were performed according to Martini et al. [21]. LipG9 was im-
mobilized by physical adsorption on polypropylene Accurel MP 1000,
according to Alnoch et al. [19].
Fig. 2. Poor substrates for Im-LipG9-mediated enzymatic kinetic resolution.
Conclusion
In conclusion, it was possible to show the enormous application
potential of Im-LipG9, a metagenomic lipase, in organic synthesis. This
unmodified enzyme presented high stereoselectivity for kinetic resolu-
tion of benzylic alcohols and esters but did not present activities for
tertiary alcohols, amines and lactones. When compared to commercial
lipases, Im-LipG9 performance was superior than Candida rugosa lipase
and slightly inferior than Candida antarctica lipase. Thus, we were able
to identify the chemo and enantioselectivity of Im-LipG9 in EKR reac-
tions and demonstrated its potential for asymmetric synthetic ap-
proaches. Moreover, further effort can be made to increase Im-LipG9
activity for improvement of its performance in order to support Im-
LipG9 as a valuable biocatalyst in the synthesis of optically active
compounds.
Characterization of substrates 1-5 and 1a-5a
Phenylethan-1-ol (1): GC–MS (70 eV), m/z (relative intensity): 122
(M•+, 34%), 107 (90%), 79 (100%), 77(54%), 51 (21%), 43 (24%). 1H
NMR (200 MHz, CDCl3, TMS), δ (ppm): 1.42 (d, J =6.5 Hz, 3 H), 4.79
(q, J =6.5 Hz, 1 H), 7.20–7.32 (m, 5 H). 13C NMR (50 MHz, CDCl3), δ
(ppm): 25.1, 70.3, 125.4, 127.4, 128.5, 145.9. IR (cm−1): 3352, 2973,
1449, 1074, 698.
(4-methoxyphenyl)ethan-1-ol (2): GC–MS (70 eV), m/z (relative in-
tensity): 152 (M•+, 30%), 137 (100%), 109 (64%), 94 (35%), 77(32%),
43 (21%). 1H NMR (200 MHz, CDCl3, TMS), δ (ppm): 1.45 (d, J
=6.5 Hz, 3 H), 3.79 (s, 3 H), 4.82 (q, J =6.5 Hz, 1 H), 6.86 (d, J
=8.6 Hz, 2 H), 7.28 (d, J =8.7 Hz, 2 H). 13C NMR (50 MHz, CDCl3), δ
(ppm): 25.0, 55.3, 69.9, 113.8, 126.7, 138.0, 158.9. IR (cm−1): 3377,
2968, 2056, 1889, 1611, 1458, 1244, 831.
Experimental section
General information
1H NMR spectra were recorded on a Bruker DPX200 MHz spectro-
meter. 13C NMR spectra were recorded on the same instrument at
50 MHz. The chemical shifts of 1H and 13C NMR were quoted relative to
internal tetramethylsilane (δTMS = 0.00) or CDCl3 (δC = 77.0). 1H NMR
data are reported as follows: chemical shift in ppm (δ), multiplicity
(s = singlet, d = doublet, t = triplet, q = quartet, sext = sextet, m =
multiplet), coupling constant (Hz) and relative intensity (integral). 13C
NMR data are reported as chemical shift in ppm (δ).
GC–MS spectra were recorded on Shimadzu model QP-5050A cro-
matograph (MS), equipped with a DB-5 capilary column (30 m ×
0.25 mm diameter, 0.25 μm film thickness) and helium as carrier gas
(1 ml min−1 constant flow). The injector temperature was set to 250 °C
for all alcohols. For alcohols 1-4 the temperature programming was
50 °C for 1 min, increasing in 7 °C min-1 up to 250 °C and kept at 250 °C
for 10 min.
Infrared spectra were recorded from KBr discs on a Bomem MB100
spectrometer coupled with FTIR. Maximum absorption (νmax) is re-
ported in wavenumber (cm−1).
Chiral GC analyses were performed on a Shimadzu model GC-17A
chromatograph (FID) equipped with CP Chirasil-DEX CB column
(25 m × 0.25 mm diameter, 0.25 μm film thickness). The split ratio was
1:50 and N2 was used as carrier gas. The injector and detector
(4-nitrophenyl)ethan-1-ol (3): GC–MS (70 eV), m/z (relative in-
tensity): 166 (M•+, 1%), 152 (100%), 122 (25%), 107(75%), 94 (41%),
77 (89%), 51 (30%), 43 (58%). 1H NMR (200 MHz, CDCl3, TMS), δ
(ppm): 1.50 (d, J =6.5 Hz, 3 H), 5.00 (q, J =6.5 Hz, 1 H), 7.52 (d, J
=8.6 Hz, 2 H), 8.15 (d, J =8.6 Hz, 2 H). 13C NMR (50 MHz, CDCl3), δ
(ppm): 25.3, 22.2, 71.2, 123.8, 126.7, 147.4, 148.9, 170.0. IR (cm−1):
3376, 1932, 1803, 1680, 1516, 1343, 1108, 855.
Phenylpropan-1-ol (4): GC–MS (70 eV), m/z (relative intensity): 136
(M•+,17%), 107 (100%), 91 (5%), 79 (79%), 77 (35%), 51 (10%). 1H
NMR (200 MHz, CDCl3, TMS), δ (ppm): 0.88 (t, J =7.4 Hz, 3 H),
1.66–1.86 (m, 2 H), 4.53 (t, J =6.6 Hz, 1 H), 7.26–7.36 (m, 5 H). 13C
NMR (50 MHz, CDCl3), δ (ppm): 10.1, 31.9, 76.0, 126.0, 127.4, 128.4,
144.6. IR (cm−1): 3330, 3029, 2873, 1496, 1454, 1210, 1014, 735,
697.
Phenylpentan-1-ol (5): GC–MS (70 eV), m/z (relative intensity): 164
(M•+, 5%); 120 (12%); 107 (100%); 104 (33%); 79 (61%); 77 (42%);
51(10%). 1H NMR (200 MHz, CDCl3, TMS), δ (ppm): 0.87 (t, J =6.8 Hz,
3 H); 1.12–1.38 (m, 4 H); 1.61–1.80 (m, 2 H); 4.61 (dd; J = 7.3; 6.1 Hz,
1 H); 7.24–7.32 (m, 5 H). 13C NMR (50 MHz, CDCl3), δ (ppm): 14.0;
22.6; 28.0; 38.8; 74.6; 125.9; 127.4; 128.4; 145.0. IR (cm−1): 3346,
2934, 1877, 1494, 1463, 1194.
1-Phenylethyl acetate (1a): GC–MS (70 eV), m/z (relative intensity):
4

J.C. Thomas, et al.
MolecularCatalysis473(2019)110402
164 (M•+, 23%), 122 (100%), 105 (67%), 104 (89%), 107 (36%), 51
(15%), 43 (55%). 1H NMR (200 MHz, CDCl3, TMS), δ (ppm): 1.53 (d, J
=6.6 Hz, 3 H), 2.06 (s, 3 H), 5.88 (q, J =6.6 Hz, 1 H), 7.24–7.31 (m,
5 H). 13C NMR (50 MHz - CDCl3), δ (ppm): 21.3, 22.2, 72.3, 126.1,
127.8, 128.5, 141.7, 170.3. IR (cm−1): 2983, 1743, 1374, 1232, 1069,
760, 696.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.mcat.2019.110402.
References
1-(4-methoxyphenyl)ethyl acetate (2a): GC–MS (70 eV), m/z (relative
intensity): 194 (M•+, 27%), 134 (100%), 119 (40%), 105 (22%), 91
(33%), 77 (20%), 43 (34%). 1H NMR (200 MHz, CDCl3, TMS), δ (ppm):
1.51 (d, J =6.5 Hz, 3 H), 2.03 (s, 3 H), 3.77 (s, 3 H), 5.84 (q, J =6.5 Hz,
1 H), 6.87 (d, J =8.7 Hz, 2 H), 7.29 (d, J =8.7 Hz, 2 H). 13C NMR
(50 MHz - CDCl3), δ (ppm): 21.3, 21.9, 55.2, 72.0, 113.8, 127.6, 133.7,
159.3, 170.3. IR (cm−1): 2981, 2838, 2062, 1885, 1733, 1612, 1513,
1242, 829.
1-(4-nitrophenyl)ethyl acetate (3a): GC–MS (70 eV), m/z (relative
intensity): 209 (M•+, 1%), 167 (56%), 150 (16%), 119 (23%), 103
(24%), 91 (29%), 77 (30%), 43 (100%). 1H NMR (200 MHz, CDCl3,
TMS), δ (ppm): 1.56 (d, J =6.7 Hz, 3 H), 2.12 (s, 3 H), 5.93 (q, J
=6.7 Hz, 1 H), 7.51 (d, J =8.8 Hz, 2 H), 8.22 (d, J =8.8 Hz, 2 H). 13C
NMR (50 MHz - CDCl3), δ (ppm): 21.1, 22.2, 71.2, 123.8, 126.7, 147.4,
149.0, 170.0. IR (cm−1): 2966, 1734, 1602, 1517, 1346, 1242, 1068,
855.
1-Phenylpropyl acetate (4a): GC–MS (70 eV), m/z (relative intensity):
178 (M•+, 15%), 149 (25%), 136 (51%), 117 (50%), 107 (100%), 91
(37%), 79 (22%), 77 (20%), 43 (65%). 1H NMR (200 MHz, CDCl3), δ
(ppm): 0.88 (t, J =7.4 Hz, 3 H), 1.76–1.97 (m, 2 H), 2.07 (s, 1 H), 5.66
(t, J =6.9 Hz, 1 H), 7.24–7.36 (m, 5 H). 13C NMR (50 MHz, CDCl3), δ
(ppm): 9.9, 21.2, 29.3, 76.4, 126.6, 127.8, 128.4, 140.5, 170.4. IR
(cm−1): 3031, 2958, 2934, 2862, 1739, 1457, 1373, 1239, 1020, 759,
697.
1-Phenylpentyl acetate (5a): GC–MS (70 eV), m/z (relative intensity):
206 (M•+, 10%); 164 (43%); 149 (30%); 117 (39%); 107 (100%); 104
(23%); 91 (30%); 79 (16%); 77 (13%); 43 (64%). 1H NMR (200 MHz,
CDCl3), δ (ppm): 0.87 (t; J =6.7 Hz; 3 H); 1.20–1.39 (m, 4 H);
1.70–1.95 (m, 2 H); 2.06 (s, 3 H); 5.72 (dd; J = 7.6; 6.5 Hz, 1 H);
7.24–7.35 (m, 5 H). 13C NMR (50 MHz - CDCl3), δ (ppm): 13.9; 21.2;
22.4; 27.7; 36.0; 76.1; 126.5; 127.8; 128.4; 140.9; 170.3. IR (cm−1):
3032, 2954, 1733, 1458, 1370, 1241, 1022.
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Acknowledgements
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The authors would like to thank National Council for Scientific and
Technological Development (CNPq, Brazil, Proc. 456834/2014) and
Coordination for the Improvement of Higher Level Personnel (CAPES)
for financial support and fellowships.
5