Water-Soluble Molecularly Imprinted Nanoparticle Receptors with Hydrogen-Bond-Assisted Hydrophobic Binding

Article abstract of DOI:10.1021/acs.joc.6b01191

Molecularly imprinted nanoparticles (MINPs) were prepared when surfactants with a tripropargylammonium headgroup and a methacrylate-functionalized hydrophobic tail were cross-linked in the micelle form on the surface and in the core in the presence of hydrophobic template molecules. With the surfactants containing an amide bond near the headgroup, the MINPs had a layer of hydrogen-bonding groups in the interior that strongly influenced their molecular recognition. Templates/guests with strong hydrogen-bonding groups in the midsection of the molecule benefited most, especially if the hydrophobe of the template could penetrate the amide layer to reach the hydrophobic core of the cross-linked micelles. The location and the orientation of the hydrophilic groups were also important, as they determined how the template interacted with the surfactant micelles and, ultimately, with the MINP receptors.

Full text of DOI:10.1021/acs.joc.6b01191

Subscriber access provided by La Trobe University Library
Article
Water-Soluble Molecularly Imprinted Nanoparticle Receptors
with Hydrogen-Bond-Assisted Hydrophobic Binding
Md Arifuzzaman, and Yan Zhao
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b01191 • Publication Date (Web): 27 Jul 2016
Downloaded from http://pubs.acs.org on July 29, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
The Journal of Organic Chemistry is published by the American Chemical Society.
1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 29
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
Water-Soluble Molecularly Imprinted Nanoparticle
Receptors with Hydrogen-Bond-Assisted Hydrophobic
Binding
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
MD Arifuzzaman and Yan Zhao*
Department of Chemistry, Iowa State University, Ames, Iowa 50011-3111
zhaoy@iastate.edu
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
RECEIVED DATE
ABSTRACT. Molecularly imprinted nanoparticles (MINPs) were prepared when surfactants with a
tripropargylammonium headgroup and a methacrylate-functionalized hydrophobic tail were cross-linked
in the micelle form on the surface and in the core in the presence of hydrophobic template molecules.
With the surfactants containing an amide bond near the headgroup, the MINPs had a layer of hydrogen-
bonding groups in the interior that strongly influenced their molecular recognition. Templates/guests
with strong hydrogen-bonding groups in the midsection of the molecule benefited most, especially if the
hydrophobe of the template could penetrate the amide layer to reach the hydrophobic core of the cross-
linked micelles. The location and the orientation of the hydrophilic groups were also important, as they
determined how the template interacted with the surfactant micelles and, ultimately, with the MINP
receptors.
1
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 2 of 29
Introduction
1
2
3
4
5
6
7
8
9
Molecular recognition is at the heart of nearly every biological process, be it enzymatic catalysis,
ligand–receptor binding, selective transport of nutrients across membranes, or gene expression. To
recognize a guest molecule with high affinity and selectivity, the host needs to possess a binding
interface complementary to the guest in size, shape, and distribution of functional groups. Over the last
several decades, a great number of synthetic hosts with such features have been prepared, often through
molecular synthesis.1-3 The benefit of molecular synthesis is that well-defined host molecules can be
obtained and numerous methods (spectroscopic or otherwise) may be used to study the host–guest
complexes. On the other hand, molecular hosts, typically larger and more complex than the guest
molecules, require significant synthetic efforts to prepare. For guest molecules with complex
functionality and shape, designing hosts with good (let alone perfect) complementarity can be extremely
challenging.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Molecular imprinting is a conceptually different method to create guest-complementary hosts.4-14 In a
typical procedure, free radical polymerization is induced in a mixture of template molecules, functional
monomers (FMs) that bind the templates by noncovalent or covalent bonds, and cross-linkers to
maintain rigidity of the resulting polymer. The cross-linked material, after removal of the templates by
washing or bond cleavage, is left with cavities complementary to the templates and thus considered
“molecularly imprinted” with the templates. The method has been adopted by numerous researchers for
a wide range of applications in molecular recognition, separation, enzyme-mimetic catalysis, and
chemical sensing. In addition to traditional macroporous polymers, imprinting could occur on surface
and even unimolecularly within dendrimers.15,16
An extremely attractive feature of molecular imprinting is the simplicity in the design and preparation
of molecularly imprinted polymers (MIPs). Free radical polymerization is easy to perform and guest-
complementary binding sites are created by the imprinting process, without the need of custom design
for each individual guest. As a result, the method can be used by scientists without substantial training
in supramolecular chemistry and organic synthesis.
2
ACS Paragon Plus Environment

Page 3 of 29
The Journal of Organic Chemistry
Nonetheless, there remain a number of challenges in this technique. For example, it is generally
1
2
3
4
5
6
7
8
9
accepted that binding sites in MIPs are heterogeneous and poorly defined in structure.4-6,8-12,17,18
Conventional MIPs are intractable macroporous polymers and limited methods are available to
understand their structure and binding properties.19 Template molecules are frequently trapped deep
inside the cross-linked and polymeric matrix and difficult to be removed. Although soluble nanoparticle
MIPs have been reported,20-28 aqueous compatibility remains a challenge.29
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
We have been interested in the design and synthesis of functional receptors through biomimetic
strategies.30,31 Recently, we reported a method to imprint within cross-linked surfactant micelles to
create molecularly imprinted nanoparticles (MINPs). Our method worked well for a number of water-
soluble molecules including bile salt derivatives,32 aromatic carboxylates and sulfonates,33-35 and
nonsteroidal anti-inflammatory drugs (NSAIDs).36 In addition, we could install specific functional
groups in the MINP binding site and chemically modify the functional groups.34 Because MINPs are
fully soluble in water and similar to protein in size (40–60 KD in MW), we could study their binding by
techniques used for molecular receptors such as fluorescence titration, isothermal titration calorimetry
(ITC), and chemical derivatization. They are similar to water-soluble proteins in topology, having a
hydrophilic exterior and a hydrophobic core with guest-complementary binding sites.
The most important building block in the MINP synthesis is the cross-linkable surfactant that serves
multiple roles in the molecular imprinting. Its micellization defines the boundary for polymerization and
cross-linking, and enables the highly cross-linked MINPs to be fully soluble due to their nanosize and
solubilizing ligands on the surface. It serves as the FM to bind the template (mainly through
hydrophobic and electrostatic interactions), as well as one of the cross-linkers to maintain integrity of
the binding site. Its alkynyl headgroup allows facile surface-decoration of the nanoparticles with
different functional groups.
In this paper, we report two new cross-linkable surfactants synthesized from more readily available,
less expensive starting materials than the previously reported surfactant. Importantly, their amide group
within the structure enables the surfactants to interact with the template or guest molecules by hydrogen
3
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 4 of 29
bonds, in addition to hydrophobic and electrostatic interactions. Our study also yielded interesting
insight into how the position of the amide bond affects the imprinting process and molecular recognition
of the MINPs. The binding pockets in the MINPs were found to be highly discriminating, even toward
very similar guest molecules.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Results and Discussion
Design, Syntheses, and Characterization of Cross-Linkable Surfactants. Our previously reported
MINPs were prepared using cross-linkable surfactant 1.32-34,36 Its tripropargylammonium headgroup
enables facile cross-linking and decoration of the micellar surface by the alkyne–azide click reaction
(Scheme 1).37,38 The hydrophobic C12 chain provides necessary amphiphilicity to the surfactant. The
methacrylate allows the surfactant to be cross-linked in the core (with DVB) by free radical
polymerization. Dynamic light scattering (DLS) showed that each MINP contained approximately 50
(cross-linked) surfactant molecules. Thus, with a surfactant/template ratio of 50:1 in the preparation, the
MINPs obtained typically have on average one binding site per nanoparticle. Doubling the amount of
template resulted in two binding sites per nanoparticle, as demonstrated in a previous study.32
4
ACS Paragon Plus Environment

Page 5 of 29
The Journal of Organic Chemistry
Scheme 1. Preparation of MINP by surface-cross-linking of micelle of 1, surface decoration of resulting
alkynyl-surface-cross-linked micelle (alkynyl-SCM) by ligand 3, and core-cross-linking of the resulting
material to afford MINP with an internal binding site complementary to the template molecule.
1
2
3
4
5
6
7
8
9
Surfactant
1
was synthesized in
a
three-step reaction from 1,12-dodecanediol—i.e.,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
monomethacrylation of the diol, conversion of the remaining alcohol to trifluoromethansulfonate
(triflate) with triflic anhydride, and nucleophilic substitution of the triflate with tripropargylamine.
Although the synthetic route is short, it requires two expensive reagents (i.e., triflic anhydride and
tripropargylamine), and a final ion exchange must be done to replace triflate with a more soluble anion
such as bromide.32
In this study, we designed two new cross-linkable surfactants prepared from commercially available
δ-undecalactone (Scheme 2). Ring opening of the lactone by 5 equiv ethylenediamine or
butylenediamine at room temperature afforded amine 5a and 5b, respectively. The amine could be used
in the next step simply after removing the excess ethylenediamine or butylenediamine by co-
evaporation with methanol under vacuum. Propargylation of 5a or 5b occurred readily in acetonitrile
with propargyl bromide and sodium bicarbonate, thus avoiding the more expensive tripropargylamine.
The ammonium salt (6a or 6b) was treated with methacryloyl chloride, which converted the hydroxyl
group on the hydrophobic tail into methacrylate to yield the final surfactant (4a and 4b).
Scheme 2. Syntheses of cross-linkable surfactants 4a and 4b from δ-undecalactone.
5
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 6 of 29
In addition to easier synthesis from less expensive starting materials, the new surfactants (4a and 4b)
have an internal amide bond on the hydrophobic backbone. Although one might think hydrogen bonds
in aqueous solution does not contribute too much to guest-binding, the hydrogen bonds in our case are
located within the micelle and ultimately within the hydrophobic core of the MINP. The local
hydrophobicity around the amide bonds should increase their strength and make them potentially
important to guest binding. Surfactants 4a and 4b differ in the number of carbons between the amide
bond and the tripropargylammonium headgroup. Because the ammonium headgroup has to stay on the
surface of the micelle/MINP, in contact with water, the hydrogen bonds are deeper within the
hydrophobic core of MINP4b (i.e., MINP prepared with 4b as the cross-linkable surfactant) than within
the core of MINP4a.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
One of the most important properties of a surfactant is its critical micelle concentration (CMC),
above which micelles begin to form. To determine the CMC, we used the method of pyrene
solubilization.39 The method in our hands afforded similar CMC values as those determined from the
reduction of surface tension for analogous surfactants.37,40 Typically, we prepared aqueous solutions of a
surfactant in different concentrations, with 0.1 µM of pyrene in water. Pyrene is an environmentally
sensitive fluorescent probe with five vibronic bands. The first band (I1) near 372 nm becomes stronger
in a more polar environment and the third (I3) near 384 nm changes little. The I3/I1 ratio thus increases
with decreasing environmental polarity.
As shown in Figure 1a,b, the I3/I1 ratio initially showed very small change but began to rise sharply as
the surfactant concentration increased beyond a certain point. The probe apparently was in the aqueous
phase in the beginning but entered a nonpolar environment at higher surfactant concentrations. The
inflection point of the curve normally is considered the CMC of the surfactant, and was 0.41 mM for 4a
and 0.27 mM for 4b. The lower CMC for the latter was reasonable given its higher hydrophobicity. It is
also possible that the deeper location of the amide in the hydrophobic core of the micelle strengthens the
hydrogen bonds among the amide groups and stabilizes the micelle in the meantime.
6
ACS Paragon Plus Environment

Page 7 of 29
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Figure 1. (a) Pyrene I3/I1 ratio as a function of [4a]. (b) Pyrene I3/I1 ratio as a function of [4b]. [pyrene]
= 0.1 μM
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Contribution of Hydrogen-Bonds to Molecular Imprinting and Guest-Binding. To understand
whether the amide group in 4a and 4b could enhance the guest binding of MINPs by hydrogen bonds,
we designed three template molecules 7–9 whose syntheses are shown in Scheme 3. The templates all
have a fluorescent dansyl group connected to an aminobenzoate derivative through sulfonamide. The
benefit of using dansyl as the hydrophobic group is its environmentally sensitive emission that enables
us to study the binding by fluorescence spectroscopy (vide infra), in addition to ITC. The choice of
having a carboxylate in the template is two-fold. First, its anionic nature makes it electrostatically
attracted to the cationic micelle and the final MINP. Second, incomplete template removal is frequently
a problem in conventional imprinting.19 Being ionic, the carboxylate has to stay on the surface of the
micelle to be solvated by water, while the dansyl group prefers to stay inside the micelle due to its
hydrophobicity. The carboxylate then serves as an anchor to keep the template near the micelle surface,
making the binding site easily vacated after imprinting and readily accessible to guest molecules during
re-binding.32
7
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 8 of 29
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 3. Syntheses of templates 7–9 from the corresponding aminobenzoic acids.
The three templates differ in the substitution of the aminobenzoate moiety. The different substitutions
keep the carboxylate and the sulfonamide in different distances. Since we expect the carboxylate of the
template to interact with the ammonium headgroup of the surfactant electrostatically and the
sulfonamide of the template with the amide of the surfactant by hydrogen bonds, it might be beneficial
to match the distance between the ionic and the hydrogen-bonding functional group in the two.
Preparation of MINPs followed Scheme 1 and detailed procedures are found in the Experimental
1
Section. In general, the surface-cross-linking and core-polymerization were monitored by H NMR
spectroscopy and DLS.32 1H NMR spectroscopy normally shows broadening/disappearance of
characteristic signals as the surfactant and DVB (core-cross-linker) undergo free radical polymerization.
DLS gives the size of the nanoparticles and allows us to estimate the molecular weight of the MINP and
the number of (cross-linked) surfactants within a MINP. The surface-cross-linked micelles previously
were characterized also by transmission electron microscopy (TEM) and mass spectrometry (after
cleaving the surface-cross-linkages).37
Figure 2a shows the emission spectra of template 7 upon titration with MINP1(7), i.e., MINP
prepared with cross-linkable surfactant 1 and template 7. The addition of MINP caused a large blue shift
in the emission maximum of dansyl from ~550 to ~470 nm, while greatly enhancing the emission
8
ACS Paragon Plus Environment

Page 9 of 29
The Journal of Organic Chemistry
intensity. Dansyl is known to fluoresce weakly in water but strongly in nonpolar environments; its
emission wavelength typically increases with increasing environmental polarity.41 The blue shift and
stronger emission indicate that the probe entered a nonpolar microenvironment during titration, in
agreement with its binding by MINP. The fluorescence data fit well to a 1:1 binding model and afforded
a binding constant (Ka) of (15.3 ± 1.1) × 104 M-1 in water (Figure 2b).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(b)
(a) 1,000
1,000
800
600
400
200
0
0-5.1 µM
800
600
400
200
0
400
500
600
0
1
2
3
4
5
λ (nm)
[MINP] (µM)
Figure 2. (a) Emission spectra of compound 7 in the presence of 0–5.1 µM of MINP1(7) in Millipore
water. [7] = 0.2 µM. λex = 340 nm. (b) Nonlinear least squares curve fitting of the fluorescence intensity
at 463 nm to a 1:1 binding isotherm.
The binding was also studied by ITC, one of the most reliable ways to study intermolecular
interactions.42 By measuring the heat change during the titration, ITC yields a wealth of information on
the binding, including the binding constant (Ka), enthalpy (ΔH), and the number of binding sites per
particle (N). The binding free energy (ΔG) can be calculated from Ka using equation -ΔG = RTln(Ka),
and ΔS can be calculated from ΔG and ΔH. As shown in Figure 3, the binding exhibited a
negative/favorable enthalpy, with Ka = (12.8 ± 0.3) × 104 M-1. The binding constant agreed well with the
value obtained by fluorescence titration. The average number of binding site per nanoparticle (N) was
1.3 ± 0.2, also consistent with the 1:1 binding model.
9
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 10 of 29
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. ITC curve obtained at 298.15 K from titration of MINP1(7) with 7 in water. MINP1(7) = 10
µM in the cell. The concentration of 7 in the syringe was 0.2 mM.
Table 1 summarizes the binding data obtained for the three model templates by different MINPs.
Entries 1–3 compare the effectiveness of molecular imprinting of the same template (7) by the three
different surfactants. The binding data, whether those from ITC or fluorescence titration, all point to 4a
as the most effective cross-linkable surfactant among the three. MINP4a(7) bound template 7 nearly one
order of magnitude stronger than MINP1(7) prepared from the original cross-linkable surfactant.
Although MINP4b(7) showed weaker binding than MINP4a(7), both amide-functionalized surfactants
clearly outperformed 1 as far as the binding affinity of the MINP was concerned.
10
ACS Paragon Plus Environment

Page 11 of 29
The Journal of Organic Chemistry
Table 1. Binding data for MINPs (obtained by ITC unless indicated otherwise)a
1
2
3
4
5
6
7
8
9
-∆G
(kcal/mol)
6.8
-∆H
T∆S
Entry
1
MINPb
Guest
N
Ka (104 M-1)
(kcal/mol) (kcal/mol)
MINP1(7)
7
1.3 ± 0.2
12.8 ± 0.3
(15.3 ± 1.1)
4.7 ± 0.9
7.99 ± 0.95
4.66 ± 0.1
3.15 ± 1.0
2.45 ± 0.24
1.36 ± 0.4
2.15 ± 0.6
3.28 ± 0.6
3.15 ± 0.92
9.54 ± 0.15
3.80 ± 0.05
1.3 ± 0.04
0.70 ± 0.03
-
2.2
0.1
3.1
2.8
5.2
5.9
4.8
4.3
3.9
-1.8
3.7
5.7
6.1
-
2
3
MINP4a(7)
MINP4b(7)
MINP1(8)
7
7
8
8
8
9
9
9
7
7
7
8
9c
7
9
7c
8.1
7.2
6.6
7.7
6.2
6.7
6.2
7.0
7.8
7.5
6.9
6.8
-
1.0 ± 0.1
1.5 ± 0.1
1.0 ± 0.3
0.5 ± 0.1
0.8 ± 0.1
0.8 ± 0.1
1.1 ± 0.1
0.8 ± 0.2
0.7 ± 0.1
1.5 ± 0.1
1.4 ± 0.1
1.6 ± 0.1
-
90.4 ± 2.8
(99.3 ± 1.6)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
46.6 ± 3.9
(40.2 ± 2.6)
4
7.70 ± 0.30
(8.2 ± 1.0)
5
MINP4a(8)
MINP4b(8)
MINP1(9)
42.6 ± 0.8
(35.6 ± 2.1)
6
22.3 ± 1.2
(25.7 ± 1.9)
7
12.0 ± 1.2
(11.0± 1.3)
8
MINP4a(9)
MINP4b(9)
MINP1:4a=1:3(7)
MINP1:4a=1:1 (7)
MINP1:4a=3:1 (7)
MINP4a(7)
MINP4a(7)
MINP4a(8)
MINP4a(8)
MINP4a(9)
34.0 ± 1.2
(33.0 ± 1.7)
9
14.7 ± 0.5
(21.7 ± 1.8)
10
11
12
13
14
15
16
17
51.6 ± 3.5
32.5 ± 2.7
12.6 ± 1.4
9.40 ± 0.17
-
6.2
6.5
-
1.6 ± 0.1
0.7 ± 0.1
-
9.07 ± 1.36
5.72 ± 0.13
-
0.25 ± 0.02
1.6 ± 0.09
-
6.5
4.9
-
11
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 12 of 29
18
MINP4a(9)
8
5.2
1.4 ± 0.3
7.87 ± 0.95
7.4 ± 0.2
0.6
1
2
3
4
5
6
a
The titrations were generally performed in duplicates in Millipore water and the errors between the
runs were <20%. The binding constants in parentheses were from fluorescence titration and thus the
b
number of binding sites and binding enthalpy/entropy were not available. The subscript denotes the
7
8
9
cross-linkable surfactant used in the MINP synthesis and the number in parentheses the template
molecule. c Binding was too weak to be detected by ITC.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The same trend was maintained for templates 8 and 9 (Table 1, entries 4–6 and 7–9), with the binding
affinity displaying a consistent order of MINP4a > MINP4b > MINP1. Thus, the two amide-functionalized
surfactants clearly worked better than the original 1 for the model templates, regardless of their
substitution pattern. The data also suggest that hydrogen bonds between the template and the cross-
linkable surfactants were important to the binding.
We also prepared nonimprinted nanoparticles from surfactant 1, 4a, and 4b, respectively. The same
synthetic procedure was followed except no template was used. ITC showed that none of the guest
molecules (7–9) could bind the nonimprinted nanoparticles (Figure 12S–14S). The results further
confirmed the molecular imprinting and ruled out nonspecific interactions between the MINPs and the
guests.
If we compare the binding affinities of MINPs prepared from the same surfactant for different
templates, MINP1 was practically insensitive to the substitution pattern of the template, with ITC-
measured Ka = 12.8, 7.70, and 12.0 × 104 M-1 for 7, 8, and 9, respectively (Table 1, entries 1, 4, and 7).
MINP4a and MINP4b, on the other hand, showed a consistent trend in their affinities, i.e., 7 > 8 > 9
(Table 1, compare entries 2, 5, 8, or 3, 6, 9). Note that the different Ka values do NOT reflect the binding
selectivity of the MINPs (which will be discussed later), as they are the binding constants between three
different template molecules and their corresponding MINPs.
Overall, two consistent trends were observed for the amide-functionalized MINPs regarding their
binding affinity: MINP4a > MINP4b for all three templates (7–9) and templates 7 > 8 > 9 for both
MINPs. These results, first of all, suggest there is no special benefit in matching the distance between
the ionic and the hydrogen-bonding functional group in the surfactant and the template. This is because
12
ACS Paragon Plus Environment

Page 13 of 29
The Journal of Organic Chemistry
the better surfactant (4a) of the two has a shorter distance between these groups and the best template
(7) among the three has the longest distance.
1
2
3
4
5
6
7
8
One possible reason for the observed binding trend of 7 > 8 > 9 is the depth of the hydrophobic
dansyl group. Compound 7 has the dansyl and carboxylate para to each other. Since the carboxylate has
to stay on the surface of the micelle/MINP, 7 is expected to have its dansyl deeper in the MINP
hydrophobic core than either the meta (8) or ortho (9) derivative. When both hydrogen-bonding and
hydrophobic interactions are involved in the guest binding, we need to maximize both interactions to
have the highest binding affinity.43 To engage in hydrogen-bonds, the sulfonamide of the template needs
to be close to the amide groups in the final MINP. Such an arrangement seems easily achievable for all
three templates, by their tilting to different degrees with the carboxylate anchored on the MINP surface.
To maximize the hydrophobic interactions, however, dansyl needs to penetrate the amide layer and
reach into the hydrocarbon core, and template 7 seems to have a clear advantage over the other two due
to its para substitution.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Why didn’t the substitution pattern of the template influence the binding of MINP1, prepared from
the amide-free surfactant? The most likely reason is the solvation of the sulfonamide group. Being
highly hydrophilic, the sulfonamide can be “solvated” properly either by water or through interactions
with the amide bonds of the surfactants. Since there are no amide groups in the micellar core of MINP1,
the only way for the sulfonamide to be properly solvated is to stay on the micellar surface, exposed to
water. When both the carboxylate and sulfonamide need to stay on the micellar surface, the
aminobenzoate moiety of the template is exposed to water and thus contributes little to binding. What is
most important to the binding under such a circumstance are the hydrophobic interactions from burying
the dansyl from solvent exposure and the electrostatic interactions between the oppositely charged host
and guest—both are fairly constant among the three templates.
We also prepared MINPs using a mixture of 1 and 4a in different ratios (Table 1, entries 10–12). The
original idea was that, in doing so, the hydrogen bonds between 4a and the template would be formed in
a “background” (or microenvironment) of hydrocarbon and might be stronger. Essentially, surfactant 1
13
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 14 of 29
would create a hydrocarbon-based micellar core, which could be drier and helpful to the hydrogen-
bonding interactions between 4a and the template. Instead, our data showed a monotonous decrease of
Ka with decreasing 4a in the MINP formulation (compare entries 2, 10, 11, 12, and 1), suggesting that
the hypothesis was incorrect.
1
2
3
4
5
6
7
8
9
A key feature of molecularly imprinted polymers is their binding selectivity toward structural
analogues. To understand this property, we titrated our MINPs with the “wrong” guest molecules, e.g.,
MINP4a(7) with 8 and 9 and likewise for the other MINPs. Our imprinted cross-linked micelles showed
good selectivity consistently. For example, MINP4a(7) bound its template 7 most strongly among the
three isomeric guests (entry 2). The binding constant decreased by nearly 10-fold for the meta derivative
8 (entry 13) and was undetectable for the ortho guest 9 (entry 14). For MINP4a(9) imprinted against the
ortho derivative, the strongest binding was observed for 9 as expected (entry 8). Binding for the meta
derivative 8 was about 1/4 as strong (entry 18) and undetectable for the para derivative 7 (entry 17). For
MINP4a(8) imprinted against the meta derivative, its best guest was certainly its own template (entry 5)
but, interestingly, both the para and ortho derivatives showed binding, albeit with a lower affinity
(entries 15 and 16). These data suggest the MINPs overall were very selective and bind their own
templates the best. In their binding of the “wrong” constitutional isomers, a mismatch of substitution by
1 (from 1,4 to 1,3; from 1,2 to 1,3; or from 1,3 to 1,2 or 1,4) gave weaker but measurable binding,
whereas a mismatch by 2 (from 1,2 to 1,4 or vice versa) could not be tolerated at all.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Binding of Other Guests. The above studies gave us a good understanding of how amide-
functionalized MINPs bind model guests containing hydrogen-bonding groups in the structure. Both
MINP4a and MINP4b clearly outperformed the original MINP1 without internal hydrogen-bonding
capabilities. To see whether the advantage is maintained with other templates, we generated MINPs
using 4a (the best surfactant in this study) for four other templates (10–13). All four templates have
been imprinted previously with surfactant 1. Among the four templates, bile salt 10 had multiple
hydrogen-bonding groups (hydroxyl and sulfonamide) arranged in a facially amphiphilic fashion.32 1-
Naphthoic acid sodium salt 11 had a hydrocarbon aryl hydrophobe but no (heteroatom) hydrogen-
14
ACS Paragon Plus Environment

Page 15 of 29
The Journal of Organic Chemistry
bonding functionalities in the hydrophobe (other than the carboxylate).33 Naproxen and Indomethacin
are both nonsteroidal anti-inflammatory drugs (NSAIDs), with Indomethacin 13 having a stronger
hydrogen-bonding amide group in the midsection of the molecule than Naproxen 12, which contains an
ether.36
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A quick glance of the binding data in Table 2 shows that the amide-containing surfactant (4a) was
not always a winner. For the two NSAIDs (12 and 13), MINP4a showed 30–40% stronger binding than
MINP1, but for templates 10 and 11, the opposite was true. Overall, the binding data are consistent with
the earlier notion that templates with internal hydrogen-bonding functionalities benefit most from the
amide-containing 4a. For example, as far as internal hydrogen-bonding capabilities are concerned,
templates 11–13 should follow the order of 11 < 12 < 13, as the template possesses an increasing
number of ether and amide. The relative binding constant of MINP4a to MINP1 (i.e., Krel in Table 2)
increased steadily from 0.2 to 1.3 to 1.4 for the three templates.
Table 2. Binding data for MINPs obtained by ITCa
-∆G
(kcal/mol)
8.3
-∆H
T∆S
b
Entry
1
MINP
Guest
N
Ka (104 M-1) Krel
(kcal/mol) (kcal/mol)
MINP4a(10)
10
0.9 ± 0.1
126 ± 6
347 ± 23
9.8 ± 0.6
42.9 ± 1.3
0.4
7.67 ± 0.3
33.1 ± 2.6
5.67 ± 0.9
6.7 ± 0.2
0.6
-24.2
1.1
MINP1(10)
MINP4a(11)
MINP1(11)
2
3
4
10
11
11
8.9
6.8
7.7
1.0 ± 0.1
0.7 ± 0.1
1.1 ± 0.1
1
0.2
1
1.0
15
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 16 of 29
MINP4a(12)
MINP1(12)
MINP4a(13)
MINP1(13)
5
6
7
8
12
12
13
13
8.3
8.1
8.4
8.2
0.7 ± 0.1
0.6 ± 0.1
0.5 ± 0.1
1.1 ± 0.1
115 ± 5
91 ± 4
140 ± 9
98 ± 5
1.3
1
1.20 ± 0.14
28.1 ± 6.0
1.48 ± 0.18
26.9 ± 1.0
7.1
-20.0
6.9
1
2
3
4
5
6
7
8
1.4
1
-18.7
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Binding was measured in 50 mM Tris buffer (pH = 7.4). The titrations were generally performed in
duplicates and the errors between the runs were <20%. The binding data for MINP4a were obtained in
the current study and those for MINP1 were taken from previous publications for templates 10,32 11,33
and 12–13.36
b Krel is the binding affinity of MINP4a relative to MINP1 for the same template.
Our model compounds 7–9 have stronger internal hydrogen-bonding capabilities than 11–13, with
the sulfonamide group possessing both hydrogen-bond donors and acceptors. Indeed, Krel was even
higher and was calculated to be 2.8 for 9, 5.5 for 8, and 7.1 for 7 from the binding data in Table 1. The
continuous increase of Krel from the ortho to meta to para template indicates that the template benefiting
most from the amide-containing surfactant had its hydrogen-bonding sulfonamide deepest in the MINP
hydrophobic core. The result is very reasonable: the deeper the hydrogen-bonding group into the
hydrophobic core, the more it can be shielded from water and the stronger the hydrogen bonds will be.
Once the above picture is made clear, it becomes quite obvious why the amide-functionalized
surfactant did not help bile salt derivative 10. Although the template has many hydrogen-bonding
groups, they are all on the α face of the cholate, opposite to the hydrophobic β face. Because
hydrophobic interactions are key to the incorporation of the hydrophobic template into the micelle, 10
might lie flat on the micellar surface, having the hydrophilic face toward water and hydrophobic face
toward the interior of the micelle. With such an orientation, the amide groups inside the micelle of 4a
would have little chance interacting with the template through hydrogen bonds. To make things worse,
the layer of amide bonds near the surface of the micelle would weaken the hydrophobic interactions
between 10 and the micelle (ultimately MINP4a). This could be the reason why MINP4a(10) displayed
much weaker binding toward the template than MINP1(10) (Table 2, entries 1 and 2). The same could
the reason why template 11 was bound more strongly by MINP1(11) than MINP4a(11).
16
ACS Paragon Plus Environment

Page 17 of 29
The Journal of Organic Chemistry
Conclusions
1
2
3
4
5
6
7
8
9
Our study so far gives a consistent mechanistic picture in the binding of amide-functionalized MINPs.
Surfactant 4a with two methylene groups between the tripropargylammonium headgroup and the amide
outperformed surfactant 4b with four methylene groups. In general, 4a was most effective in the
molecular imprinting of template molecules with strong hydrogen-bonding functionalities but the
location of these functional groups was also critical. Overall, the binding between amide-functionalized
MINPs and the templates are driven by a combination of hydrophobic and hydrogen-bonding
interactions (in addition to electrostatic interactions in our case). To maximize the hydrophobic
interactions, the template/guest molecule needs to possess a sizable hydrophobe but it is best for the
hydrophobe to penetrate the amide layer to reach into the hydrophobic core of the MINP. To maximize
the hydrogen-bonding interactions, the templates should possess strong hydrogen-bonding groups that
can favorably interact with the internal amide bonds geometrically.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The most significant learning in this work is a detailed understanding of how different intermolecular
interactions can be used rationally to enhance the binding of molecularly imprinted nanoparticle
receptors. MIPs are traditionally intractable cross-linked polymers that prohibit detailed study of their
binding mechanisms. The water-solubility, nanodimension, and fine-tunability of our MINPs gave us
tremendous opportunities to examine the details of binding. The knowledge generated will enable better
designs of these protein-mimetic “plastic antibodies” that could find many applications in chemistry and
biology.
Experimental Section
Syntheses of compounds 1, 2, 3, 4, and 10 were previously reported.32
N-(2-Aminoethyl)-5-hydroxyundecanamide (5a). δ-Undecalactone (1.0 mL, 5 mmol) and
ethylenediamine (1.5 g, 25 mmol) were stirred at room temperature for 4 h. The excess amine was
removed by co-evaporation with methanol (5 × 2 mL) in a rotary evaporator. The residual was
combined with ether (50 mL) and kept overnight. The precipitate was recovered by vacuum filtration
17
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 18 of 29
1
and washed with ether (3 × 30 mL) to afford a white powder (1.10 g, 84 %). H NMR (400 MHz,
1
2
3
CDCl3, δ): 6.11 (br, 1H, N-H), 3.62 (m, 1H), 3.30 (t, J = 8.0 Hz, 2H), 2.84 (t, J = 8.0 Hz, 2H), 2.37 (t, J
4
5
6
13
= 8.0 Hz, 2H), 1.88 (m, 2H), 1.67 (m, 2H), 1.43 (m, 2H), 1.26 (m, 8H), 0.85 (t, J = 8.0 Hz, 3H). C
7
8
9
NMR (100 MHz, CD3OD, δ): 176.5, 71.7, 42.0, 41.6, 38.3, 33.9, 33.3, 32.8, 30.6, 30.3, 26.6, 23.5, 14.2.
ESI-QTOF-HRMS (m/z): [M+H]+ calcd for C13H28N2O2 245.2224; found, 245.2219.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N-(2-(5-hydroxyundecanamido)ethyl)-N,N-di(prop-2-yn-1-yl)prop-2-yn-1-aminium
bromide
(6a). A mixture of 5a (0.80 g, 3.3 mmol) and NaHCO3 (0.87 g, 10.82 mmol) in CH3CN (2.00 mL) was
stirred at 70 °C while a solution of propargyl bromide (1.0 mL, 10.82 mmol) in dry CH3CN (2.0 mL)
was added dropwise over 10 min. The mixture was stirred for 16 h at 70 °C before another batch of
propargyl bromide (0.33 mL) and NaHCO3 (0.30 g) were added. The mixture was stirred for another 4
h. The inorganic salts were removed by filtration. After the solvent was removed by rotary evaporation,
the residue was purified by column chromatography over silica gel using 30:1
1
dichloromethane/methanol as the eluent to afford a yellowish oil (0.65 g, 77 %). H NMR (400 MHz,
CDCl3, δ): 8.52 (br, 1H, N-H), 4.82 (m, 1H), 4.78 (s, 6H), 3.93 (t, J = 8.0 Hz, 2H), 3.84 (t, J = 8.0 Hz,
2H), 3.59 (t, J = 8.0 Hz, 2H), 3.13 (s, 3H), 2.36 (m, 2H), 1.77 (m, 2H), 1.46 (m, 4H), 1.26 (m, 6H), 0.88
(t, J = 8.0 Hz, 3H) ppm. 13C NMR (100 MHz, CDCl3, δ): 175.2, 83.1, 71, 70, 58.5, 50.8, 37.6, 36.7, 36,
34.1, 31.8 29.4, 25.8, 22.64, 21.5, 14.1 ppm. ESI-QTOF-HRMS (m/z): [M-Br]+ calcd for C22H35N2O2,
359.2693; found, 359.2696.
N-(2-(5-(methacryloyloxy)undecanamido)ethyl)-N,N-di(prop-2-yn-1-yl)prop-2-yn-1-aminium
chloride (4a). A solution of methacryloyl chloride (0.1 mL, 1.0 mmol) in dry CH2Cl2 (0.5 mL) was
added dropwise over 10 min to a solution of 6a (0.193 g, 0.60 mmol) and diisopropylethylamine
(DIPEA , 0.14 mL. 1.5 mmol) in dry CH2Cl2 (10 mL) at 0 °C. After 30 min at 0 °C, the mixture was
stirred for 5 h at room temperature before a second batch of methacryloyl chloride (0.05 mL, 0.5 mmol)
was added. A third batch of methacryloyl chloride (0.05 mL, 0.5 mmol) was added after another 5 h and
the mixture was stirred at 40 °C for additional 5 h. After the solvent was removed by rotary evaporation,
18
ACS Paragon Plus Environment

Page 19 of 29
The Journal of Organic Chemistry
the residue was purified by column chromatography over silica gel using 4:1 ethyl acetate/methanol as
the eluent to afford a yellowish oil (0.10 g, 45%).1H NMR (400 MHz, CDCl3, δ): 8.44 (br, 1H, N-H),
6.1(s,1H), 5.5 (s, 1H), 4.90 (m, 1H), 4.80 (s, 6H), 4.10 (t, J = 8.0 Hz, 2H), 3.90 (t, J = 8.0 Hz, 2H), 3.84
(t, J = 8.0 Hz, 2H),3.03 (s, 3H), 2.30 (m, 2H), 2.04 (m, 2H), 1.92 (s, 3H), 1.62 (m, 4H), 1.26 (m, 6H),
0.88 (t, J = 8.0 Hz, 3H) ppm. 13C NMR (100 MHz, CDCl3, δ): 174.5, 167.3, 136.6, 125.2, 82.9, 74.3,
69.8, 60.4, 58.8, 50.8, 37.8, 35.9, 34, 31.7, 29.2, 25.2, 22.5,21.3, 18.4, 14 ppm. ESI-QTOF-HRMS
(m/z): [M-Cl]+ calcd for C26H39N2O3, 427.2955; found, 427.2961.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N-(4-aminobutyl)-5-hydroxyundecanamide (5b). δ-Undecalactone (3.0 mL, 15 mmol) and 1,4-
diaminobutane (6.6 g, 75 mmol) were stirred for 4 h at room temperature. The excess amine was
removed by co-evaporation with methanol (5 × 4 mL) in a rotary evaporator. The residual was
combined with ether (100 mL) and kept overnight. The precipitate was recovered by vacuum filtration
and washed with ether (3 × 30 mL) to afford a white powder (3.5 g, 80 %). 1H NMR (400 MHz, CDCl3,
δ): 6.0 (br, 1H, N-H), 3.50 (m, 1H), 3.18 (t, J = 8.0 Hz, 2H), 2.66 (t, J = 8.0 Hz, 2H), 2.14 (t, J = 8.0
Hz, 2H), 1.67 (m, 6H), 1.44 (m, 4H), 1.35 (m, 4H), 1.20 (m, 4H), 0.80 (t, J = 8.0 Hz, 3H) ppm. 13C
NMR (100 MHz, CD3OD, δ): 175.9, 71.8, 41.3, 39.7, 38.2, 37.6,36.9, 32.9, 30.3, 29.1, 27.5, 26.6, 23.5,
23.2, 14.2 ppm. ESI-QTOF-HRMS (m/z): [M-Br]+ calcd for C26H39N2O3, 273.2537; found, 273.2541.
4-(5-Hydroxyundecanamido)-N,N,N-tri(prop-2-yn-1-yl)butan-1-aminium bromide (6b).
A
mixture of 5b (0.40 g, 1.4 mmol) and NaHCO3 (0.41 g, 5.7 mmol) in CH3CN (5.00 mL) was stirred at
70 °C while a solution of propargyl bromide (0.46 mL, 5.7 mmol) in dry CH3CN (2.0 mL) was added
dropwise over 10 min. The mixture was stirred for 16 h at 70 °C before another batch of propargyl
bromide (0.2 mL) and NaHCO3 (0.30 g) were added. The mixture was stirred for another 4 h. The
inorganic salts were removed by filtration. After the solvent was removed by rotary evaporation, the
residue was purified by column chromatography over silica gel using 7:3 ethyl acetate/methanol as the
eluent to afford a yellowish oil (0.40 g, 72 %). 1H NMR (400 MHz, CDCl3, δ): 7.68 (br, 1H, N-H), 4.72
(s, 6H), 3.71 (t, J = 8.0 Hz, 2H), 3.60 (t, J = 8.0 Hz, 2H), 3.58 (m, 1H), 3.2 (m, 4H), 3.2 (m,4H), 3.03 (s,
19
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 20 of 29
3H), 2.15 (m, 2H), 1.76 (m, 2H), 1.70 (m, 4H), 1.40 (m, 4H), 0.86 (t, J = 8.0 Hz, 3H) ppm. 13C NMR
(100 MHz, CDCl3, δ): 175.5, 82.6, 71, 70.5, 69.9, 60.5, 50.3, 37.8, 37.3, 36.5, 36.1, 31.7, 29.5, 29.3,
25.9, 25.7, 22.5, 21.9, 3.9 ppm. ESI-QTOF-HRMS (m/z): [M-Br]+ calcd for C24H39N2O2 387.3006;
found, 387.2999.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4-(5-(Methacryloyloxy)undecanamido)-N,N,N-tri(prop-2-yn-1-yl)butan-1-aminium chloride (4b).
A solution of methacryloyl chloride (0.5 mL, 4.12 mmol) in dry CH2Cl2 (0.5 mL) was added dropwise
over 10 min to a solution of 6b (0.71 g, 1.82 mmol) and DIPEA (0.45 mL, 4.5 mmol) in dry CH2Cl2 (30
mL) at 0 °C. After 30 min at 0 °C, the mixture was stirred for 5 h at room temperature before a second
batch of methacryloyl chloride (0.23 mL, 1.88 mmol) was added. The mixture was stirred at 40 °C for
additional 8h. After the solvent was removed by rotary evaporation, the residue was purified by column
chromatography over silica gel using 4:1 ethyl acetate/methanol as the eluent to afford a yellowish oil
(0.47 g, 56%).1H NMR (400 MHz, CDCl3 δ): 6.0 (s,1H), 5.5 (s, 1H), 4.77 (s, 6H), 4.08 (t, J = 8.0 Hz,
2H), 3.60 (m, 2H), 2.81 (s, 3H) ,1.98 (m, 4H), 1.89 (s, 3H), 1.61 (m, 4H), 1.58 (m, 6H), 1.52 (m, 8H),
0.88 (t, J = 8.0 Hz, 3H) ppm. 13C NMR (100 MHz, CDCl3 & CH3OD, δ): 180.30, 171.57, 145.8, 129.4,
86.0, 78.1, 73.7, 58.6, 54.2, 46.8, 42.0, 37.8, 35.8, 33.0, 30.0, 29.1, 26.4, 24.3, 22.7, 22.4, 21.1, 17.8
ppm. ESI-QTOF-HRMS (m/z): [M-Cl]+ calcd for C28H43N2O3 455.3268; found, 455.3255.
Methyl 4-aminobenzoate (7a).44 To a solution of 4-amino benzoic acid (2.0 g, 14.6 mmol) in 40 mL
MeOH, concentrated H2SO4 (1.75 mL, 33 mmol) was added dropwise. After the mixture was heated to
reflux for 8 h, the solvent was removed under reduced pressure. Water (25 mL) was added and the
solution pH was adjusted to 3 with 2 M NaOH. The precipitate formed was collected by filtration and
washed with water (20 mL) to afford a white solid (1.94 g, 86%). 1H NMR (400 MHz, CDCl3, δ): 7.85
(dd, J = 8 & 4 Hz, 2H), 6.64 (dd, J = 8 & 4 Hz, 2H), 4.03 (br, NH2), 3.85 (s, 3H).
Methyl 4-((5-(dimethylamino)naphthalene)-1-sulfonamido)benzoate (7b).45 To a solution of
dansyl chloride (0.09 g, 0.33 mmol) in dry dichloromethane (20 mL) was added to a solution of
20
ACS Paragon Plus Environment

Page 21 of 29
The Journal of Organic Chemistry
compound 7a (0.05 g, 0.33 mmol) and triethylamine (0.07 mL, 0.66 mmol) in 3 mL dry
dichloromethane. The mixture was stirred at room temperature for 6 h under N2. After the solvent was
removed by rotary evaporation, the residue was purified by column chromatography over silica gel
using 2:1 ethyl acetate/hexane as the eluent to afford a white powder (0.10 g, 77%).1H NMR (400
MHz, CDCl3, δ): 8.53 (d, J = 8 Hz, 1H), 8.21 (d, J = 8 Hz, 1H), 7.77 (dd, J = 8 & 4 Hz, 2H), 7.41 (dd, J
= 8 & 4 Hz, 2H), 3.84 (s, 3H), 2.81 (s, 6H).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4-((5-(Dimethylamino)naphthalene)-1-sulfonamido)benzoic acid (7c).46 LiOH (5.0 mL, 2 M) was
added to a solution of compound 7b (0.17 g, 0.44 mmol) in MeOH (15 mL).The mixture was stirred for
12 h at room temperature. The mixture was concentrated under reduced pressure and acidified by 2 M
HCl. The precipitate formed was collected by filtration and dried in air to afford a white powder (0.10 g,
77%). To obtain the sodium salt of this compound, the above acid was mixed with saturated sodium
bicarbonate (2.0 mL) and methanol (5 mL). The reaction mixture was stirred for 2 h. After the solvents
were removed by rotary evaporation, the residue was dissolved in methanol (5 mL). The solution was
filtered and then concentrated by rotary evaporation to give the sodium salt as a white powder. 1H NMR
(400 MHz, CD3OD, δ): 8.81 (d, J = 8 Hz, 1H), 8.67 (d, J = 8 Hz, 1H), 8.56 (d, J = 8 Hz, 1H), 8.03 (dd,
J = 8 & 4 Hz, 2H), 7.85 (m, 2H),7.55 (d, J = 8 Hz, 1H), 7.35 (dd, J = 8 & 4 Hz, 2H), 3.13 (s, 6H).
Methyl 3-aminobenzoate (8a).47 To a solution of 3-amino benzoic acid (3.0 g, 22 mmol) in 40 mL
MeOH, concentrated H2SO4 (2.5 mL, 47 mmol) was added dropwise. After the mixture was heated to
reflux for 8 h, the solvent was removed under reduced pressure. Water (25 mL) was added and the
solution pH was adjusted to 3 with 2 M NaOH. The precipitate formed was collected by filtration and
washed with water (20 mL) to afford a white solid (2.71g, 82%).1H NMR (400 MHz, CDCl3, δ): 7.42
(dd, J = 8 & 4 Hz, 2H), 7.35 (s, 1H), 7.21 (t, J = 8 Hz, 2H), 6.85 (dd, J = 8 & 4 Hz, 2H), 3.89 (s, 3H).
Methyl 3-((5-(dimethylamino)naphthalene)-1-sulfonamido)benzoate (8b).48 To a solution of
dansyl chloride (0.3 g, 1.1 mmol) in dry dichloromethane (20 mL) was added to a solution of compound
21
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 22 of 29
8a (0.15 g, 1.0 mmol) and triethylamine (0.22 mL, 2.0 mmol) in 3 mL dry dichloromethane. The
mixture was stirred at room temperature for 6 h under N2. After the solvent was removed by rotary
evaporation, the residue was purified by column chromatography over silica gel using 2:1 ethyl
acetate/hexane as the eluent to afford a white powder (0.10 g, 77%). 1H NMR (400 MHz, CDCl3, δ): 8.6
(d, J = 8 Hz, 1H), 8.3 (d, J = 8 Hz, 1H), 7.01 (m, 2H), 7.79 (d, J = 8 Hz, 1H), 7.56 (dd, J = 8 & 4 Hz,
1H), 7.46 (s, 1H), 7.20 (m, 1H), 7.20 (m, 2H), 5.30 (s, 3H), 2.89 (s, 6H).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3-((5-(Dimethylamino)naphthalene)-1-sulfonamido)benzoic acid (8c).47 LiOH (5.0 mL, 2 M) was
added to a solution of compound 8b (0.17 g, 0.44 mmol) in MeOH (15 mL). The mixture was stirred for
12 h at room temperature. The mixture was concentrated under reduced pressure and acidified by 2 M
HCl. The precipitate was collected by filtration and dried in air to afford a white powder (0.14 g, 93%).
To obtain the sodium salt of this compound, the above acid was mixed with saturated sodium
bicarbonate (2.0 mL) and methanol (5 mL). The reaction mixture was stirred for 2 h. After the solvents
were removed by rotary evaporation, the residue was dissolved in methanol (5 mL). The solution was
filtered and then concentrated by rotary evaporation to give the sodium salt as a white powder.1H NMR
(600 MHz, CD3OD, δ): 8.83 (d, J = 8 Hz, 1H), 8.57 (d, J = 8 Hz, 1H), 8.41 (m, 1H), 8.21 (m, 2H), 7.49
(m, 2H), 7.24 (m, 2H), 7.06 (m, 1H), 6.95 (t, J = 8 Hz, 1H), 2.89 (s, 3H), 2.86(s, 3H).
Methyl 2-aminobenzoate (9a).49 To a solution of 2-amino benzoic acid (2.0 g, 14.6 mmol) in 30 mL
MeOH, concentrated H2SO4 (1.6 mL, 30 mmol) was added dropwise. After the mixture was heated to
reflux for 8 h, the solvent was removed under reduced pressure. Water (20 mL) was added and the
solution pH was adjusted to 3 with 2 M NaOH. The precipitate formed was collected by filtration and
washed with water (20 mL) to afford a white solid (2.10 g, 95%). 1H NMR (400 MHz, CDCl3, δ): 7.79
(d, J = 8 Hz, 2H), 7.18 (t, J = 8 Hz, 1H), 6.53 (m, 2H), 3.72 (s, 3H).
Methyl 2-((5-(dimethylamino)naphthalene)-1-sulfonamido)benzoate (9b).49 To a solution of
dansyl chloride (0.22 g, 0.8 mmol) in dry dichloromethane (15 mL) was added to a solution of
22
ACS Paragon Plus Environment

Page 23 of 29
The Journal of Organic Chemistry
compound 9a (0.12 g, 0.8 mmol) and triethylamine (0.17 mL, 1.6 mmol) in 3 mL dry dichloromethane.
The mixture was stirred at room temperature for 6 h under N2. After the solvent was removed by rotary
evaporation, the residue was purified by column chromatography over silica gel using 2:1 ethyl
1
2
3
4
5
6
7
8
1
acetate/hexane as the eluent to afford a white powder (0.25 g, 83%). H NMR (400 MHz, CDCl3,
9
δ):11.1 (br, 1H), 8.5 (m, 1H), 8.35 (3, 2H), 7.84 (m, 1H), 7.59 (m, 1H), 7.50 (m, 2H), 7.36 (m, 1H),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7.20 (m, 1H), 6.92 (m, 1H), 3.85 (s, 6H), 2.87 (s, 3H).
2-((5-(Dimethylamino)naphthalene)-1-sulfonamido)benzoic acid (9c).49 LiOH (5.0 mL, 2 M) was
added to a solution of compound 9b (0.2 g, 0.40 mmol) in MeOH (15 mL). The mixture was stirred for
12 h at room temperature. The mixture was concentrated under reduced pressure and acidified by 2 M
HCl. The precipitate was collected by filtration and dried in air to afford a white powder (0.14 g, 93%).
To obtain the sodium salt of this compound, the above acid was mixed with saturated sodium
bicarbonate (2.0 mL) and methanol (5 mL). The reaction mixture was stirred for 2 h. After the solvents
were removed by rotary evaporation, the residue was dissolved in methanol (5 mL). The solution was
filtered and then concentrated by rotary evaporation to give the sodium salt as a white powder. 1H NMR
(600 MHz, CDCl3, δ): 11.1 (br, 1H), 8.5 (m, 1H), 8.35 (3, 2H), 7.84 (m, 1H), 7.59 (m, 1H), 7.50 (m,
2H), 7.36 (m, 1H), 7.20 (m, 1H), 6.92 (m, 1H), 3.85 (s, 6H).
Preparation of Molecularly Imprinted Nanoparticles (MINPs). To a micellar solution of
compound 4a (10.2 mg, 0.02 mmol) in D2O (2.0 mL), divinylbenzene (DVB, 2.8 μL, 0.02 mmol),
compound 7 in D2O (10 μL of a solution of 15.68 mg/mL, 0.0004 mmol), and 2,2-dimethoxy-2-
phenylacetophenone (DMPA,10 μL of a 12.8 mg/mL solution in DMSO, 0.0005 mmol) were added.
(D2O instead of H2O was used in the preparation so that the cross-linking and polymerization of the
1
micelles could by monitored by H NMR spectroscopy) The mixture was subjected to ultrasonication
for 10 min before compound 2 (4.13 mg, 0.024 mmol), CuCl2 (10 μL of a 6.7 mg/mL solution in D2O,
0.0005 mmol), and sodium ascorbate (10 μL of a 99 mg/mL solution in D2O, 0.005 mmol) were added.
After the reaction mixture was stirred slowly at room temperature for 12 h, compound 3 (10.6 mg, 0.04
23
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 24 of 29
mmol), CuCl2 (10 μL of a 6.7 mg/mL solution in D2O, 0.0005 mmol l), and sodium ascorbate (10 μL of
a 99 mg/mL solution in D2O, 0.005 mmol) were added. After being stirred for another 6 h at room
temperature, the reaction mixture was transferred to a glass vial, purged with nitrogen for 15 min, sealed
1
2
3
4
5
6
7
8
1
with a rubber stopper, and irradiated in a Rayonet reactor for 12 h. H NMR spectroscopy was used to
9
monitor the progress of reaction. The reaction mixture was poured into acetone (8 mL). The precipitate
was collected by centrifugation and washed with a mixture of acetone/water (5 mL/1 mL) three times.
The crude produce was washed by methanol/acetic acid (5 mL/0.1 mL) three times until the emission
peak at 480 nm (for the dansyl) disappeared and then with excess methanol. The off white powder was
dried in air to afford the final MINPs (12 mg, 70%).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Determination of Critical Micelle Concentration (CMC) of Surfactants. A typical procedure is as
follows. A stock solution was prepared by adding surfactant 4a (10.2 mg, 0.02 mmol) to 2.0 mL of an
aqueous solution of pyrene (1.0 × 10-7 M). To 11 separate vials, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15,
10, and 5 μL of the above stock solution were added. Millipore water was added to each vial to make
the total volume 2.0 mL. Fluorescence spectra were recorded with the excitation wavelength at 336 nm.
The final results were based on duplicate experiments with separately prepared solutions.
Determination of Binding Constants by Fluorescence Titration. A typical procedure is as follows.
A stock solution containing MINP1(7) (150 µM) was prepared in Millipore water. Aliquots (2.0 µL) of
the MINP stock solution were added to 2.00 mL of the solution of 7 in Millipore water (0.2 µM). After
each addition, the sample was allowed to sit for 1 min at room temperature before the fluorescence
spectrum was collected. The excitation wavelength (λex) was 340 nm. The excitation slit width was 10
nm, and the emission slit width was 10 nm.
Determination of Binding Constants by ITC. The determination of binding constants by ITC
followed standard procedures.50-52 In general, a solution of an appropriate guest in Millipore water was
injected in equal steps into 1.43 mL of the corresponding MINP in the same solution. The top panel
shows the raw calorimetric data. The area under each peak represents the amount of heat generated at
each ejection and is plotted against the molar ratio of the MINP to the guest. The smooth solid line is the
24
ACS Paragon Plus Environment

Page 25 of 29
The Journal of Organic Chemistry
best fit of the experimental data to the sequential binding of N binding site on the MINP. The heat of
dilution for the guest, obtained by titration carried out beyond the saturation point, was subtracted from
the heat released during the binding. Binding parameters were auto-generated after curve fitting using
Microcal Origin 7.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website.
ITC titration curves, additional figures, and NMR spectra of key compounds (PDF).
AUTHOR INFORMATION
Corresponding Author
*E-mail: zhaoy@iastate.edu
Acknowledgments
We thank the National Institute of General Medical Sciences of the National Institutes of Health
(R01GM113883) and NSF (DMR-1464927) for financial support of this research
Notes
The authors declare no competing financial interest.
References
(1)
York, 1996.
(2)
Atwood, J. L.; Lehn, J. M. Comprehensive Supramolecular Chemistry; Pergamon: New
Steed, J. W.; Gale, P. A. Supramolecular Chemistry: From Molecules to Nanomaterials;
Wiley: Weinheim, 2012.
25
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 26 of 29
(3)
Schneider, H.-J.; Yatsimirsky, A. K. Principles and methods in supramolecular
1
2
3
chemistry; Wiley: New York, 2000.
4
5
6
7
8
(4)
(5)
(6)
(7)
(8)
(9)
Wulff, G. Angew. Chem. Int. Ed. Engl. 1995, 34, 1812.
Wulff, G. Chem. Rev. 2001, 102, 1.
9
Haupt, K.; Mosbach, K. Chem. Rev. 2000, 100, 2495.
Ye, L.; Mosbach, K. Chem. Mater. 2008, 20, 859.
Shea, K. J. Trends Polym. Sci. 1994, 2, 166.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Sellergren, B. Molecularly imprinted polymers: man-made mimics of antibodies and
their applications in analytical chemistry; Elsevier: Amsterdam, 2001.
(10) Komiyama, M. Molecular imprinting: from fundamentals to applications; Wiley-VCH:
Weinheim, 2003.
(11) Yan, M.; Ramström, O. Molecularly imprinted materials: science and technology;
Marcel Dekker: New York, 2005.
(12) Alexander, C.; Andersson, H. S.; Andersson, L. I.; Ansell, R. J.; Kirsch, N.; Nicholls, I.
A.; O'Mahony, J.; Whitcombe, M. J. J. Mol. Recognit. 2006, 19, 106.
(13) Sellergren, B.; Hall, A. J. In Supramol Chem; John Wiley & Sons, Ltd: 2012.
(14) Haupt, K.; Ayela, C. Molecular Imprinting; Springer: Heidelberg ; New York, 2012.
(15) Zimmerman, S. C.; Wendland, M. S.; Rakow, N. A.; Zharov, I.; Suslick, K. S. Nature
2002, 418, 399.
(16) Zimmerman, S. C.; Zharov, I.; Wendland, M. S.; Rakow, N. A.; Suslick, K. S. J. Am.
Chem. Soc. 2003, 125, 13504.
(17) Sellergren, B. Angew. Chem. Int. Ed. 2000, 39, 1031.
(18) Wu, X.; Carroll, W. R.; Shimizu, K. D. Chem. Mater. 2008, 20, 4335.
(19) Zimmerman, S. C.; Lemcoff, N. G. Chem. Commun. 2004, 5.
(20) Li, Z.; Ding, J.; Day, M.; Tao, Y. Macromolecules 2006, 39, 2629.
(21) Hoshino, Y.; Kodama, T.; Okahata, Y.; Shea, K. J. J. Am. Chem. Soc. 2008, 130, 15242.
26
ACS Paragon Plus Environment

Page 27 of 29
The Journal of Organic Chemistry
(22) Priego-Capote, F.; Ye, L.; Shakil, S.; Shamsi, S. A.; Nilsson, S. Anal. Chem. 2008, 80,
1
2
3
2881.
4
5
6
7
8
(23) Cutivet, A.; Schembri, C.; Kovensky, J.; Haupt, K. J. Am. Chem. Soc. 2009, 131, 14699.
(24) Yang, K. G.; Berg, M. M.; Zhao, C. S.; Ye, L. Macromolecules 2009, 42, 8739.
(25) Zeng, Z. Y.; Patel, J.; Lee, S. H.; McCallum, M.; Tyagi, A.; Yan, M. D.; Shea, K. J. J.
Am. Chem. Soc. 2012, 134, 2681.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(26) Ma, Y.; Pan, G. Q.; Zhang, Y.; Guo, X. Z.; Zhang, H. Q. Angew. Chem. Int. Ed. 2013,
52, 1511.
(27) Çakir, P.; Cutivet, A.; Resmini, M.; Bui, B. T.; Haupt, K. Adv. Mater. 2013, 25, 1048.
(28) Zhang, Y.; Deng, C.; Liu, S.; Wu, J.; Chen, Z.; Li, C.; Lu, W. Angew. Chem. Int. Ed.
2015, 54, 5157.
(29) Chen, L. X.; Xu, S. F.; Li, J. H. Chem. Soc. Rev. 2011, 40, 2922.
(30) Gunasekara, R. W.; Zhao, Y. J. Am. Chem. Soc. 2015, 137, 843.
(31) Gunasekara, R. W.; Zhao, Y. Chem. Commun. 2016, 52, 4345.
(32) Awino, J. K.; Zhao, Y. J. Am. Chem. Soc. 2013, 135, 12552.
(33) Awino, J. K.; Zhao, Y. Chem. Commun. 2014, 50, 5752.
(34) Awino, J. K.; Zhao, Y. Chem.-Eur. J. 2015, 21, 655.
(35) Awino, J. K.; Hu, L.; Zhao, Y. Org. Lett. 2016, 18, 1650.
(36) Awino, J. K.; Zhao, Y. ACS Biomater. Sci. Eng. 2015, 1, 425.
(37) Zhang, S.; Zhao, Y. Macromolecules 2010, 43, 4020.
(38) Peng, H.-Q.; Chen, Y.-Z.; Zhao, Y.; Yang, Q.-Z.; Wu, L.-Z.; Tung, C.-H.; Zhang, L.-P.;
Tong, Q.-X. Angew. Chem. Int. Ed. 2012, 51, 2088.
(39) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99, 2039.
(40) Zhang, S.; Zhao, Y. J. Am. Chem. Soc. 2010, 132, 10642.
(41) Li, Y. H.; Chan, L. M.; Tyer, L.; Moody, R. T.; Himel, C. M.; Hercules, D. M. J. Am.
Chem. Soc. 1975, 97, 3118.
27
ACS Paragon Plus Environment

The Journal of Organic Chemistry
(42) Schmidtchen, F. P. In Supramol Chem; John Wiley & Sons, Ltd: 2012.
(43) Electrostatic interactions between the positively charged MINP and the negatively
charged template should be fairly constant within the series and thus not discussed here.
(44) Takamatsu, D.; Fukui, K.-i.; Aroua, S.; Yamakoshi, Y. Org. Biomol. Chem. 2010, 8,
3655.
Page 28 of 29
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(45) Wu, H.; Zhou, P.; Wang, J.; Zhao, L.; Duan, C. New J. Chem. 2009, 33, 653.
(46) Himel, C. M.; Aboul-Saad, W. G.; Uk, S. J. Agric. Food Chem. 1971, 19, 1175.
(47) Hejesen, C.; Petersen, L. K.; Hansen, N. J. V.; Gothelf, K. V. Org. Biomol. Chem. 2013,
11, 2493.
(48) Sosic, I.; Turk, S.; Sinreih, M.; Trost, N.; Verlaine, O.; Amoroso, A.; Zervosen, A.;
Luxen, A.; Joris, B.; Gobec, S. Acta Chim. Slov. 2012, 59, 380.
(49) Carrillo-Arcos, U. A.; Rojas-Ocampo, J.; Porcel, S. Dalton transactions (Cambridge,
England : 2003) 2016, 45, 479.
(50) Wiseman, T.; Williston, S.; Brandts, J. F.; Lin, L. N. Anal. Biochem. 1989, 179, 131.
(51) Jelesarov, I.; Bosshard, H. R. J. Mol. Recognit. 1999, 12, 3.
(52) Velazquez-Campoy, A.; Leavitt, S. A.; Freire, E. Methods Mol. Biol. 2004, 261, 35.
28
ACS Paragon Plus Environment

Page 29 of 29
The Journal of Organic Chemistry
1
2
3
TOC graphic
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
29
ACS Paragon Plus Environment