Enantioselective hydrolysis of amino acid esters by non-chiral copper complexes equipped with bis (β-cyclodextrin)s

Article abstract of DOI:10.1016/j.molcata.2016.09.009

Two non-chiral copper(II) complexes equipped with bis(β-cyclodextrin)s (bisCDs) were explored as hydrolase models for the enantioselective hydrolysis of two pairs of alkyl chain-possessing amino acid ester enantiomers. The two bisCD complexes are pyridine-linked with different CD cavity orientations, denoted as CuL¹ (L¹?=?2,6-bis(6-mono-amino-β-cyclodextrin-methyl)-pyridine) and CuL² (L²?=?2,6-bis(3-mono-amino-β-cyclodextrin-methyl)-pyridine). Kinetic studies indicated that the “back-to-back” bisCD complex CuL¹ showed higher catalytic efficiency and more pronounced enantioselectivity for all substrates than the “face-to-face” bisCD complex CuL². Overall preference of L-isomers was observed for both complexes. In the presence of CuL¹, the formation of catalyst-substrate Michaelis complexes during the hydrolysis was demonstrated by saturation kinetic study and Electrospray ionization mass spectrometry (ESI–MS) analysis. Enantiomer selectivity (v_max^L/v_max^D) value for N-Boc-N'-Boc-Lysine 4-nitrophenyl esters (Boc-Lys(Boc)-ONp), the longer alkyl-chain analogs, is twice of that for N-Boc-Alanine 4-nitrophenyl esters (Boc-Ala-ONp). The enantioselective hydrolysis of Boc-Lys(Boc)-ONp promoted by CuL¹ was confirmed by chiral high-performance liquid chromatography (HPLC) analysis. The participation of CD cavities during enantioselective hydrolysis was investigated through inhibition assay. The enantioselectivity in hydrolyzing different amino acid esters promoted by CuL¹ was compared. The mechanism involved in the cooperation of two adjacent CD cavities of bisCD was proposed.

Full text of DOI:10.1016/j.molcata.2016.09.009

Journal of Molecular Catalysis A: Chemical 424 (2016) 297–303
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Editor’s choice paper
Enantioselective hydrolysis of amino acid esters by non-chiral copper
complexes equipped with bis (␤-cyclodextrin)s
Shan-Shan Xue, Meng Zhao, Jing-Xing Lan, Rui-Rong Ye, Yi Li, Liang-Nian Ji,
Zong-Wan Mao∗
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275,
PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 July 2016
Received in revised form 5 September 2016
Accepted 6 September 2016
Available online 8 September 2016
Two non-chiral copper(II) complexes equipped with bis(␤-cyclodextrin)s (bisCDs) were explored as
hydrolase models for the enantioselective hydrolysis of two pairs of alkyl chain-possessing amino acid
ester enantiomers. The two bisCD complexes are pyridine-linked with different CD cavity orientations,
denoted as CuL1 (L1 = 2,6-bis(6-mono-amino-␤-cyclodextrin-methyl)-pyridine) and CuL2 (L2 = 2,6-bis(3-
mono-amino-␤-cyclodextrin-methyl)-pyridine). Kinetic studies indicated that the “back-to-back” bisCD
complex CuL1 showed higher catalytic efficiency and more pronounced enantioselectivity for all sub-
strates than the “face-to-face” bisCD complex CuL2. Overall preference of l-isomers was observed for
both complexes. In the presence of CuL1, the formation of catalyst-substrate Michaelis complexes dur-
ing the hydrolysis was demonstrated by saturation kinetic study and Electrospray ionization mass
spectrometry (ESI–MS) analysis. Enantiomer selectivity (vmaxL/vmaxD) value for N-Boc-N’-Boc-Lysine 4-
nitrophenyl esters (Boc-Lys(Boc)-ONp), the longer alkyl-chain analogs, is twice of that for N-Boc-Alanine
4-nitrophenyl esters (Boc-Ala-ONp). The enantioselective hydrolysis of Boc-Lys(Boc)-ONp promoted by
CuL1 was confirmed by chiral high-performance liquid chromatography (HPLC) analysis. The participa-
tion of CD cavities during enantioselective hydrolysis was investigated through inhibition assay. The
enantioselectivity in hydrolyzing different amino acid esters promoted by CuL1 was compared. The
mechanism involved in the cooperation of two adjacent CD cavities of bisCD was proposed.
© 2016 Published by Elsevier B.V.
Keywords:
Cyclodextrin
Hydrolase
Amino acid esters
Enantioselective
hydrolysis
Cooperative effect
1. Introduction
interactions to regulate the nature of the metal ion, which was
proved to be a promising approach [7–11].
Native metalloenzymes possess impressive catalytic efficiency
and exquisite substrate specificity, regio- and enantio-selectivity,
owing to the cooperation of the catalytic centre and the
second coordination sphere [1,2]. For example, the (S)-2-
hydroxypropylphosphonate ((S)-2-HPP) epoxidase (HppE) reaction
course is governed by the stereochemistry of substrates, as the
substrate enantiomers proceeded totally different reactions [3,4];
␣-chymotrypsin catalyzes the hydrolysis of peptide bonds at the
carboxyl side of specific amino acid residues in proteins, and has
marked enantioselectivity in hydrolyzing certain amino acid esters
[5]. Biomimetic study could help understand the origins of effi-
ciency and specificity of enzymes [5,6]. Introducing supramolecules
as the second coordination sphere brings multiple non-covalent
Cyclodextrins (CDs) have been widely exploited in biomimetic
study since 1970s [7,12–14]. Increased substrate binding affin-
ity was achieved owing to the hydrophobic microenvironment of
CD cavity [7,13,15,16]. The intramolecular cooperative effect of
bridged bisCD was also observed [7,17–19]. On the other hand,
appropriately modified CDs have been used in some enantiose-
lectivity studies because of their inherently chiral cavities, such
as stereoselectively molecular recognition [20,21] and asymmetric
catalysis reactions [22–27]. CDs play an important role in preorga-
nizing substrates by means of the noncovalent interactions brought
by the hydrophobic chiral cavities. Ueoka [28,29], Ueno [30,31], and
others [32–35] reported the deacylation of amino acid or dipeptide
esters with high enantioselectivity, catalyzed by unmodified CDs
or functional group-containing CD monomers. However, few study
focused on the cooperation between the metal cofactor and the sec-
ond coordination sphere effect for enantioselective deacylation of
amino acid esters [36].
∗
Corresponding author.
E-mail address: cesmzw@mail.sysu.edu.cn (Z.-W. Mao).
http://dx.doi.org/10.1016/j.molcata.2016.09.009
1381-1169/© 2016 Published by Elsevier B.V.

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S.-S. Xue et al. / Journal of Molecular Catalysis A: Chemical 424 (2016) 297–303
Scheme 1. Schematic representations of the structures of catalysts and hydrolysis pathway of substrates.
Table 1
Recently, we constructed a series of metallohydrolase mim-
The initial rate constants for substrates (2.5 ␮M) promoted by different catalysts
(50.0 ␮M) in a 10% MeCN solution in HEPES buffer (pH 7.2, 50 mM) at 298 0.1 K.
ics and superoxide dismutase mimics with CD domains [7,36–44].
Among the hydrolase models, the secondary face pyridine-linked
“face-to-face” [45] bisCD complex CuL2 (L2 = 2,6-bis(3-mono-
amino-␤-cyclodextrin-methyl)-pyridine) [38] was demonstrated
to possess higher activity for bis(4-nitrophenyl)phosphate (BNPP)
cleavage than the “back-to-back” analog CuL1 (L1 = 2,6-bis(6-
mono-amino-␤-cyclodextrin-methyl)-pyridine) [40]. Meanwhile,
our latest result proved the two hydrolase models possess dif-
ferent enantioselectivity in hydrolyzing the aromatic amino acid
esters [36]. With the promotion of CuL1, the hydrolysis of
N-Boc-phenylalanine 4-nitrophenyl ester enantiomers achieved
pronounced enantioselectivity. Interestingly, changing CD orienta-
tions of CuL1 resulted in dramatically changed enantioselectivity.
The three amino acid esters contain aromatic moieties that can be
adapted in CD cavity. Herein, further evaluation of the two hydro-
lase models in enantioselective hydrolysis was performed, with
two pairs of alkyl chain-containing amino acid esters enantiomers,
N-Boc-N’-Boc-lysine 4-nitrophenyl esters (Boc-Lys(Boc)-ONp) and
N-Boc-Alanine 4-nitrophenyl esters (Boc-Ala-ONp), as substrates
(Scheme 1). The Lys alkyl-chain of Boc-Lys(Boc)-ONp encapsulated
into the CD cavity [46], contributes to the enantioselective hydrol-
ysis.
Catalyst
kin (10−5 s−1
)
Boc-Lys(Boc)-ONp
Boc-Ala-ONp
l−
d−
l−
d−
CuL1
CuL2
L1
13.9
1.6
3.5
6.8 × 10−1
2.0 × 10−1
6.0 × 10−2
8.5 × 10−2
1.3 × 10−1
2.5 × 10−1
2.6 × 10−1
7.7 × 10−2
2.9 × 10−2
7.1 × 10−2
2.9 × 10−1
6.4 × 10−2
2.8 × 10−1
2.1 × 10−1
6.5 × 10−2
7.3 × 10−2
2.8 × 10−2
L2
Cu(ClO4)2
Buffer
2.2. Hydrolysis of Boc-protected amino acid esters
2.2.1. Comparison between CuL1 and CuL2
Kinetic study for the hydrolysis of Boc-Lys(Boc)-ONp and
Boc-Ala-ONp were performed at pH 7.2 and 298 0.1 K (50 mM
2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES)
buffer containing 10% CH3CN) [36]. The reactions were also mon-
itored by ultraviolet-visible (UV–vis) spectroscopy, detecting the
hydrolytic product 4-nitrophenolate (NP) at 400 nm. The initial
rate constants, kin (s−1) of the substrates cleavage events were
calculated using initial rate method, based on plots of the NP
concentration versus time (Supplementary Fig. S3), and listed in
Table 1.
2. Results and discussion
As shown in Table 1, The spontaneous hydrolysis rates for
substrates were very slow, with kin 2.9 × 10−7 s−1 for Boc-Lys(Boc)-
ONp enantiomers and 2.8 × 10−7 s−1 for Boc-Ala-ONp enantiomers,
respectively. Little rate enhancement was observed when cop-
per(II), L1 or L2 separately mixed with the substrates. The
primary face linked “back-to-back” bisCD complex CuL1 exhibited
2.1. Synthesis and characterization
The two copper(II) complexes, CuL1 and CuL2, were synthesized
and characterized according to our previously reported procedures
[36,38,40,42].

S.-S. Xue et al. / Journal of Molecular Catalysis A: Chemical 424 (2016) 297–303
299
Fig. 1. Hydrolysis of single enantiomer of the substrates (2.5 ␮M) a) Boc-Lys(Boc)-ONp, b) Boc-Ala-ONp promoted by CuL1 (Red circles), by CuL2 (blue circles), by L1 (red
triangles), by L2 (blue triangles), by Cu(ClO4)2 (black squares). Solid ones are for the l-isomer and hollow ones are for the d-isomer for all the cases. The reactions were
performed in a 10% MeCN solution in HEPES buffer (pH 7.2, 50 mM) promoted by 50 ␮M different catalysts at 298 0.1 K. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
a higher catalytic efficiency and more pronounced enantios-
electivity than the secondary face linked “face-to-face” bisCD
complex CuL2 for both pairs of enantiomers. In the presence of
CuL1 (50.0 ␮M), Boc-l-Lys(Boc)-ONp achieved the highest hydrol-
ysis rate (kinL = 13.9 × 10−5 s−1) among all the tested substrates
(Table 1), which is twice of the largest value of our previous study
[36]. The value is 479-fold and 10-fold higher than the spontaneous
and d-isomer hydrolysis, respectively (Fig. 1a, Table 1). Boc-Ala-
ONp enantiomers resulted in moderate hydrolytic efficiency and
enantioselectivity compared with Boc-Lys(Boc)-ONp promoted by
CuL1 (Fig. 1b). On the other hand, CuL2 hydrolyzed Boc-Lys(Boc)-
ONp with a lower or comparative rate compared with L2 (Table 1).
We consider that the coordination of copper(II) ion shortened the
linker length of L2, and the spaced distance was adjusted too nar-
row, which is detrimental to the hydrolysis reactions, especially for
the long alkyl chain-possessing substrates [19,46].
amino acid esters using the same catalyst [36]. The decrease of
enantioselectivity can be related to the hydrophobic interaction
between alkyl chain and the CDs is weaker than that between
aromatic moiety and the CDs. The value is also moderate among
the reported values towards similar substrates [5,28,47,50–52],
which can also be resulted from weaker interaction between
alkyl chain and the CD cavity. As for Boc-Ala-ONp, the short alkyl
chain analogs, the enantiomer selectivity value was decreased
to 3.6. The value is smaller than the imidazolyl-␤-CD promoted
ones [30], but the preferred isomer is opposite. The Km values for
Boc-Ala-ONp are higher than that for Boc-Lys(Boc)-ONp, indicating
weaker binding capability of CuL1. These data suggested that the
alkyl chains of Boc-Lys(Boc)-ONp participate in governing the
enantioselective hydrolysis. On the other hand, similar Km values
for Boc-Lys(Boc)-ONp enantiomers were observed, with (6.5 1.4)
for l-isomer and (6.0 1.2) for d-isomer, indicating the similar
binding affinity for the two enantiomers with CuL1. The hydrolysis
enantioselectivity was proposed coming from the different geome-
tries of the substrates that were regulated by the closely linked
CD chiral cavities [36]. Electrospray ionization mass spectrometry
(ESI-MS) analysis of CuL1 in solution mixed with each enantiomer
of Boc-Lys(Boc)-ONp and Boc-Ala-ONp individually was performed
(Supplementary Figs. S4–S7). The catalyst-substrate (1:1) com-
plexes were detected by ESI-MS for all cases, which confirmed the
presence of Michalis intermediates along the reaction pathway.
However, for Boc-Ala-ONp enantiomers, the ESI-MS signals of
catalyst-substrate (1:1) complexes were much weaker than that
for Boc-Lys(Boc)-ONp enantiomers, indicating weaker interaction
with CuL1. The hydrolytic product NP and Boc-Lys(Boc)-OH or
Boc-Ala-OH were also detected, see Supplementary Figs. S4–S7.
The “back-to-back” bisCD complex CuL1 favoured the enantios-
elective hydrolysis of the amino acid-containing substrates. For
either CuL1 or CuL2, the chiral CD cavities in our models have
an overall preference for the l-isomers of the amino acid esters.
Additionally, changing the CD orientation of catalyst reduced the
enantioselectivity more than that induced by changing the sub-
strate, suggesting that the CD orientation plays a vital role in the
enantioselective hydrolysis. Thus, the initial kinetic study indicated
consistent results with the study using the same catalysts with
aromatic amino acid ester analogs as substrates [36].
2.2.2. Saturation kinetics promoted by CuL1
Further kinetic studies of the hydrolysis of Boc-Lys(Boc)-ONp
and Boc-Ala-ONp promoted by CuL1 were performed. Fig. 2 shows
the dependence of the rate constants on the CuL1 concentra-
tion [28,29,47–49]. By increasing the initial catalyst concentration
from 5.0 to 100.0 ␮M, deviation from linear increase was observed
(Fig. 2), which implied that the catalyst-substrate complex formed
in prior to the catalytic reaction. The kinetic parameters were
deduced by fitting the data to the Michaelis–Menten equation, and
gathered in Table 2.
The highest acceleration factor (vmax/vuncat = 1049) was
observed in the case of Boc-l-Lys(Boc)-ONp hydrolysis pro-
moted by CuL1. The value was among the largest ones which were
gained by using CD-based [29–31] or other surfactants containing
complex [49,50] as artificial mimics, and are close to those cases
by using enzymes or modified enzymes [5,47,50,51], for the
hydrolysis of amino acid esters or peptide esters. The hydrolysis
of Boc-Lys(Boc)-ONp enantiomers promoted by CuL1 resulted in
more pronounced enantioselectivity than that of Boc-Ala-ONp,
with an enantiomer selectivity of 8.8. The enantiomer selectiv-
ity value is smaller than the largest value (15.7) for aromatic
2.3. Chiral HPLC analysis
To further confirm that the l-isomer was preferentially
hydrolysed over the d-isomer, a chiral high-performance liquid
chromatography (HPLC) analysis was performed with racemic
Boc-Lys(Boc)-ONp (Supplementary Figs. S8, S9). Reaction aliquots
were taken and subjected to HPLC analysis with CHIRALPAK®
IC as the chiral stationary phase [36,47]. As shown in Supple-
mentary Fig. S9, when 38% and 47% of Boc-Lys(Boc)-ONp was
consumed, the remaining L:D ratios were 28:72 and 31:69, respec-
tively (Table 3). Based on these conversions and the remaining
substrate enantiomeric ratios (e.r.), the consumption ratios of the
Boc-l-Lys(Boc)-ONp and Boc-d-Lys(Boc)-ONp were calculated to
be 86:14 and 76:24, respectively, indicating that the correspond-
ing hydrolyzed product (Boc-Lys(Boc)-OH) were present at e.r. of
86:14 and 76:24 (L/D) (Table 3). The results demonstrated that CuL1
possessed high enantioselectivity for the hydrolysis of racemic

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S.-S. Xue et al. / Journal of Molecular Catalysis A: Chemical 424 (2016) 297–303
Fig. 2. Saturation kinetics for the hydrolysis of different substrates (2.5 ␮M) promoted by CuL1 (5.0 − 100.0 ␮M) in a 10% MeCN solution in HEPES buffer (pH 7.2, 50 mM) at
298 0.1 K. The solid lines show the fits of data to Michaelis–Menten equation.
Table 2
Kinetic parameters for Boc-Lys(Boc)-ONp (2.5 ␮M) and Boc-Ala-ONp (2.5 ␮M) hydrolysis fitting from Michaelis–Menten equation, in the presence of CuL1 (5.0 − 100.0 ␮M)
in a 10% MeCN solution of HEPES buffer (50 mM, pH = 7.2) at (298 0.1) K.
D
vmax (10−11 M s−1
)
Km (10−2 mM)
v
max/Km (10−6 s−1
)
vuncat (10−13 M s−1
)
v
max/vuncat
vmaxL/vmax
Boc-Lys(Boc)-ONp
Boc-Ala-ONp
l−
d−
l−
d−
75.0 8.5
8.5 0.7
22.7 2.2
6.3 0.7
6.5 1.4
6.0 1.2
7.6 1.3
12.6 2.6
11.5
1.4
3.0
7.3
1049
133
325
8.8
7.0
3.6
0.5
89.3
Table 3
Chiral HPLC analysis of remaining Boc-Lys(Boc)-ONp enantiomers under different conversions.
Entry
Conversion (%)
Remaining e.r. of Boc-Lys(Boc)-ONp (L:D)a
e.r. of Boc-Lys(Boc)-OH (L:D)b
1
2
3
0
38
47
50:50
28:72
31:69
0
86:14
76:24
a
The enantiomeric ratio (e.r.) values of remaining substrates were determined by HPLC analysis with a chiral stationary phase.
e.r. values of products were calculated from the result of HPLC analysis.
b
Table 4
dramatically decreased in the presence of DBBA, with the
inhibition constant Ki (6.3 0.2) mM for l-isomer, (6.1 0.6)
mM for d-isomer, suggesting that the CD cavities of CuL1 played
The inhibition constants (Ki) for different substrates (2.5 ␮M) promoted by CuL1
(12.5 ␮M) in the presence of DBBA were obtained from Eq. (1) fitting.
Boc-Lys(Boc)-ONp
Boc-Ala-ONp
a
crucial role during catalyzing Boc-Lys(Boc)-ONp hydrolysis
(Fig. 3a,b). Besides, the similar Ki values indicate the CD cavities
encapsulated Boc-Lys(Boc)-ONp enantiomers with the similar
binding affinity. The Ki values for Boc-Ala-ONp enantiomers are
larger than Boc-Lys(Boc)-ONp, with (13.3 0.6) mM for Boc-l-Ala-
ONp and (39.2 4.7) mM for Boc-d-Ala-ONp, indicating that the
alkyl chains of both Boc-Lys(Boc)-ONp enantiomers involved in
the enantioselective hydrolysis through being encapsulated into
CD cavity [46]. Meanwhile, the different Ki values indicate DBBA
have different inhibition effect for Boc-Ala-ONp enantiomers.
Considering the Boc group could be encapsulated by CD cavity
[53], we proposed that the CD cavity of CuL1 encapsulted the Boc
group of Boc-l-Ala-ONp during the reaction courses, while failed
for the d-isomer. The Km values deduced from Michaelis-Menten
equation have similar tendency with Ki values (Table 2). These
results confirmed that CD cavities of CuL1 played a vital role in
performing enantioselective hydrolysis and the cooperation of two
adjacent CD cavities of bisCD existed.
l−
d−
6.1 0.6
l−
d−
Ki (mM)
6.3 0.2
13.3 0.6
39.2 4.7
Boc-Lys(Boc)-ONp and showed a preference for the l-isomer. The
enantioselectivity toward Boc-Ala-ONp enantiomers was weak-
ened under same hydrolysis condition, which is consistent with
the results of saturation kinetic study.
2.4. Catalytic inhibition assay
To further investigate the role of the CD cavity, di(p-
tert-butylbenzyl) amine (DBBA) was applied as an inhibitor
[36,39,41,43]. CD cavities of CuL1 could be occupied by DBBA. The
possibility that DBBA exerts its inhibitory effect through coordi-
nation with the metal ions was excluded [41]. In each run, DBBA
was added to the reaction system at increased concentrations. As
a result, the inhibiting effect varied among the substrates (Fig. 3).
The data were fit to Eq. (1), where the substrates and inhibitor com-
peted for binding to the hydrophobic cavities, and the inhibition
constants (Ki) were listed in Table 4 [41].
2.5. CuL1-mediated enantioselective hydrolysis of different amino
acid esters
Consequently, the “back-to-back” bisCD complex CuL1 exhibits
much better catalytic efficiency and enantioselectivity than the
“face-to-face” analog CuL2 for all the amino acid esters we have
studied. The vmax/vuncat values for the amino acid esters pro-
moted by CuL1 are shown in Fig. 4. The enantioselectivity of
kin
k0
Ki
=
(1)
(Ki + [DBBA])
As shown in Table 4, the initial rate of the CuL1-catalyzed
hydrolysis of both Boc-Lys(Boc)-ONp enantiomers was

S.-S. Xue et al. / Journal of Molecular Catalysis A: Chemical 424 (2016) 297–303
301
Fig. 3. DBBA-concentration dependence of normalized initial rate constants of substrates (2.5 mM) cleavage catalyzed by CuL1 (12.5 mM) in HEPES buffer (pH 7.2, 50 mM)
containing 10% MeCN at (298 0.1) K, a) Boc-l-Lys(Boc)-ONp, b) Boc-d-Lys(Boc)-ONp, c) Boc-l-Ala-ONp and d) Boc-d-Ala-ONp, where k0 is the initial rate constant without
inhibition. The solid lines are from fitting Eq. (1) to the data where the complex binds one molecule of inhibitor.
cavities to form effective reaction intermediates, and the coopera-
tive effect of the two adjacent CD cavities was also achieved.
2.6. Catalytic mechanism
Based on the above experimental results, the hydrolytic mech-
anisms were proposed. For Boc-Lys(Boc)-ONp, NP and the Lys
alkyl-chain encapsulated into the CD cavities of CuL1 during the
hydrolysis. Benefitting from the cooperating regulation of adjacent
CD chiral cavities of bisCD, pronounced enantioselectivity for Boc-
Lys(Boc)-ONp was achieved. Meanwhile, the cooperative effect of
the two closely linked CD cavities for hydrolyzing Boc-Ala-ONp was
diminished, which induced inferior enantioselectivity. The enan-
tioselectivity for the two pairs of substrate enantiomers originated
from the different geometries of the substrates during the reaction
that were regulated by the hydrophobic interaction of CD chiral
cavities [36].
Fig. 4. vmax/vuncat values for amino acid esters (2.5 ␮M) hydrolysis in the presence
of CuL1. The values for Boc-Phg-ONp, Boc-Phe-ONp and Boc-Trp-ONp are from Ref.
[36].
3. Conclusion
In conclusion, our study presented two non-chiral copper(II)
complexes equipped with bisCDs as hydrolase models for enan-
tioselective hydrolysis of two pairs of alkyl-chain containing
amino acid ester enantiomers under physiological conditions
(pH = 7.2). The chiral CD cavities of the two complexes had an over-
all preference for l-isomers. The “back-to-back” bisCD complex
CuL1 exhibited higher catalytic efficiency and more pronounced
enantioselectivity towards both substrates than the “face-to-
face” analog CuL2. CuL1 catalyzed the long alkyl-chain analog
Boc-Lys(Boc)-ONp with larger enantiomer selectivity value than
Boc-Ala-ONp, indicating the alkyl chain played a role during the
enantioselective hydrolysis. Inhibition assay confirmed the vital
role of CD cavities during the hydrolysis. In line with previous
study, this work systematically studies the bisCD metallohydro-
lase model on enantioselective hydrolysis of amino acid esters,
CuL1-mediated hydrolysis was found to be very sensitive to the
aromatic moieties of aromatic amino acid esters, while the Benzyl
group of Boc-Phe-ONp was replaced with Phenyl or Indolyl group,
the hydrolysis enantioselectivity was considerably decreased [36].
The result indicates that the cavities of CuL1 could selectively
induce certain aromatic amino acid esters to form effective reaction
intermediates. With a long alkyl-chain, Boc-Lys(Boc)-ONp resulted
in larger enantioselectivity than Boc-Ala-ONp, which emphasized
the cooperative effect of adjacent CD cavities of CuL1 in enan-
tioselective hydrolysis. Meanwhile, inferior enantioselectivity for
Boc-Lys(Boc)-ONp than Boc-Phe-ONp confirmed the vital role of
the inherently CD chiral cavities in enantioselective hydrolysis.
Hence, CuL1 could mediate the enantioselective hydrolysis through
regulating substrates by the hydrophobic interaction of CD chiral

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S.-S. Xue et al. / Journal of Molecular Catalysis A: Chemical 424 (2016) 297–303
featuring the cooperative effect of two adjacent CD cavities. Our
results can help understand the origins of enantioselectivity of
enzymes and also provides hints for guiding further exploration of
CD-based hydrolase models in enantioselective hydrolysis study.
4.4. Kinetic resolution of racemic Boc-Lys(Boc)-ONp
The hydrolysis of racemic Boc-Lys(Boc)-ONp was performed
using a method similar to that described in the previous section.
Racemic Boc-Lys(Boc)-ONp (5.0 ␮M) was mixed with CuL1 (50 ␮M)
under the same conditions as those used in the kinetic studies.
The reaction process was monitored with a UV/vis spectrometer.
Then, upon mixing of the reaction mixture with ethyl acetate,
the products (Boc-Lys(Boc)-OH and 4-nitrophenolate (NP)) and
remaining Boc-Lys(Boc)-ONp were completely extracted into the
organic phase [47]. The organic phase was evaporated to dryness
under reduced pressure, and the resulting residue was dissolved in
ethanol for HPLC analysis. The chiral HPLC analyses were performed
with a CHIRALPAK® IC 250 × 4.6 mm column and monitored by a
UV–vis detector at 254 nm; the elution solvent was 45/55H2O (0.1%
formic acid)/CH3CN, and the flow rate was 1.0 mL/min.
4. Experimental
4.1. Materials
Reagent-grade ␤-CD was recrystallized twice from H2O and
dried in vacuo for 12 h at 373 K. All of the amino acid esters
or precursor enantiomers, N-Boc-N’-Boc-lysine 4-nitrophenyl
ester (Boc-Lys(Boc)-ONp) enantiomers and N-Boc-Alanine 4-
nitrophenyl ester (Boc-Ala-ONp) enantiomers were purchased
from GL Biochem (Shanghai) Ltd. Di(p-tert-butylbenzyl) amine
(DBBA) was synthesized with previously reported methods [43].
DMF was superdry grade and stored over a molecular sieve. Com-
mon organic reagents were reagent grade and redistilled before
use. Milli-Q water was used in all physical measurements. CuL1 and
CuL2 were synthesized and characterized as described in previous
studies [36,38,40,42], and the 1H NMR spectra for L1, L2 are shown
as Supplementary Figs. S1, S2. All compounds were confirmed by
elemental analyses, ESI-MS, and 1H NMR spectra.
Acknowledgements
This work is supported by National Natural Science Foundation
of China (Nos. 21231007 and J1103305), the Ministry of Education
of China (Nos. IRT1298 and 313058), the National Basic Research
Program of China (973 Program No. 2014CB845604) and the Fun-
damental Research Funds for the Central Universities.
Appendix A. Supplementary data
4.2. General methods
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.molcata.2016.09.
009.
1H NMR spectra were recorded on Mercury plus 300 spectrom-
eters. The elemental contents were analysed with a Perkin-Elmer
240 elemental analyser. The ESI-MS spectra were collected on
a Thermo LCQ-DECA-XP spectrometer. The UV/vis spectra were
monitored with a Varian Cary 100 UV/vis spectrophotometer
equipped with a temperature controller ( 0.1 K). The HPLC anal-
yses were performed on an Agilent 1200 HPLC with CHIRALPAK®
IC 250 × 4.6 mm column.
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