A study of the strength of a template molecule - A functional monomer interaction that affects the performance of molecularly imprinted polymers and its application to chiral amplification

Article abstract of DOI:10.1248/cpb.c16-00974

A novel type of molecularly imprinted polymer (MIP), N-benzoyl-(S)-valine anilide-imprinted polymer (IP-2), was prepared using hydrogen-bonding interactions as a main force in the pre-polymerization step. The performance of the IP-2 was evaluated via batch procedure and compared with a (S)-valine anilideimprinted polymer (IP-1) that was prepared using an ionic interaction that is stronger than hydrogen bonding. Although both polymers showed a preferential adsorbability for (S)-amino acid derivatives, different performances were observed in terms of adsorbability and enantioselectivity. In addition, the IP-2 was able to recognize the enantiomer of a valine-derived chiral catalyst. This phenomenon was applied to a chiral amplification reaction, and a highly selective asymmetric Mannich-type amination was achieved using the combination of a racemic catalyst and a MIP.

Full text of DOI:10.1248/cpb.c16-00974

Chemical and Pharmaceutical Bulletin Advance Publication by J-STAGE
DOI:10.1248/cpb.c16-00974
Advance Publication
February 4, 2017
Chem. Pharm. Bull.
Regular article
A study of the strength of a template molecule - a functional monomer
interaction that affects the performance of molecularly imprinted polymers
and its application to chiral amplification
Takuro Yasuyama, Hirofumi Matsunaga,* Shin Ando, Tadao Ishizuka*
Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto,
862-0972, Japan
*To whom correspondence should be addressed.
e-mail: mtc@gpo.kumamoto-u.ac.jp (H.M.)
tstone@gpo.kumamoto-u.ac.jp (T.I.)
Ⓒ 2017 The Pharmaceutical Society of Japan

Summary
A novel type of molecularly imprinted polymer (MIP), N-benzoyl-(S)-valine
anilide-imprinted polymer (IP-2), was prepared using hydrogen-bonding interactions as a
main force in the pre-polymerization step. The performance of the IP-2 was evaluated via
batch procedure and compared with a (S)-valine anilide-imprinted polymer (IP-1) that was
prepared using an ionic interaction that is stronger than hydrogen bonding. Although both
polymers showed a preferential adsorbability for (S)-amino acid derivatives, different
performances were observed in terms of adsorbability and enantioselectivity. In addition, the
IP-2 was able to recognize the enantiomer of a valine-derived chiral catalyst. This
phenomenon was applied to a chiral amplification reaction, and a highly selective asymmetric
Mannich-type amination was achieved using the combination of a racemic catalyst and a
MIP.
Keyword: molecularly imprinted polymer, chiral recognition, (S)-benzoyl amino acid anilide,
asymmetric amplification
Chemical and Pharmaceutical Bulletin Advance Publication

Introduction
Molecularly imprinted polymers (MIPs)1-3) have been energetically pursued in the field
of molecular recognition, and various types of applications such as a stationary phase of
HPLC,4,5) solid-state extraction,6) and chemical sensors7,8) have been reported due to qualities
such as synthetic simplicity, stability, and a high level of recognition selectivity.
There are several methods for the preparation of imprinted polymers, including acid-base
interaction, simple hydrogen bonding, dipole-dipole interaction, and hydrophobic interaction
at the pre-polymerization step. Among these, the acid-base interaction4,5,9) and/or hydrogen
bonding10,11) are mainly used due to the effectiveness of bonding strength in regards to the
tailor-made construction of three-dimentional binding sites.
Therefore, MIPs prepared using an acid-base interaction appear to show a higher level of
performance than those without such an ionic interaction. However, unexpectedly, only a few
reports12) described the comparison of the different performances of MIPs between the
presence of acid-base interaction and the absence of such a strong interaction. Therefore, it
would be desirable to establish a new methodology, which would enable a convenient and
precise comparison of the performance of MIPs that could lead to the development of a new
type of MIP.
We previously reported13) the evaluation of basic (S)-amino acid anilide (such as
(S)-1)-imprinted MIPs that were prepared using an acid-base interaction by batch procedure
that allowed independent observation of adsorbability and selectivity, which were not
precisely measured by a HPLC column system.
In this paper we describe the evaluation of the performance of neutral (S)-N-benzoyl
amino acid anilide (such as (S)-5)-imprinted MIPs, which were prepared in the presence of a
weaker hydrogen-bonding interaction in the pre-polymerization step, by batch procedure. We
also report a MIP with a (S)-N-benzoyl valine anilide cavity and its application to chiral
amplification.
Chemical and Pharmaceutical Bulletin Advance Publication

O
H
N
H
N
H2N
N
H
O
(S)-1
O
(S)-5
Fig. 1. Structures of (S)-1 and (S)-5.
Experimental
General Ethylene glycol dimethacrylate (EGDM), methacrylic acid (MAA) and
chloroform were freshly distilled prior to use. (RS)-alanine, (S)- and (RS)-valine,
(RS)-phenylalanine, (RS)-tert-leucine, methacryloyl chloride, 2,2′-azobis(isobutyronitrile)
(AIBN),
2-aminoethanol,
p-toluenesulfonyl
chloride,
cyclohexane
(c-hex),
diethylcyanophosphonate (DEPC), o-anisidine, o-anisic acid, La(NO3)3 ･ 6H2O and
H-D-Val-OtBu were purchased and used without further purification. n-hexane (n-hex),
2-propanol, ethanol (EtOH), and acetonitrile were all HPLC grade and used without further
purification. Tetrahydrofuran (THF; dehydrated) and dichloromethane (CH2Cl2; dehydrated)
were purchased (Kanto Chemical, Co., Inc.), stored under an argon atmosphere and used
without further purification. Diethyl ether (Et2O), ethyl acetate (EtOAc),
N,N’-dimethylformamide (DMF) and methanol (MeOH) were purified by distillation.
The preparation of (S)-valine anilide-imprinted polymers (IP-1), using an acid-base
interaction, and the adsorbability of various types of amino acid anilides onto IP-1 have been
reported.13)
Synthesis
(S)-N-benzoyl-valine anilide ((S)-5); general procedure for the preparation of
N-benzoyl amino acids: (S)-valine anilide ((S)-1) (1.34 g, 7.00 mmol) and benzoic acid
(0.900 g, 7.35 mmol) were dissolved in DMF (41.7 mL). To the mixed solution was added
Et3N (1.10 mL, 7.89 mmol) and DEPC (1.10 mL, 7.35 mmol), and the solution was stirred at
o
0 C for 45 min. The resultant mixture was diluted with EtOAc (300 mL), and the organic
layer was washed with satd. NaHCO3 aq. (50 mL x 3) and satd. NaCl aq. (50 mL x 3) and
dried over anhyd. Na2SO4. After concentration of the organic layer in vacuo., the residue was
Chemical and Pharmaceutical Bulletin Advance Publication

purified by recrystallization (hexane-CH2Cl2) to give (S)-5 (1.60 g, 5.38 mmol) as a colorless
solid: m.p. 216-218 oC; [α]D22 -46.9 (c 0.52, CHCl3); 1H-NMR (300 MHz/CDCl3) δ: 9.54 (1H,
br s), 7.82-7.80 (2H, m), 7.51-7.45 (4H, m), 7.36-7.33 (2H, m), 7.05-7.02 (1H, m), 4.98 (1H,
t, J = 8.4Hz), 2.41-2.36 (1H, m), 1.16 (3H, d, J = 7.0 Hz), 1.14 (3H, d, J = 7.0 Hz); 13C-NMR
(75 MHz/CDCl3) δ: 170.5, 168.1, 137.8, 133.9, 131.7, 128.7, 128.5, 127.3, 124.2, 120.3, 60.0,
31.7, 19.3, 18.9; IR (KBr) 3278, 1668, 1633, 1603, 1531, 1446 cm-1; HRMS (FAB) m/z calcd.
for C18H21N2O2 [M+H]+ 297.1603, found 297.1586.
Racemic N-benzoyl amino acids, (RS)-6, 7 and 8 were synthesized by the same
procedure as (S)-5 from corresponding amino acid derivatives. Then, (RS)-6 and (RS)-8 were
purified by recrystallization from hexane-CH2Cl2, and (RS)-7 was purified by
recrystallization from EtOAc.
o
1
(RS)-N-benzoyl-alanine anilide ((RS)-6): Colorless solid; m.p. 169-171 C; H-NMR (300
MHz/CDCl3) δ: 9.42 (1H, br s), 7.87-7.84 (2H, m), 7.60 (2H, d, J = 7.7 Hz), 7.52 (1H, dd, J =
9.5, 7.7 Hz), 7.44-7.40 (3H, m), 7.31-7.25 (2H, m), 7.11-7.06 (1H, m), 5.15-5.05 (1H, m),
13
1.58 (3H, d, J = 7.0 Hz); C-NMR (75 MHz/CDCl3) δ: 170.9, 167.7, 138.0, 133.5, 132.0,
128.9, 128.6, 127.2, 124.3, 120.0, 50.1, 18.5; IR (KBr) 3297, 1686, 1633, 1606, 1548, 1489,
1442 cm-1; HRMS (FAB) m/z calcd for C16H17N2O2 [M+H]+ 269.1290, found 269.1284.
(RS)-N-benzoyl-phenylalanine anilide ((RS)-7): Colorless solid; m.p. 236-239 oC;
1H-NMR (300 MHz/DMSO-d6) δ: 10.20 (1H, br s), 8.72 (1H, d, J = 8.1 Hz), 7.83 (2H, d, J =
6.6 Hz), 7.62 (2H, d, J = 8.1 Hz), 7.54-7.40 (5H, m), 7.34-7.25 (4H, m), 7.17 (1H, dd, J = 7.7,
6.6 Hz), 7.08-7.03 (1H, m), 4.87-4.82 (1H, m), 3.17-3.11 (2H, m); 13C-NMR (75
MHz/DMSO-d6) δ: 170.4, 166.5, 138.9, 138.1, 133.9, 131.3, 129.2, 128.7, 128.1, 128.1,
128.0, 127.4, 126.3, 123.3, 119.3, 55.8, 37.2; IR (KBr) 3292, 3268, 1676, 1635, 1551, 1535,
1446 cm-1; HRMS (FAB) m/z calcd for C22H21N2O2 [M+H]+ 345.1603, found 345.1583.
o
1
(RS)-N-benzoyl-tert-leucine anilide ((RS)-8): Colorless solid; m.p. 226-228 C; H-NMR
Chemical and Pharmaceutical Bulletin Advance Publication

(300 MHz/CDCl3) δ: 9.50 (1H, br s), 7.87-7.84 (2H, m), 7.57-7.52 (3H, m), 7.48-7.43 (2H,
13
m), 7.27-7.20 (3H, m), 7.09-7.04 (1H, m), 5.17 (1H, d, J = 9.2 Hz), 1.14 (9H, s); C-NMR
(75 MHz/CDCl3) δ: 169.4, 167.5, 137.9, 134.1, 131.9, 128.8, 128.72, 128.71, 127.1, 124.2,
120.0, 61.2, 35.9, 26.7; IR (KBr) 3305, 3267, 2962, 1678, 1627, 1554, 1539, 1490, 1446
cm-1; HRMS (FAB) m/z calcd for C19H23N2O2 [M+H]+ 311.1760, found 311.1741.
(RS)-N-(2-hydroxyphenyl)-2-(2-hydroxybenzoylamino)-3-methylbutylamide
((RS)-9):
(RS)-9 was synthesized by the same procedure as previously reported.14) Colorless solid; m.p.
195-197 oC; 1H-NMR (300 MHz/DMSO-d6) δ: 11.81 (1H, s), 9.76 (1H, s), 9.41 (1H, s), 8.87
(1H, d, J = 8.2 Hz), 7.98 (1H, d, J = 7.9 Hz), 7.75 (1H, d, J = 7.9 Hz), 7.42-7.38 (1H, m),
6.95-6.85 (4H, m), 6.79-6.74 (1H, m), 4.69 (1H, dd, J = 7.7, 7.3 Hz), 2.28-2.21 (1H, m), 1.00
(3H, d, J = 5.7 Hz), 0.98 (3H, d, J = 5.9 Hz).
(S)-N-(2-methoxyphenyl)-2-(2-methoxybenzoylamino)-3-methylbutyramide ((S)-10): To
a mixture of (S)-N-Boc-Val-OH (2.50 g, 11.5 mmol) and Et3N (1.60 mL, 11.5 mmol) in THF
(30 mL) was added ethyl chloroformate (1.10 mL, 11.5 mmol) in THF (13 mL) dropwise
o
over 15 minutes at -15 C. After stirring for 15 minutes, o-anisidine (1.36 mL, 12.1 mmol)
was subsequently added and stirred at r.t. for 15 hours. The resultant mixture was diluted with
EtOAc (300 mL), then washed with 10% KHSO4 aq. (50 mL x 3), satd. NaHCO3 aq. (50 mL
x 3), satd. NaCl aq. (50 mL x 3) and dried over anhyd. Na2SO4. After concentration in vacuo,
the residual solid was washed with a mixture of hexane- Et2O. To a solution of the washed
solid (2.38 g) in CH2Cl2 was added Amberlyst 15 (19.6 g) with stirring at r.t. for 15 hours.
The resultant mixture was filtered, and the resin was washed with hexane, MeOH and THF.
The resin was poured into NH3/iPrOH (197 mL) and stirred at r.t. for 40 minutes. The resin
was filtered off, and the filtrate was concentrated in vacuo. The residue was dissolved in
DMF (35 mL) and o-anisic acid (0.930 g, 6.10 mmol); Et3N (0.85 mL, 6.10 mmol) and DEPC
o
o
(0.870 mL, 5.80 mmol) were sequentially added at 0 C followed by stirring at 0 C for 30
min. The resultant mixture was diluted with EtOAc (300 mL), washed (satd. NaHCO3 aq. (50
mL x 3) and satd. NaCl aq. (50 mL x 3)) and dried over anhyd. Na2SO4. After concentration
Chemical and Pharmaceutical Bulletin Advance Publication

in vacuo, the residue was purified by silica gel column chromatography (CH2Cl2 to
CH2Cl2/EtOAc (97.5: 7.5)) to give (S)-10 as a colorless solid (1.60 g, 4.49 mmol); m.p.
108-110 oC; [α]D22 +39.9 (c 0.70, CHCl3); 1H-NMR (300 MHz/CDCl3) δ: 8.51 (1H, d, J = 8.4
Hz), 8.38 (1H, br s), 8.35 (1H, dd, J = 7.9, 1.6 Hz), 8.21 (1H, dd, J = 7.7, 1.8 Hz), 7.50-7.44
(1H, m), 7.11-6.83 (5H, m), 4.71 (1H, dd, J = 8.4, 5.9 Hz), 4.01 (3H, s), 3.80 (3H, s),
2.44-2.40 (1H, m), 1.09 (3H, d, J = 6.8 Hz), 1.07 (3H, d, J = 6.8 Hz); 13C-NMR (75
MHz/CDCl3) δ: 169.4, 165.4, 157.6, 148.1, 133.0, 132.3, 127.3, 123.9, 121.2, 121.0, 120.9,
119.9, 111.4, 110.0, 59.9, 56.1, 55.7, 30.8, 19.4, 18.0; IR (KBr) 3305, 2953, 1668, 1630, 1535,
1493, 1461, 1432 cm-1; HRMS (FAB) m/z calcd for C20H25N2O4 [M+H]+ 357.1814, found
357.1812.
Synthesis of (S)-N-phenyl-2-(2-methoxybenzoylamino)-3-methylbutyramide ((S)-11):
(S)-valine anilide ((S)-11) (200 mg, 1.04 mmol) and o-anisic acid (166 mg, 1.09 mmol) were
dissolved in DMF (6.20 mL). Et3N (0.150 mL, 1.10 mmol) and DEPC (0.160 mL, 1.00
mmol) were added sequentially to the solution at 0 oC, and the mixture was stirred at 0 oC for
45 min. The resultant mixture was diluted with EtOAc (150 mL) and washed with
satd.NaHCO3 aq. (50 mLx3), satd. NaCl aq. (50 mLx3) and dried over anhyd. Na2SO4. After
the organic layer was concentrated in vacuo, the residue was purified by silica gel column
chromatography (CH2Cl2 to CH2Cl2/EtOAc (87.5: 12.5)) to give a colorless solid; m.p.
o
22
1
103-105 C; [α]D -30.6 (c 0.54, CHCl3); H-NMR (300 MHz/CDCl3) δ: 8.82 (1H, br s),
8.54 (1H, d, J = 8.2 Hz), 8.20 (1H, dd, J = 7.9, 1.5 Hz), 7.54-7.46 (3H, m), 7.27-7.24 (2H, m),
7.08-7.03 (3H, m), 4.74 (1H, dd, J = 8.2, 6.9 Hz), 4.02 (3H, s), 2.50-2.38 (1H, m), 1.12 (3H,
13
d, J = 6.8 Hz), 1.10 (3H, d, J = 6.8 Hz); C-NMR (75 MHz/CDCl3) δ: 170.0, 165.8, 138.0,
133.2, 132.2, 128.7, 124.0, 121.2, 121.0, 120.1, 111.5, 59.8, 56.1, 30.9, 19.5, 18.4; IR (KBr)
3284, 2958, 1635, 1601, 1535, 1489, 1444 cm-1; HRMS (FAB) m/z calcd for C19H23N2O3
[M+H]+ 327.1709, found 327.1719.
Chemical and Pharmaceutical Bulletin Advance Publication

R
O
R
H
N
H
N
1: R= i-Pr
2: R= Me
3: R= Bn
4: R= t-Bu
5: R= i-Pr
6: R= Me
7: R= Bn
8: R= t-Bu
H2N
N
H
O
O
OH O
OH
OMe O
OMe
OMe O
H
N
H
N
H
N
N
H
N
H
N
H
O
O
O
O
9
10
11
Boc
O
O
O
H
N
N
H2N
OEt
HO
Ts
H2N
OEt
Ph
NBoc
N
Ph
Boc
NHBoc
12
13
14
15
Fig. 2. Structures of the compounds.
General Procedure for the Preparation of Polymers:
Preparation of a Molecular-Imprinted Polymer (MIP) (Table 1): We prepared several
types of polymers as follows. A N-benzoyl-(S)-valine anilide ((S)-5)-imprinted MIP (IP-2)
was prepared by the polymerization of a mixture of (S)-5, ethylene glycol dimethacrylate
(EGDM), methacrylic acid (MAA), and AIBN in chloroform under irradiation followed by an
appropriate amount of grinding and washing. In a similar manner, IP-3 and IP-4 were
prepared by following the IP-2 procedure. In a glass vial, (S)-5 (889 mg, 3.00 mmol) was
dissolved in chloroform (12.0 mL). To this solution were added MAA (1.00 mL, 12.0 mmol),
EGDM (9.00 mL, 48.0 mmol), and AIBN (99.6 mg, 0.610 mmol). After sufficient mixing, the
solution was separated into four borosilicate screw-capped test tubes, and each test tube was
flushed with argon gas followed by irradiation with a 100 W high-pressure mercury vapor
lamp (5°C, 24 h) to form a polymer. Bulk polymers were ground with a mill (OSAKA
chemical, Wonder Blender, WB-1) and a ball mill (Nitto kagaku, ANZ-50S). These were then
sieved through a 500 µm mesh filter, and particles of <500 µm were collected. The polymer
was washed (by soxhlet extractor (MeOH/AcOH (7: 3)), 20 h), filtered (Kiriyama funnel
(filter paper: No. 5B)), immersed in THF, and refluxed for 5 hours. The polymer was
Chemical and Pharmaceutical Bulletin Advance Publication

collected by filtration (Kiriyama funnel (filter paper: No. 5B)), washed again (CH2Cl2), and
dried in vacuo.
The preparation of a (S)-valine anilide ((S)-1)-imprinted MIP (IP-1) has been reported
previously.13)
Table 1. Composition of the MIPs.
Entry Polymer Template Template (mmol) MAA (mmol) EGDM (mmol) CHCl3 (mL)
1
2
3
4
IP-1a)
IP-2
IP-3
IP-4
(S)-1
(S)-5
10
1
4
12
1
16
48
4
4
12
1
3
0.25
0.25
1
4
1
11
a) See ref. 13.
General Procedure for Evaluating the Ability of a MIP Using the “Batch Procedure”: In
a glass vial, (RS)-5 (2.60 µmol) was dissolved in a mixture of CH2Cl2 and n-hexane (10 mL;
CH2Cl2/ hexane (1: 1)). This concentration was defined as [5]0, which was an average of [5]S0
(concentration value of (S)-form) and [5]R0 (concentration value of (R)-form). To this solution
was added a MIP (46.0 mg, 13.0 µmol as recognizing sites), followed by stirring at 24 °C for
2 h. The MIP was filtered off, and the concentrate of this filtrate was defined as [5]S and [5]R.
[5]0 and [5] were determined by HPLC15) using N-p-toluenesulfonyl-2-aminoethanol
(Ts-C2AA; 12)16) as an internal standard. The adsorbed amount and selectivity were
calculated using the following equations:
The adsorbed amount of the S form:
Sad (%) = {([5]0 - [5]S ) / [5]0} x 100
The adsorbed amount of the R form:
Rad (%) = {([5]0 - [5]R ) / [5]0} x 100
The enantiomeric excess in solution (% e.e.s.)
= {([5]S - [5]R ) / ([5]S + [5]R )} x 100
Chemical and Pharmaceutical Bulletin Advance Publication

The enantiomeric excess in the polymer (% e.e.p.)
= {(Sad- Rad)/(Sad+ Rad)}x 100
Sad and Rad were used as the indices of adsorbability, and e.e.p. was used as an index of
selectivity.
The number of recognition sites in the MIP was calculated by dividing the number of
template molecules (mmol) by the assumption of 100% of the yield of MIP (g), i.e.,
3 (mmol) / 10.54 (g) ≈ 0.285 (mmol/g)
In this paper, we calculated the equivalents of the MIPs based on this value.
Application: asymmetric amination
Preparation of cat. A and cat. B: (RS)-9 (65.7 mg, 0.200 mmol) was dissolved in a mixture
of cyclohexane, CH2Cl2 and EtOAc (153 mL; c-hex/ CH2Cl2/ EtOAc = 7/ 2/ 1). To this
solution was added IP-2 (3.50 g), which was stirred at r.t. for 30 min. The IP-2 was filtered
off (Kiriyama funnel (filter paper: No. 5B)) and washed with cyclohexane (15 mL). The
combined filtrates were concentrated in vacuo to obtain unadsorbed 9, with a slight incline to
the (R)-isomer (7% e.e.) as a colorless solid. To the solid was added a mixed solvent of
n-hexane and CH2Cl2 (4 mL; hexane/ CH2Cl2 (6: 4)), followed by filtration (minisalt SRP 15)
to remove the racemate ((RS)-9) as a solid (95% recovered yield). The filtrate was
concentrated in vacuo to afford (R)-9 (1.5 mg) with 94% e.e. as a colorless solid that was
used as cat. A.
The filtered MIP was washed with MeOH (15 mL x 3) and CH2Cl2 (15 mL x 1). The
eluent was concentrated in vacuo, followed by the same procedure as the filtered solution
described above to give (S)-9 (1.5 mg) with 94% e.e. as a colorless solid that was used as cat.
B.
The recovered MIP was reused after desiccation in vacuo, without further operation.
Chemical and Pharmaceutical Bulletin Advance Publication

Asymmetric amination14,17)
The cat. A and La(NO3)3･6H2O (4.40 mg, 10.0 mmol) were dissolved in EtOAc under an
argon atmosphere. To the solution was added H-D-Val-OtBu in EtOAc (60.0 mL, 30.0 mmol)
and ethyl 3-amino-3-oxo-2-phenylpropanoate 13 at r.t., and the reaction mixture was cooled
to 0 °C. To the mixture was added di-tert-butyl azodicarboxylate 14 (55.3 mg, 0.240 mmol)
in EtOAc (0.20 mL) at 0 oC with stirring for 48 hr. The reaction was quenched by the addition
of 1M HCl aq. (1.0 mL), and the resultant mixture was extracted with CH2Cl2 (5.0 mL x 3).
The organic layer was washed with satd. NaHCO3 aq. (5.0 mL x 3), satd. NaCl aq. (5.0 mL x
3), and dried over anhyd. Na2SO4. After concentration in vacuo, the residue was purified by
silica gel column chromatography (hexane/ EtOAc = 5: 1 to 3 :1) to give (R)-15 (80.1 mg,
0.183 mmol, 92 %, 97 % e.e.) as a colorless foam: 1H-NMR (300 MHz/CDCl3) δ: 8.65 - 8.53
(1H, br m), 7.68 - 7.57 (2H, m), 7.47 - 7.28 (3H, m), 6.66 (1H, br s), 6.29 - 6.17 (1H, br m),
4.34 - 4.29 (1H, br m), 1.49 - 1.09 (21 H, m).
The same experiment using cat. B instead of cat. A gave (S)-15 (86.6 mg, 0.198 mmol,
99 %, 97 % e.e.) as a colorless foam.
The enantiomeric excess of the obtained products was determined via HPLC; DAICEL
o
CHIRALPAK AD-H, hexane/ MeOH= 95/ 5, 1.0 mL/min, 40 C, 254 nm, tR= 7.8 min (S),
10.4 min (R). Spectroscopic data was consistent with that previously reported.17)
Result and discussion
Evaluation of IP-2 and the solvent effect
We first evaluated the adsorbability of the racemic N-benzoyl-valine anilide ((RS)-5)
onto IP-2 in dichloromethane via batch procedure (Table 2). As described above, IP-2 was
prepared using hydrogen-bonding interactions as a main force in the pre-polymerization step
and IP-1 was similarly prepared using an acid-base interaction.13) Despite the lack of a
stronger acid-base interaction that was advantageous for molding the recognition sites,
surprisingly, IP-2 showed a higher stereoselectivity than expected (entry 1). In CH2Cl2,
N-benzoylvaline anilide exhibited lower adsorbability onto IP-2 than that of a valine anilide
onto IP-1 (see Table 3, entry 2). Such a weak point for IP-2, however, was overcome by
Chemical and Pharmaceutical Bulletin Advance Publication

decreasing the polarity of the solvent, and adequate adsorbability of IP-2 was achieved.
Comparing the results of adsorbability and enantioselectivity, we chose a mixed-solvent
system that consisted of dichloromethane and n-hexane (1: 1) for further research (entry 2).
Table 2. The effects of solvents for the adsorbability of (RS)-5 onto IP-2.
Entry Solvent
Sad (%)
25
Rad (%)
12
e.e.p. (%)
e.e.s. (%)
1
2
3
CH2Cl2
35
21
8
8
CH2Cl2 : c-Hex = 50 : 50
CH2Cl2 : c-Hex = 25 : 75
46
30
13
12
64
54
a) Conditions: (RS)-5, 0.26 mM, IP-2 (5 eq), 24 oC, 2 hr.
Comparison of substrates
In our previous report, we found that IP-1 synthesized using (S)-valine anilide as a
template molecule, with the aid of a stronger acid-base interaction than hydrogen bonding,
could recognize not only the enantiomers of a template molecule but also those of similar
amino acid derivatives (Ala, Phe and t-Leu anilides), which showed similar
enantioselectivities for all substrates, but also variations in the amount of adsorbed substrates
(Table 3).13)
Table 3. Enantioselective adsorption of amino acid anilides 1-4 by IP-1.a,b)
Entry
Substrate
Sad (%)
Rad (%)
e.e.p. (%) e.e.s. (%)
1
36
25
18
20
8
2
45
30
12
Chemical and Pharmaceutical Bulletin Advance Publication

3
4
22
38
15
26
20
18
4
9
a) Conditions: 0.26 mM, CH2Cl2, IP-1 (3 eq), 24 oC, 2 hr.
b) See ref. 13.
Based on such curious results, we next evaluated the adsorbabilities and
enantioselectivities of IP-2 toward various types of N-benzoyl amino acid anilides (Table 4).
Table 4. Enantioselective adsorption of N-benzoyl amino acid anilides 5-8 by IP-2.a)
Entry
Substrate
Sad (%)
Rad (%)
e.e.p. (%) e.e.s. (%)
1
40
28
18
21
9
2
3
46
36
30
27
13
14
19
7
9
4
38
26
a) Conditions: 0.26 mM, CH2Cl2/c-Hex = 1/1, IP-2 (5 eq), 24 oC, 2hr.
Chemical and Pharmaceutical Bulletin Advance Publication

To our delight, as shown in Table 4, IP-2 could effectively differentiate not only the
enantiomers of template molecules, N-benzoyl valine anilide 5, but also those of similar
N-benzoyl amino acid derivatives 6, 7, and 8 to yield slightly (R)-enriched solutions, along
with preferentially (S)-adsorbed IP-2. N-benzoyl phenylalanine anilide 7 possessed benzyl
moieties that were larger than those of methyl, isopropyl, or tert-butyl groups in the amino
acid scaffold and, intriguingly, it was adsorbed by IP-2 in a ratio similar to other N-benzoyl
amino acid anilides, but with an inferior level of enantioselectivity. The trend in these results
concerning the adsorption of phenylalanine anilide onto IP-2 was apparently different than
the results with IP-1, which showed lower adsorbability and a similar enantioselectivity in
the presence of acid-base interactions (see Table 3).
According to observations from the present study, the different properties of IP-1 and
IP-2 with regard to adsorbability and enantioselectivity can be summarized as follows. The
adsorbability of IP-1 was strongly influenced by the basicity of amino acids. Phenylalanine
has a lower basicity than other amino acids such as alanine, valine or tert-leucine18), which is
suspected to be caused by sterical bulkiness, and, therefore, the weaker acid-base interaction
between the acidic moiety in the mold of a MIP and phenylalanine showed adsorption that
was inferior to those of other amino acid anilides. However, once the amino acid anilides
were attracted to the recognition sites of a MIP, the stronger acid-base interaction dominated
the steric repulsion between the mold and the substrates, which then resulted in similar
enantioselectivities for all the amino acid anilides we tested.
On the other hand, weak hydrogen-bonding attracting interactions and steric repulsion
existed between the mold of IP-2 and the N-benzoyl amino acid anilide substrates. Therefore,
the steric repulsion between IP-2 and the side chains of amino acids, particularly the benzyl
group of phenylalanine, would affect the enantioselectivities for the adsorbance of IP-2 in a
more apparent fashion than that for IP-1, which introduces a slight decrease in the
adsorbance of (S)-enantiomers to show a lower e.e.p. value. However, the strength of the
hydrogen bonding between IP-2 and N-benzoyl amino acid anilides seemed comparable,
which would lead to similar adsorbabilities for amino acid derivatives toward IP-2.
Chemical and Pharmaceutical Bulletin Advance Publication

Application
From our delightful results showing that MIPs molded using hydrogen bonding as a main
attracting force caused an incline in the enantiomeric ratio, we envisaged the application of
such a MIP to chiral amplification.19-21) Thus, treatment of a solution of racemic catalyst to a
MIP would cause a slight lean in the ratio of enantiomers, and the use of such an
“enantioenriched solution” for asymmetric synthesis via chiral amplification could produce
enantiomerically pure compounds. It is noteworthy that a racemic catalyst would produce
optically pure compounds with the help of a MIP that would be easily recovered and
recyclable.
Kumagai and Shibasaki, et al., previously reported the non-linear effect of chiral catalyst
917) for an asymmetric Mannich-type reaction. In their report, the catalyst was prepared by
intentional mixing of the pure enantiomers of (S)-9 and (R)-9. Considering the structural
similarities of 9 and 5, we attempted to apply the above-mentioned methodology to the
racemic solution of 9 for the generation of an enantioenriched solution of 9, followed by
application to an asymmetric Mannich-type reaction.
We first tried to prepare several types of MIPs for the enantioselective recognition of 9,
using various N-benzoyl-(S)-valine anilide derivatives (10, 11) as chiral templates. Except for
the use of 9,22) two types of MIPs, IP-3 and IP-4, using 10 and 11 as template molecules,
respectively, were newly synthesized (see Table 1).
Table 5. The effects of concentration and composition of the solvents for the enantioselective
adsorption of (RS)-9 by IP-2–IP-4.a)
Solvents (%)
Conc.
(mM)
0.26
0.26
0.26
1.3
MIP
Sad
(%)
40
Rad
(%)
31
e.e.p.
e.e.s.
Entry
(eq)
(%)
13
-
(%)
7
c-Hex
75
CH2Cl2 EtOAcb)
1
2
3
4
5
IP-2 (5)
IP-3 (5)
IP-4 (5)
IP-2 (1)
IP-2 (3)
20
20
20
20
20
5
5
5
5
5
75
20
20
-
75
24
22
5
1
75
23
20
7
2
1.3
75
53
48
4
4
Chemical and Pharmaceutical Bulletin Advance Publication

6
7
8
1.3
1.3
1.3
IP-2 (5)
IP-2 (5)
IP-2 (5)
75
70
75
20
20
15
5
69
47
52
64
41
47
4
7
5
8
6
5
10
10
a) Conditions: Substrate was used (RS)-9, 24 oC, 2 hr.
b) See ref. 23.
The adsorbability and enantioselectivity of IP-3 and IP-4, along with IP-2, was
evaluated in the presence of (RS)-9 as a substrate (Table 5, entries 1-3). As a result, contrary
to our expectations, only IP-2 gave satisfactory levels of adsorbability and enantioselectivity
to a racemic solution of 9, although its effect as a substrate was inferior to that of (RS)-5.
We next optimized the adsorption conditions (Table 5, entries 4-8). A greater
concentration of the substrate (RS)-5 led to a greater level of adsorption (entries 1 and 6). We
also concluded that the addition of a 3-5 equivalent amount of IP-2 was optimal for the
production of an adequate e.e.s. value (entries 4-6). The optimization of the composition of a
solvent system was also determined, and an increase in the proportion of EtOAc decreased
the amount of adsorption (entries 6-8).
Based on the results assembled in Table 5, the condition shown in entry 7 seemed to be
the optimized choice for an appropriate adsorbability that would be compatible with
enantioselectivity, and we applied this condition to the actual asymmetric Mannich-type
amination system. Thus, the treatment of a racemic solution of 9 with 5 equivalents of IP-2 at
room temperature for 2 hours followed by separation gave the filtrate including a slightly
(R)-inclined 9, which was transformed into (R)-enriched 9 with 94% e.e. by single
reprecipitation of the racemate. Moreover, adsorbed 9, which was slightly inclined to an
(S)-form, was also collected via the washing of IP-2 with methanol, followed by the same
procedure as the filtered solution described above to give (S)-enriched 9 with a 94% e.e. Both
of the gained enantiomers, (S)-9 and (R)-9, were applied to an asymmetric Mannich-type
amination reaction, and, to our delight, both enantiomers gave the aminated products, (R)-15
and (S)-15, respectively, in high yields with high enantioselectivities (Chart 1).
Chemical and Pharmaceutical Bulletin Advance Publication

single reprecipitation
cat. a
(94 %e.e., R)
filtration
filtration
firtrate
(7 %e.e.)
(RS)-9
+
IP-2
(5 eq)
r.t.
filtration
30 min
washed and
firtrate was single reprecipitated
cat. b
(94 %e.e., S)
MIP
O
O
cat. a (R)
La(NO3)3•6H2O (5 mol%)
-Val-OtBu (15 mol%)
H2N
OEt
Ph NBoc
yield 92 %
97 % e.e. (R)
H-
D
EtOAc
O
O
Boc
NHBoc
0 oC, 48 hr
N
N
+
H2N
OEt
cat. b (S)
O
O
Boc
Ph
La(NO3)3•6H2O (5 mol%)
yield 99 %
97 % e.e. (S)
-Val-OtBu (15 mol%)
H2N
OEt
Ph NBoc
H-
L
EtOAc
0 oC, 48 hr
NHBoc
Chart 1. Application to chiral amplification.
Conclusions
We prepared a novel type of N-benzoyl-(S)-valine anilide-imprinted polymer, IP-2, using
hydrogen-bonding interactions as a main force for molding and evaluated the details of the
performance of IP-2 via batch procedure. It appeared that IP-2 could differentiate not only
the enantiomer of a template molecule but also those of other types of N-benzoyl amino acid
anilides, which led to the preferential adsorption of N-benzoyl-(S)-amino acid anilides in
good order. Interestingly, the properties of IP-2 differed from those of IP-1, which was
synthesized using an acid-base interaction for the molding of adsorbability and
enantioselectivity.
Based on such curious findings, we also found that IP-2 could recognize the enantiomers
of valine-derived bis(2-hydroxyphenyl)diamide 9, the analogue of N-benzoylvaline anilide 5,
and that both (R)- and (S)-enriched 9 with 94% e.e. were easily afforded from the filtrate and
filtered MIP, respectively, with the help of a single reprecipitation of the racemate. The (R)-
and (S)-enriched 9 was applied to an asymmetric Mannich-type amination reaction, and the
Chemical and Pharmaceutical Bulletin Advance Publication

corresponding aminated products were obtained in high yields with high enantioselectivities.
These intriguing results clearly prove that the treatment of a racemic catalyst with MIP
triggered a highly stereoselective asymmetric synthesis via chiral amplification. Furthermore,
adsorbed MIP was reusable after an appropriate work-up (washing with polar solvent,
substitution to a non-polar solvent and desiccation).24) Thus, we proved the great potential of
a MIP for application to asymmetric syntheses and to recyclability for environmental
friendliness.
Further, and more precise, mechanistic studies and synthetic applications to various
asymmetric reactions are currently in progress.
Conflict of Interest
The authors declare no conflict of interest.
Chemical and Pharmaceutical Bulletin Advance Publication

References and notes
1) “Molecularly Imprinted Polymers: Man-Made Mimics of Antibodies and Their Application
in Analytical Chemistry,” ed. By Sellergren B., Elsevier, 2001.
2) Takeuchi T., Matsui J., Acta Polym., 47, 471- 480 (1996).
3) Kempe M., Mosbach K., J. Chromatogr. A, 694, 3- 13 (1995).
4) Matsui J., Nicholls I. A., Takeuchi T., Tetrahedron Asymmetry, 7, 1357- 1361 (1996).
5) Simon R., Collins M. E., Spivak D. A., Anal. Chim. Acta., 591, 7- 16 (2007).
6) Xu X., Liang S., Meng X., Zhang M., Chen Y., Zhao D., Li Y., J. Chromatogr. B, 988, 182-
186 (2015).
7) Stringer C. R., Gangopadhyay S., Grant A. S., Anal. Chem., 82, 4015- 4019 (2010).
8) Liang R.-N., Song D.-A., Zhang R.-M., Qin W., Angew. Chem. Int. ed., 49, 2556- 2559
(2010).
9) Dong X., Sun H., Lii X., Wang H., Liu S., Wang N., Analyst, 127, 1427- 1432 (2002).
10) Yin J., Yang G., Chen Y., J. Chromatogr. A., 1090, 68- 75 (2005).
11) Jin X., Wang Y., Bai W., Zhao D., Song X., Xu X., J. Macromol. Sci. Part A: Pure Appl.
Chem., 52, 316-321 (2015).
12) Scorrano S., Mergola L., Del Sole R., Vasapollo G., Int. J. Mol. Sci., 12, 1735- 1743
(2011).
13) Yasuyama T., Matsunaga H., Shin A., Ishizuka T., Chem. Pharm. Bull., 61, 546- 550
(2013).
14) Mashiko T., Kumagai N., Shibasaki M., J. Am. Chem. Soc., 131, 14990- 14999 (2009).
15) HPLC condition were as follows; Table 2, table 4, entry 2: DAICEL CHIRALPAK IA,
hexane-EtOH= 85: 15, 0.75 mL/min, 254 nm, 12 as internal standard, tR= 8.8 min (R), 18.4
min (S), 27.8 min (internal standard); Table 4, entry 1: DAICEL CHIRALPAK IA,
hexane-EtOH= 85: 15, 1.0 mL/min, 254 nm, 12 as internal standard, tR= 9.2 min (R), 13.0
min (S), 19.6 min (internal standard); Entry 3: DAICEL CHIRALPAK IA, hexane-EtOH= 85:
15, 1.0 mL/min, 254 nm, 12 as internal standard, tR= 13.7 min (S), 16.1 min (R), 19.4 min
(internal standard); Entry 4: DAICEL CHIRALPAK IA, hexane-EtOH= 85: 15, 0.75 mL/min,
254 nm, 12 as internal standard, tR= 6.7 min (R), 9.6 min (S), 25.7 min (internal standard);
Chemical and Pharmaceutical Bulletin Advance Publication

Table 5: DAICEL CHIRALPAK IC, hexane-EtOH= 95/5, 0.75 mL/min, 254 nm,
N-benzoyl-2-phenethylamine as internal standard, tR= 11.8 min (S), 13.4 min (R), 26.1 min
(internal standard).
16) Fukudome Y., Naito H., Hata T., Urabe H., J. Am. Chem. Soc., 130, 1820- 1821 (2008).
17) Matsuzawa A., Nojiri A., Kumagai N., Shibasaki M., Chem. Eur. J., 16, 5036- 5042
(2010).
18) Predicted pKa values of the protonated amines shown in SciFinder (calculated by
ACD/Labs); phenylalanine: 9.20±0.15; alanine: 9.61±0.13; valine: 10.21±0.25; tert-leucine:
10.51±0.38.
19) Girard C., Kagan B. H., Angew. Chem. Int. Ed., 37, 2922- 2959 (1998).
20) Ishizuka T., Yakugaku Zasshi, 117, 339- 352 (1997).
21) Hui Y., Jiang J., Wang W., Chen W., Cai Y., Lin L., Liu X., Feng X., Angew. Chem. Int.
Ed., 49, 4290- 4293 (2010).
22) Unfortunately, compound (RS)-9 did not tolerated radical polymerization condition and
we could not prepare the corresponding MIP.
23) (RS)-9 could not dissolve in non-polar solvent without polar co-solvent. For this reason,
ethyl acetate was added in the presence of (RS)-9 as a substrate.
24) The recyclability of IP-2 is shown in Table 6. These results were part of the adsorption
experiments of (RS)-9 using IP-2, as shown in Chart 1.
Table 6. Recyclability of IP-2a)
Run
1
2
Sad (%)
46
Rad(%)
39
e.e.s. (%) e.e.p. (%)
8
9
9
7
7
6
44
44
36
37
3
a) Conditions: (RS)-9 (0.20 mmol), IP-2 (5 eq), c-Hex/ CH2Cl2/ EtOAc = 7/ 2/ 1, r.t., 30
min.
Through the recycling process, none of the turbidity by the pulverization of the polymer
particle in the filtrate was found. Considering this observation and the results of Table 6, we
concluded that IP-2 was reusable.
Chemical and Pharmaceutical Bulletin Advance Publication