# Amidinium salt bridges for real-world applications of molecularly imprinted polymers

Bernadette Tse Sum Bui, Karsten Haupt

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Bernadette Tse Sum Bui, Karsten Haupt. Amidinium salt bridges for real-world applications of molecularly imprinted polymers. Trends in Analytical Chemistry, 2026, 194, pp.118509. 10.1016/j.trac.2025.118509. hal-05406860

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# Amidinium salt bridges for real-world applications of molecularly imprinted polymers

Bernadette Tse Sum Bui<sup>a, *</sup>, Karsten Haupt<sup>a,b,**</sup> <sup>©</sup>

## ARTICLE INFO

In memory of our friends and colleagues Klaus Mosbach and Günter Wulff, the fathers of molecular imprinting.

**Keywords:**

Amidinium

Guanidinium

Molecularly imprinted polymer

Oxoanion

Salt bridge interaction

Stoichiometric noncovalent binding

## ABSTRACT

Günter Wulff and Klaus Mosbach pioneered the molecular imprinting technique in the 70s and 80s, respectively, using complementary approaches. An important development in noncovalent molecular imprinting was the use by Wulff of strong salt bridge interactions displayed by amidinium-phosphate and amidinium-phosphonate complexes in catalytically active enzyme-mimicking molecularly imprinted polymers (MIPs). Inspired by their work, others used amidine monomers and their strong pairing with carboxylate groups in the aim of generating molecular imprints in polar organic solvents and in aqueous solutions. The strong directional salt bridge association between amidine and carboxylate template yields MIPs with high affinity and specificity for their target, allowing applications even in challenging complex environments such as human sweat, urine, and cellular and tissue extracts. Herein, we review the use of amidine monomers as well as related guanidine monomers in molecular imprinting, from the time of Wulff to nowadays, highlighting recent advances and applications from the literature, and discuss future directions in this area.

## 1. Introduction

Amidine groups, like guanidine groups (the side chain of the amino acid arginine) are superbases with pKₐ (H₂O) of 12.4 and 13.6, respectively. The high basicity of these amine derivatives is the effect of resonance-stabilized cations caused by the effective network conjugation after their protonation under reversible conditions [1] (Fig. 1). Due to their high pKₐs, amidinium and guanidinium cations remain protonated over a wide range of pHs and importantly can form salt bridges with oxoanion substrates at physiological pH [2].

Salt bridges are noncovalent interactions that involve both hydrogen bonding and ion pairing when a cationic H-bond donor (amidine, guanidine) interacts with an anionic H-bond acceptor (carboxylate, phosphate, phosphonate, sulfate) [3,4]. Amidinium-carboxylate, amidinium-phosphate and amidinium-phosphonate salt bridges were brought to light at the end of the 1990s by Günter Wulff who introduced the concept of stoichiometric noncovalent interactions in molecularly imprinted polymers (MIPs) [5,6]. His group synthesized a large variety of polymerizable amidines [7], among which N,N'-diethyl-4-vinylbenzamidine (Fig. 2) readily formed stoichiometric

**Abbreviations:** 3H3MH, 3-hydroxy-3-methyl-hexanoic acid; 3M2H, (E)-3-methyl-2-hexenoic acid; AB, N-acryloyl-4-aminobenzamidine acetate; AEM, N-(2-aminoethyl) methacrylamide; APM, N-(3-aminopropyl) methacrylamide; ATC, (3-acrylamidopropyl)trimethylammonium chloride; BIS, N,N'-methylenebisacrylamide; cj3H3MH, Nₐ-3-hydroxy-3-methylhexanoyl-glutamine; cj3M2H, Nₐ-3-methyl-2-hexenoyl-glutamine; CM, critical monomer concentration; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; EGDMA, ethylene glycol dimethacrylate; GBs, glass beads; GlcA, D-glucuronic acid; GPS, glyphosate; GUA, N-(3-guanidino-propyl)-2-methacrylamide; HA, hyaluronic acid; HRTEM, high-resolution transmission electron microscopy; Kₐ, association constant; Kᵢ, inhibition constant; KIM-1, kidney injury molecule-1; LCST, lower critical solution temperature; LOD, limit of detection; MAG, N-(2-methyl-acryloyl)-guanidine; MIP, molecularly imprinted polymer; NANA, N-acetyl-D-neuraminic acid; NG, nanogel; NIP, non-imprinted polymer; NIPAM, N-isopropyacrylamide; NMR, nuclear magnetic resonance; NP, nanoparticle; NUPR1, nuclear protein 1; PAA, phenyl acrylamide; pI, isoelectric point; QCM, quartz crystal microbalance; QD, quantum dot; STEM, scanning transmission electron microscopy; TBA, tetrabutylammonium; TNF-α, tumor necrosis factor alpha; THF, tetrahydrofuran; TSA, transition state analogue.

This article is part of a special issue entitled: molecularly imprinted polymers published in *Trends in Analytical Chemistry*.

* Corresponding author.

** Corresponding author. Université de Technologie de Compiegne, CNRS Laboratory for Enzyme and Cell Engineering, Rue du Docteur Schweitzer, CS 60319, Compiegne, 60203, Cedex, France.

E-mail addresses: jeanne.tse-sum-bui@utc.fr (B. Tse Sum Bui), karsten.haupt@utc.fr (K. Haupt).

<footnote><sup>a</sup> Université de Technologie de Compiegne, CNRS Laboratory for Enzyme and Cell Engineering, Rue du Docteur Schweitzer, CS 60319, Compiegne, 60203, Cedex, France</footnote>

<footnote><sup>b</sup> Institut Universitaire de France, Paris, France</footnote>
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Conjugation of amidines and guanidines.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Chemical structures of amidine monomers.</figcaption></figure>

noncovalent interactions with carboxylates ($K_a$: $1.2 \times 10^4 \text{ M}^{-1}$), phosphates ($K_a$: $4.6 \times 10^3 \text{ M}^{-1}$) and phosphonates ($K_a$: $8.7 \times 10^3 \text{ M}^{-1}$) in acetonitrile-d3 at 25 °C [6]. Association constants ($K_a \ge 10^3 \text{ M}^{-1}$) are considered as high and thus stoichiometric noncovalent interactions are considered to be strong. As a comparison, the $K_a$ of hydrogen bond for instance of a 1:1 complex of methacrylic acid (MAA)-phenylalanine anilide [8] and MAA-bisphenol A [9] in acetonitrile-d3 and at a similar temperature is $30 \text{ M}^{-1}$ and $0.05 \text{ M}^{-1}$, respectively.

Particularly, Wulff and co-workers exploited stoichiometric noncovalent interactions to develop MIPs as enzyme analogues (catalytic MIPs) [10,11]. Since it is essential to have the catalytic functional groups exclusively inside the active site, a strong interaction of template with functional monomer is a prerequisite. This cannot be achieved with weak noncovalent interactions such as H bonds, which necessitate the use of at least 4-fold molar excess of functional monomers with regard to the template, resulting in the majority of functional groups after polymerization and removal of template to be randomly distributed in the polymer. To design the catalytic MIPs, the authors adopted the concept of transition state stabilization. In fact, it is the tight binding of the enzyme to the transition state of a reactant (rather than to the substrate) that lowers the activation energy of the reaction, providing strong catalytic activity. The process consists in imprinting an analogue of the transition state of a reaction, in the presence of an amidine-based functional monomer and a crosslinker, which after removal of the transition state analogue from the polymer matrix, gives rise to cavities with amidinium sites for binding and catalytic transformation (hydrolyses) of the substrate (esters and carbonates). In this review, we will start by presenting a super MIP catalyst, mimicking carboxypeptidase A [12,13]. All the catalytic MIPs were mostly prepared in organic solvents for solubility issues of *N*,*N*'-diethyl-4-vinylbenzamidine. With the advent of water-soluble amidine monomers, biocompatible MIP microgels [14,15] and nanogels [16–18] were prepared in buffer or aqueous mixtures. The resulting MIPs could operate directly in fully aqueous medium, for instance, as MIP inhibitors [14,19], and fluorescent MIPs for sensing the enzyme, trypsin [20]. Following this, we will report on the exploitation of amidinium-carboxylate interaction in the imprinting of small organic molecules with a carboxylate moiety [15,21], and peptides bearing aspartic and glutamic acids side chains [17]. The resulting MIPs were particularly performant in applications where the MIP had to bind selectively in complex media such as human sweat, urine, cellular and tissue extracts.

## 2. Amidines for imprinting transition state analogues

Catalytic MIPs [22] and the strategy of imprinting the transition state analogue (TSA) of a reaction to obtain MIPs with enzyme-like specificity and activity, was pioneered by Mosbach and co-workers [23], but Wulff and co-workers developed and brought visibility to this research. Wulff used template molecules containing phosphate groups [12,13,24,25], as well as phosphonate groups [5,26], as TSAs, and *N*,*N*'-diethyl-4-vinylbenzamidine as functional monomer for stoichiometric imprinting. Their first report dating from 1997, described very encouraging results in the sense that the TSA-MIP showed typical Michaelis-Menten kinetics and a significant rate enhancement (102 times that of the reaction in solution), albeit the turnover number ($k_{cat}$) was very low ($0.00008 \text{ min}^{-1}$) and no appropriate controls were studied in parallel [5]. During the following years, other TSA-MIPs were reported, for instance for the hydrolysis of cholesterol [24], amino acid phenyl esters [26], and carbonate substrates [12,13]. The MIP model [12] for carboxypeptidase A will be detailed as its catalytic activity and efficiency for carbonate hydrolysis, surpassed that of a catalytic antibody prepared for the same function. Fig. 3A shows the major components of the MIP; the functional monomer consists of an amidinium group to mimic the guanidinium moiety of Arg 127 in the active site of the enzyme. In addition, the monomer contains a $Cu^{2+}$ ion-binding site to stabilize the complex formed between its triamine part and the pyridine nitrogen of the TSA molecule (Fig. 3a), thus providing a well-defined orientation of the complex as well as metal ion catalysis, for the exact reproduction of the reaction of the substrate during catalysis. As shown in Fig. 3a, the tetrahedral geometry of the phosphate ester functional group mimics the tetrahedral geometry of the transition state of carbonate (Fig. 3b) hydrolysis. Due to the stabilization of the transition state reaction, the hydrolysis of the carbonate substrate is enhanced, leading to a remarkable turnover number of 2.86 as compared to the non-imprinted polymer (NIP) (turnover number 0.035). Moreover, the catalytic activity of the MIP, expressed as the ratio $k_{cat}/k_{uncat}$ ($k_{uncat} = k_{solution}$) was rather impressive ($k_{cat}/k_{uncat} = 15700$), a value considerably superior to that of a catalytic antibody for carbonate hydrolysis ($k_{cat}/k_{uncat} = 810$). The imprinting factor, i.e. the ratio of catalysis by MIP and NIP was high (76.9) and the efficiency $k_{cat}/K_m$ of the MIP catalyst compared to that of the NIP control polymers containing the same functional groups was 537-fold higher, clearly indicating a very efficient imprinting procedure.

However, this MIP as well as most TSA-MIPs were prepared by bulk polymerization with a large excess of crosslinker, giving rise to insoluble particles, a format far from that of soluble natural enzymes. The group therefore developed a new polymerization method, termed post-dilution polymerization, to prepare soluble, highly crosslinked, single molecule catalytic nanogels [25]. The catalytic system was composed of *N*, *N*'-diethyl-4-vinylbenzamidine as functional monomer and diphenyl phosphate as TSA, to mimic the catalytic mechanism of hydrolase enzymes with carbonate substrates (Fig. 3B). The polymerization started with a high concentration of monomers (favoring complex formation by noncovalent bonds and the formation of dense particles) and is stopped just prior to macrogelation by dilution with cyclopentanone, the solvent of polymerization, to keep the concentration below the critical monomer concentration ($C_M$) of 0.1–1.5 %. $C_M$ corresponds to the total mass of functional and crosslinking monomers upon total mass of solvent and monomers used for the polymerization. Above the $C_M$, macrogelation takes place. The resulting MIP nanogels showed Michaelis-Menten kinetics with a $k_{cat}/k_{uncat}$ ratio of 2990. Scanning and high-resolution transmission electron microscopy (TEM) images (Fig. 3B) showed that the nanogels were spherical in shape with diameters between 10 and 20 nm. Further analyses showed that the soluble crosslinked nanogels had a molecular weight of 39 kDa, with an average of one catalytic site per particle, approaching natural enzymes characteristics. Nowadays, work on catalytic MIPs is still being actively pursued by Zhao's group who has recently reported water-soluble artificial esterases based on nanoMIPs
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** (A) Composition and function of a catalytic MIP mimicking carboxypeptidase A [12]. Copyright 2004, American Chemical Society. (a) Molecular imprinting with the TSA molecule and monomer in the presence of Cu²⁺; (b) Intermediate substrate during catalysis. (B) Composition of MIP nanogel. Transmission electron microscopy image of nanogel measured in the (a) imaging mode (HRTEM) and (b) scanning mode (STEM) [25]. Copyright 2006, Wiley-VCH GmbH.</figcaption></figure>

polymerized in crosslinked micelles [27,28].

### 3. Amidines for imprinting small molecules

Wulff's N,N'-diethyl-4-vinylbenzamidine monomer was also used to prepare chiral imprints with optically active dicarboxylic acids as templates [29]. Chiral selectivity for the enantiomers (2R, 3R)-(-)-di-O-benzoyltartaric acid or (2S,3S)-(+)-di-O-benzoyltartaric acid (Fig. 4A) was found for MIPs incorporating two equivalents of N, N'-diethyl-4-vinylbenzamidine as binding site monomers, in tetrahydrofuran. Due to the asymmetric shape of the imprinted cavity in which the template is more easily embedded than its enantiomer (Fig. 4B), the imprinted polymers were able to discriminate between the enantiomers of the templates, whereas the reference polymer shows no difference between the two enantiomers, in aqueous solutions.

Later our group employed N-acryloyl-4-aminobenzamidine acetate (AB, Fig. 2), an unsubstituted version of Wulff's amidine monomer to prepare MIPs for several small molecules and peptides bearing carboxylic groups. AB is soluble in polar organic solvents (DMSO, ethanol), and more importantly in phosphate buffer pH 7.0, in which we perform the imprinting of peptides and proteins (vide infra). AB is easily obtained by reacting acryloyl chloride and 4-aminobenzamidine dihydrochloride, in an excess of sodium acetate in water [15,21]. The reaction product is then precipitated by adding concentrated hydrochloric acid. After dissolution in hot water, the product is recrystallized by adding HCl, yielding N-acryloyl-4-aminobenzamidine hydrochloride. The latter is then converted to its acetate salt by leaving it overnight in a saturated solution of sodium acetate. The product is collected by filtration, washed with water and dried. The yield is usually ~60 % and the monomer is generally stored at 4 °C for routine use or -20 °C for long-term conservation. No stability problem with time is encountered as verified with ¹H NMR spectroscopy. Working with the acetate salt is an advantage because the acetate ion is readily exchangeable with the carboxylate ion of the template to form the amidinium complex.

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** (A) Chemical structures of templates: enantiomer (2R,3R)-(-)-di-O-benzoyltartaric acid and (2S,3S)-(+)-di-O-benzoyltartaric acid; (B) MIP cavity without template (left) and with template (right) [29]. Copyright 2003, Springer Nature.</figcaption></figure>

AB was used in the synthesis of MIPs for D-glucuronic acid (GlcA) [30,31] and N-acetyl-D-neuraminic acid (NANA), which were then applied for the dual bioimaging of hyaluronic acid (HA) and sialic acid on human keratinocytes [21]. HA is present in the extracellular matrix of
all mammalian cells and is a linear glycosaminoglycan composed of alternating units of GlcA and N-acetyl-D-glucosamine (Fig. 5A), while sialic acid is a generic term referring to N and O-derivatives of neu- raminic acid, of which the most common member is NANA. NANA is predominantly located at the terminal ends of N- and O-glycans (Fig. 5B) of glycoproteins and glycosphingolipids. Since altered glycosylation levels and distributions of NANA and HA are indicators of pathological conditions like infection and malignancy, imaging agents based on fluorescently-labeled MIPs can be useful for their detection and locali- zation [32,33]. Both MIPs were prepared with a stoichiometric amount of AB, an excess of methacrylamide which provides an H-bond network, in DMSO (Fig. 5C and D). Prior to MIP syntheses, the stoichiometry of both AB–GlcA and AB–NANA complexes in DMSO-d6 were deduced from ¹H NMR analysis of Job plot and titration studies to be 1:1 with high association constants (Kₐ) of 7.1 × 10³ M⁻¹ (AB–GlcA) and 41 × 10³ M⁻¹ (AB–NANA), respectively. Both MIPs were selective towards their template monosaccharide in competitive binding assays as no cross-reactivity was observed with other sugars such as D-glucose, N-acetyl-D-glucosamine, D-galactose, N-acetyl-D-galactosamine, NANA (for MIPGlcA) and GlcA (for MIPNANA). Consequently, staining with green fluorescent MIPGlcA-QD (quantum dot) could selectively localize HA (Fig. 5E, top) and red fluorescent MIPNANA-QD could localize sia- lylation sites (Fig. 5E, middle) individually, or together simultaneously (Fig. 5E, bottom).

In another example, we used two equivalents of AB to form stoichiometric noncovalent interactions with a dicarboxylic acid, N<sub>α</sub>-hexanoyl-glutamic acid [15] (Fig. 6A). N<sub>α</sub>-hexanoyl-glutamic acid was employed as a generic template to prepare MIPs for targeting N<sub>α</sub>-3-hydroxy-3-methylhexanoyl-glutamine (cj3H3MH) and N<sub>α</sub>-3-methyl-2-hexenoyl-glutamine (cj3M2H) (Fig. 6B), precursors of odorous molecules present in human sweat. Both target molecules

contain one amide and one carboxylate group, which *a priori* would suggest N<sub>α</sub>-hexanoyl glutamine, as a target-analogue template (Fig. 6C). However, the resulting MIP synthesized with a template bearing only one amidine interaction, did not show strong-enough binding in a complex environment (sweat mixed with a deodorant formulation), as compared to the MIP templated with N<sub>α</sub>-hexanoyl glutamic acid, which can strongly interact with two amidines and fix the –COOH in a precise position in the MIP binding site (Fig. 6A). Upon binding of the targets, the second amidine group would then interact via hydrogen bonding with their amide group and contribute to the overall binding strength. The MIP was synthesized by precipitation polymerization in the presence of ethylene glycol dimethacrylate (EGDMA) as crosslinker, in ethanol:water (4:1). Prior to MIP synthesis, ¹H NMR analysis of Job plot and titration experiments of N<sub>α</sub>-hexanoyl glutamine and N<sub>α</sub>-hexanoyl glutamic acid with AB in CD<sub>3</sub>OD:D<sub>2</sub>O (4:1) at 25 °C was studied. Non-linear regression analysis gave a 1:1 stoichiometry and a K<sub>a</sub> of 9.4 × 10<sup>3</sup> M<sup>-1</sup> for the complex AB–hexanoyl glutamine. For the association AB–hexanoyl glutamic acid, a mixture of 1:1 and 1:2 complexes with an apparent binding constant of > 10<sup>7</sup> M<sup>-1</sup> for the latter, was deduced. It is worth noting that the K<sub>a</sub> of the monomer-template complex was still very high (≥10<sup>3</sup> M<sup>-1</sup>) even in highly competitive protic solvents.

cj3H3MH and cj3M2H are non-odorous precursors of malodorous acids (Fig. 6B) secreted by sweat glands found for example under the armpit. The objective was to apply the MIP as a specific scavenger agent to trap these precursors, thus preventing their hydrolysis by skin bacteria, into volatile malodorous fatty acids, (E)-3-methyl-2-hexenoic acid (3M2H) and 3-hydroxy-3-methyl-hexanoic acid (3H3MH). The synthesized MIPs were biocompatible as no cytotoxic or irritation (induction of proinflammatory cytokines) response was observed on human keratinocytes [34]. When the MIPs incorporated in a deodorant formulation were incubated in human sweat at body temperature, they could

<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Structure of (A) hyaluronic acid and (B) N-acetyl-D-neuraminic acid (NANA). Amidinium binding site interacting with the carboxylate of (C) GlcA and (D) NANA, complemented by a network of H-bonds between template and methacrylamide (green). (E) Confocal images on fixated keratinocytes showing the localization of MIPGlcA-QDs (green), MIPNANA-QDs (red, middle) and simultaneous dual labeling of both GlcA and NANA by MIPGlcA-QDs (green) and MIPNANA-QDs (red). Nuclear staining is performed with DAPI (blue). Scale bar: 25 µm [21]. Copyright 2016, Wiley-VCH GmbH. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** (A) Proposed complex between the template N<sub>α</sub>-hexanoyl-glutamic acid and amidine functional monomer in ethanol:water (4:1); (B) Target molecules, cj3M2H and cj3H3MH, odorless glutamine conjugates in axillary sweat are transformed to malodorous fatty acids, (E)-3-methyl-2-hexenoic acid (3M2H) and 3-hydroxy-3-methyl-hexanoic acid (3H3MH) by the action of skin bacteria [15]. Copyright 2016, Wiley-VCH GmbH; (C) Chemical structure of N<sub>α</sub>-hexanoyl glutamine originally considered as template molecule; (D) MIP and NIP binding to cj3H3MH and cj3M2H in human sweat, at 37 °C. The MIP and NIP were homogeneously suspended in a deodorant formulation. Amount of cj3H3MH and cj3M2H in human sweat were 135 ± 6.6 nmol/mL and 17 ± 1 nmol/mL, respectively [34]. Copyright 2019, American Chemical Society.</figcaption></figure>

selectively capture both cj3H3MH and cj3M2H (Fig. 6D), despite the
presence of numerous other molecules with carboxylic acid groups
(some in the mM range) present in this highly complex environment.
This is certainly due to the favorably spatial position of the amidinium
functionalities in the MIP cavities.

**4. Amidines for imprinting proteins and peptides**

**4.1. Protein**

Nowadays, molecular imprinting of protein targets still remains a
challenge, mainly due to their complex native conformation that has to
be preserved during the polymerization process [35]. Moreover, the
generation of selective imprinted cavities is extremely difficult due to
the multitude of functional groups and their large size hampers their
extraction. However, if the protein can be pre-oriented in a fixed posi-
tion by using an anchoring monomer containing its affinity ligand, the
polymerization can be confined to the close proximity of the recognition
site of the protein, and lead to the generation of selective MIPs. The
following examples illustrate a case study on trypsin and its anchoring
monomer-inhibitor.

Trypsin is a serine protease which hydrolyzes substrates containing
arginine and lysine residues. These basic residues interact with the
carboxyl group of Asp189, located at the substrate binding pocket.
Benzamidine is a well-known competitive trypsin inhibitor, which
blocks the proteolytic function by interacting with Asp189 in an
arginine-analogous manner [36]. Our group synthesized N-meth-
acryloyl-4-aminobenzamidine, to prepare MIP microgels by precipita-
tion polymerization in phosphate buffer containing a comonomer
methacrylamide and the crosslinker N,N'-ethylenebis(acrylamide).

Therein, the benzamidine monomer acted as a strong anchoring
monomer to attract the forming polymer chains to the template protein
[14] (Fig. 7). The resulting microgels bind selectively trypsin over other
proteins of similar size and molecular weight, and show competitive
inhibition of trypsin with an inhibition constant K<sub>i</sub> of 79 nM, making
them more potent inhibitors than the low molecular weight competitive
inhibitor benzamidine by almost three orders of magnitude.

Interestingly, this benzamidine monomer was later employed as
comonomer to prepare linear high-affinity polymers recognizing surface
glutamic and aspartic acids, for inhibition of trypsin [37]. According to
the authors, the linear copolymer undergoes an extensive induced-fit

<figure><img src="image_9.png" /><figcaption>**Fig. 7.** Schematic representation of the molecular imprinting of trypsin using a polymerizable benzamidine inhibitor, N-methacryloyl-4-aminobenzamidine, as an anchoring monomer [14]. Trypsin (yellow) with the anchoring monomer in its substrate binding pocket, and comonomers (A). Polymerization takes place (1) and a crosslinked polymer is molded around the substrate binding site (B). After enzyme removal (2), a specific recognition site with inhibitory properties is revealed in the MIP (C). Copyright 2009, American Chemical Society. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>
procedure across the protein surface and thus optimizes in a dynamic
process its noncovalent contacts to the highest possible number of
solvent-exposed residues. If many of these are located near the active
site, enzymatic activity is significantly reduced, because of the hin-
drance of the substrate's access to the active site.

It is worth noting that water-soluble benzamidine-based monomers such as N-acryloyl-3-aminobenzamidine, were already reported as early as 1987 and used for the synthesis of acrylamide-based polymers for the affinity purification of trypsin [38]. The synthesis method to produce our benzamidine N-acryloyl-4-aminobenzamidine monomer is inspired from their protocol. Later in 2001, Vaidya et al. used N-acryloyl-4-aminobenzamidine for the synthesis of acrylamide-based MIP macrogels in a mixture of water:dimethylformamide (DMF) (3:1), to target trypsin [39].

Continuing the exploration of the high affinity of the benzamidine
monomer for trypsin, our group employed N-acryloyl-4-amino-
benzamidine (AB) to prepare MIPs for sensing trypsin in human urine.
Previous work on 4-aminobenzamidine reported that its binding to
trypsin led to a 50-fold fluorescence enhancement, and this was due to
its interaction with the enzyme's active center [40], indicating that
4-aminobenzamidine can act as a specific fluorescence probe for trypsin.
Thus, we incorporated AB as a signaling fluorescent monomer in MIP
nanogels (MIP-NGs), for trypsin biosensing. The MIPs made use of
fluorescence enhancement in order to directly detect non-fluorescent
analyte binding by incorporating a fluorescent reporter group into the
MIP, a concept that has been originally developed by Takeuchi's group
[41]. Contrarily to the precedent MIP microgels described above, which
suffered from difficult template removal and binding site heterogeneity,
this time we prepared the MIP-NGs using solid-phase synthesis so as to
obtain binding sites located at the surface, to enable rapid trypsin
detection [20]. Fig. 8 shows the strategy that was employed to prepare
the MIP-NGs. Trypsin was immobilized on Cu²⁺-iminodiacetic acid
functionalized glass beads (GBs), via its surface histidine residue, leav-
ing the active site of the enzyme free for binding to the benzamidine
moiety of the fluorescent monomer AB. Additionally, the polymerization

mixture contained N-isopropylacrylamide (NIPAM) and N,
N'-methylenebisacrylamide (BIS) in phosphate buffer, with a total
monomer concentration of 0.5 % (w/w), i.e. under high dilution con-
ditions so as to obtain nanoparticles. NIPAM was used as the major
component in order to generate thermoresponsive MIP-NGs. pNIPAM is
known to undergo a volume-phase transition at its lower critical solution
temperature (LCST) of 32 °C. MIP-NGs were synthesized at 37 °C, above
the LCST of pNIPAM, so that the growing polymeric nanoparticles,
which are in the collapsed state at this temperature, encapsulate the
immobilized protein during polymerization. After polymerization, the
GBs were washed with buffer at 37 °C to remove unreacted reagents and
particles that polymerized distantly from the immobilized template
(low-affinity polymers). To recover the high-affinity MIP-NGs, the GBs
were cooled to 4 °C, a temperature lower than the LCST of pNIPAM,
allowing the MIP-NGs to swell and be eluted template-free. These
MIP-NGs (size ~70 nm) were endowed with improved binding site ho-
mogeneity since all binding sites have the same orientation of benza-
midine moiety. When the MIP-NGs were excited at 280 nm, fluorescence
enhancement occurred upon trypsin binding, the fluorescence intensity
being proportional to the trypsin concentration (Fig. 8C) [20].
Furthermore, in a different work, the MIP-NGs proved to be more potent
competitive inhibitors of trypsin (Kᵢ = 3.4 nM) than the MIP microgels
prepared by precipitation polymerization. Consequently, they were
applied to inhibit tryptic digestion-induced extracellular matrix lysis
and cell membrane destruction on liver cells, indicating their utility in
the treatment of active trypsin-dependent cell injury [19].

## 4.2. Protein epitopes

Even though the strategy of using an inhibitor-monomer to target
enzymes has proved useful to prepare MIPs for other proteins like lac-
tamases [42] and glycosidases [43], it is not applicable to all proteins.
Moreover, the use of whole proteins as templates will always have
limitations [35]. Cancer biomarker proteins and other proteins impli-
cated in societal diseases are very expensive, difficult to obtain in

<figure><img src="image_10.png" /><figcaption>**Fig. 8.** (A) Solid-phase synthesis of trypsin MIP-NGs containing the fluorescent signaling monomer *N*-acryloyl-4-aminobenzamidine (AB); (B) Schematic representation of fluorescence enhancement of MIP nanoprobe upon binding with trypsin; (C) Fluorescence sensing of trypsin with 0.1 mg/mL of MIP-NGs in urine (λ<sub>ex</sub>: 280 nm, λ<sub>em</sub>: 380 nm) [20]. Copyright 2018, Elsevier.</figcaption></figure>
sufficiently pure amount, are often unstable at the temperature of
polymerization, and particularly the large number of surface chemical
groups leads to inhomogeneous binding site populations. Inspired by
Nature, it has therefore been proposed to use, as an alternative, only a
fragment of the protein as the imprinting template [44–47]. Generally,
antibodies recognize their protein antigen, via a well-defined small
exposed part of the protein, called epitope. The epitope imprinting
approach is thus based on the use of a short surface-exposed fragment of
a protein as template, generating MIPs which are then capable of
recognizing the whole macromolecule. In the following section, we will
describe an example of epitope imprinting using a peptide epitope in
association with AB as functional monomer to prepare selective MIPs for
the recognition of the whole protein. The target protein, kidney injury
molecule-1 (KIM-1), a urinary biomarker of acute kidney injury, as well
as its epitope NH₂-Glu-His-Arg-Gly-Trp-Phe-Asn-Asp-COOH [17] is
shown in Fig. 9A and B. The epitope was selected using an in silico
rational approach that we recently developed in-house, with the main
guidelines being that it has a flexible solvent-exposed loop structure, it
has a 3D structure with a conformation similar to that within the native
protein, and it is unique to the protein. Since the epitope adopts a loop
conformation in the native protein, a cyclic template peptide was syn-
thesized in order to mimic closely the conformation of the epitope in its
native form. Thus, two cysteine residues were added at both ends for
cyclization. A modified lysine residue bearing an azide moiety was
included to enable the immobilization of the template peptide on
propargylated glass beads (GBs), via click chemistry. Click chemistry not
only allows for template immobilization without engaging the func-
tional groups that can be exploited for recognition properties, but also
results in a unique orientation of the template. The experimental cyclic
peptide template presents the following sequence: NH₂-Cys-Glu-Hi-
s-Arg-Gly-Trp-Phe-Asn-Asp-Cys-Lys(N₃)–COOH (Cys-Cys cyclic)

(Fig. 9B). To further fix the template in a definite spatial orientation, we employed AB to provide a strong two-point anchorage to the Glu and Asp side chains, whereas phenyl acrylamide (PAA) [48] complemented the side chains of Trp and Phe with π-π stacking interactions (Fig. 9C). NIPAM (to render the polymer thermoresponsive) and N-tert-butyl acrylamide (for hydrophobic interactions) were added as comonomers, and BIS was used as crosslinker. Polymerization was done in 25 mM phosphate buffer under high dilution conditions. Though one might speculate that AB will form salt-bridge interaction with the anionic phosphate of the buffer, its interaction with –COOH is not compromised. The presence of AB in the MIP was indeed confirmed by fluorescence spectroscopy. The resulting MIP nanogels (size ~50 nm) were completely soluble in water and showed high binding affinity and selectivity towards the epitope peptide and the protein KIM-1. The existence of specific binding sites at atomic level resolution, created by the imprinting of the template epitope in the MIP nanogel was confirmed using Saturation Transfer Difference and WaterLOGSY NMR spectroscopies. Moreover, all binding sites are generated at the surface of the MIPs, facilitating rapid binding kinetics.

In this example, we showed that imprinting with AB monomer pro-
vided two strong judiciously-positioned amidine groups in the MIP
nanogels (Fig. 9C), that are able, via salt bridge interactions to drive the
binding of the epitope template in a pre-oriented position. The selec-
tivity is additionally reinforced by the presence of PAA, thus providing
multipoint binding sites in the imprinted cavity. We believe that the use
of AB allowed to finetune the selectivity in the MIP’s binding sites,
which would not have been possible with other weak basic monomers.
The AB was essential, as its omission from the pre-polymerization
mixture, resulted in MIP-NGs that had no binding. Other groups
employed the basic monomer, N-(3-aminopropyl)methacrylamide hy-
drochloride [49–51] to target peptides in aqueous medium in similar

<figure><img src="image_11.png" /><figcaption>**Fig. 9.** (A) Crystal structure of the ectodomain of KIM-1 (PDB 5DZO) with the selected protein epitope highlighted in orange, superposed by the predicted 3D structure of the template peptide (red); (B) 1D and 3D models of the template peptide CEHRGWFNDC-K(N<sub>3</sub>) [17]. Copyright 2021, Wiley-VCH GmbH. (C) Template peptide immobilized by click chemistry onto cycloalkyne-derivatized GBs, amino acid side chains are highlighted in red for acidic, brown for hydrophobic, and blue for basic residues. Glu and Asp form salt bridges with N-acryloyl-4-aminobenzamidine and Trp and Phe form π-π stacking with phenyl acrylamide. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>
set-ups. However, in our hands, this monomer produced MIPs with no binding, whereas with AB, high affinity and selective MIPs for the recognition of Nuclear Protein 1 (NUPR1) in urine [52], and of cadherin [16] and Tumor Necrosis Factor-alpha (TNF-α) [18] in various cell cultures, were obtained.

## 5. Guanidine monomers for MIPs

Guanidines are stronger bases than amidines and the origin of their higher basicity is the higher number of resonance-stabilized cations they display [1] (Fig. 10A). As seen from Fig. 10A, the positive charge is delocalized over three nitrogen atoms, and four out of the five hydrogen bond donors present in the guanidinium group of arginine can complement bidentate oxoanion acceptors, along the two edges available [53]. Though the first guanidinium monomers for MIP preparation were described as early as 1999 [54], i.e. around the same period as Wulff’s amidinium monomer [5], there are not many associated MIPs as judged by the limited number of articles found in the literature. N-(3-methacrylamidopropyl) guanidinium chloride (GUA, Fig. 10B) has been employed a few times by Shea’s group who described its synthesis via two different synthetic routes, namely reacting N-(3-aminopropyl) methacrylamide hydrochloride (APM, Fig. 10B) with 2-ethyl-2-thiopseudourea hydrobromide [54] or with 1H-pyrazole-1-carboxamidine, under basic conditions [55]. The first protocol yielded a white oily solid with a 20 % yield after column chromatography purification, and the second protocol gave rise to a pale-yellow sticky solid, with a yield of 39 %. Importantly, the first protocol was advantageously modified by Treat et al., obtaining a clear oil with a yield of 72 % [56]. This GUA monomer was not prepared to synthesize MIPs but cell-penetrating polymers. It was reported to be soluble in acetate buffer pH 5.5, auguring its large

potential use in future preparations of water-compatible MIPs.

The first GUA-MIP was synthesized using t-BOC-L-phenylalanine as template in presence of EGDMA in DMF. It was compared to MIPs synthesized with different monomers containing basic functional groups, namely APM (as a mimic of the amino acid lysine), N-(2-methylacryloyl)-guanidine (MAG) and N-(2-aminoethyl) methacrylamide (AEM) (Fig. 10B). Surprisingly, AEM-MIP instead of GUA-MIP, showed the highest binding affinity, whereas APM-MIP (primary amine functional group as in AEM) and MAG-MIP (also containing a guanidinium group) do not bind to the template at all, as deduced from chromatographic data. In addition, neither of these polymers imprinted with optically active templates showed enantioselective recognition.

Later, Shea and co-workers [55] used GUA to prepare water-soluble acrylamide-based polymer nanoparticles (without template) for targeting fibrinogen, a negatively-charged protein (pI = 5.5) at physiological pH. GUA polymer NP was shown to have a higher affinity for fibrinogen (Fig. 10C, left), than two other NPs prepared with positively-charged monomers, namely APM and (3-acrylamidopropyl)trimethylammonium chloride (ATC) (Fig. 10B). The high affinity of GUA-NP was attributed to the guanidinium group which can form two hydrogen bonds and interact electrostatically with a carboxylate group on fibrinogen. The primary amine group in the APM-NP forms only one hydrogen bond with electrostatic interaction, and the quaternary ammonium group in ATC-NP does not form a hydrogen bond, resulting in a relatively weak interaction with fibrinogen. (Fig. 10C, right shows also that GUA-NP was more selective towards its target protein, even though there is ~35 % and 12 % of cross-reactivity with γ-globulin and HSA, respectively. Nevertheless, when added to a plasma protein mixture, the GUA-NP could selectively immunoprecipitate fibrinogen.

Another guanidinium-based monomer, acryloyl L-arginine amide.

<figure><img src="image_12.png" /><figcaption>**Fig. 10.** (A) Guanidinium group of arginine and its two possible binding modes with carboxylates. (B) Chemical structures of monomers with basic functional groups. (C) (left) QCM analysis of interactions between immobilized fibrinogen and charged NPs containing 20 % positively charged monomers; Final concentrations of NPs after each arrow are (1) 1.0, (2) 3.0, (3) 6.9, (4) 15, (5) 29, and (6) 56 µg/mL. (Right) Interaction of positively charged NPs with various proteins: Avi (avidin), RNA (ribonuclease A), Myo (myoglobin), Glo (γ-globulin), Fib (fibrinogen) and HSA (human serum albumin); Isoelectric points (pI) of each protein are indicated in brackets [55]. Copyright 2013, American Chemical Society.</figcaption></figure>
HCl, a polymerizable arginine monomer was frequently employed by Resmini's group [57,58]. This monomer seems to be easily accessible and is obtained in one-step by reacting acryloyl chloride with L-Arginine amide. 2HCl under basic conditions. Acryloyl L-arginine amide. HCl as well as its methacryloyl version [59] were used to prepare soluble acrylamide-based stoichiometric molecularly imprinted catalytic micro and nanogels in DMSO, with hydrolytic activity towards 4-nitrophenyl-4'-acetamidophenyl carbonate, respectively. The imprinting strategy was similar to that employed by Wulff; instead of an amidine monomer, an arginine monomer was used to induce a salt-bridge with the phosphate of the transition state analogue template in the active site, producing a MIP with an enhanced rate of carbonate hydrolysis (Fig. 11). Notably, acrylate and methacrylate-based guanidinium monomers, namely 2-acrylic acid 3-guanidinopropyl ester and 2-methylacrylic acid 3-guanidinopropyl ester respectively [60,61] also exist in the literature but have not been used for MIP preparation.

Two fluorescent guanidinium-based functional monomers that seem promising for detecting oxoanions are shown in Fig. 12. Titration studies of 9-(guanidinomethyl)-10-vinylanthracene with ammonium acetate showed a 1:1 complexation with an association constant of $1.2 \times 10^5$ M⁻¹ in deuteriomethanol [62]. A MIP incorporating this monomer and EGDMA was prepared in THF but no sensing experiments were shown. Recently, a polymerizable fluorescent benzoxadiazole guanidinium probe (Fig. 12) was developed and incorporated into MIP shells on silica core particles for the selective detection of the herbicide glyphosate (GPS), in chloroform and acetonitrile, which were also the solvents of polymerization [63]. Upon the titration of MIPs against GPS-TBA

(tetrabutylammonium) in both solvents, a quenching of the fluorescence response was observed. The binding of MIP to GPS-TBA fits a 1:1 stoichiometry, with high apparent association constants of $3.95 \times 10^4$ M⁻¹ and $2.01 \times 10^4$ M⁻¹ in chloroform and acetonitrile, respectively. Acceptable discrimination against other herbicides, such as 3,6-dichloro-2-methoxybenzoic acid and 2,4-dichlorophenoxyacetic acid was achieved in both solvents. According to the authors, the LOD of 0.6 µM and the broad dynamic range found in acetonitrile was promising for the development of a final assay to detect GPS in water at a concentration range for the acceptable daily intake of 5.3 µM.

## 6. Conclusion and outlook

Amidinium groups interact with oxoanionic species such as carboxylates, phosphates and phosphonates. The interaction developed between the two protagonists is based on a salt bridge, which is a combination of electrostatic and hydrogen bonding. Their association constants in both polar aprotic solvents (THF, DMSO, acetonitrile) and polar protic solvents (water, ethanol) is very high ($K_a > 10^3$ M⁻¹). The popularity of amidinium monomers originated from Wulff's 'lucky' monomer, N,N'-diethyl-4-vinylbenzamidine, which was originally employed for the synthesis of catalytic MIPs. At that time, in the late 1990s, MIPs were mostly synthesized in polar aprotic solvents and their catalytic activity was tested in aqueous mixtures of these solvents (Table S1). Though dealing with enzyme mimics, the MIPs were prepared in bulk solid form, and the recognition of the oxoanions was not done in complete aqueous media.

Later, following a logical evolution of the molecular imprinting technique, MIPs' biochemists got interested in synthesizing MIPs for protein recognition. Interestingly, benzamidine is a well-known inhibitor of trypsin from which came the idea to synthesize a polymerizable soluble form of benzamidine, N-methacryloyl-4-aminobenzamidine, which would act as an anchoring monomer, so as to produce inhibitor MIPs for trypsin. As the MIPs had to be hydrophilic and water compatible, acrylamide comonomers and crosslinkers under high dilution buffer conditions were used, resulting in potent MIP inhibitor microgels [14]. Since then, the passage from the microgel to the nanogel format, tending toward the size of antibodies, became obviously achievable. A major breakthrough came in 2010 when Hoshino and Shea reported the application of MIP nanogels for the neutralization and clearance of melittin, a peptide bee venom, from the bloodstream of mice [64].

With an objective to produce water-soluble nanosized MIPs for pathological protein biomarkers, we continued to develop MIP nanogels based on N-acryloyl-4-aminobenzamidine, a water-soluble amidine monomer that can be easily obtained synthetically in one-step, for the recognition of carboxylate groups within peptides and proteins. In this context, the MIPs were prepared in buffer and their recognition properties (affinity, specificity, selectivity) were determined using equilibrium binding assays with the free peptide and protein in buffer solution. Amidinium-based MIP nanogels were thus obtained for inhibiting trypsin digestion in cells, sensing in human urine, fluorescence bioimaging of cancer biomarkers on cells, and as therapeutic antibodies for disrupting tumor spheroids, or neutralizing inflammation in macrophages, among others. The well-structured salt bridge interactions provide extremely well-defined geometric configurations and contribute to conformational stability and specificity by positioning critical functional groups of the target molecule in a defined oriented position during imprinting. These binding site interactions were particularly useful for preparing MIPs which need to selectively bind their targets in aqueous or complex biological media where interactions involving high association constants are essential. Clearly, the amidinium groups were responsible for the improved selectivity of the MIPs.

Though the emergence of amidinium monomers stemmed from the occurrence of guanidine-based arginine residues in oxoanion binding sites in biological systems, guanidinium monomers were reported later, after the advent of Wulff's monomer, by Spivak and Shea [54]. In order

<figure><img src="image_13.png" /><figcaption>**Fig. 11.** (A) Arginine monomer flanked with its positively charged guanidinium group forms a salt bridge with the phosphate anion of the template TSA; (B) Carbonate substrate.</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>Fig. 12. Chemical structures of fluorescent guanidinium-functionalized monomers and template [62,63].</figcaption></figure>

to provide readers with a more balanced perspective of the two classes of monomers, a section has been devoted to guanidinium-based MIPs. Table S1 summarizes the different amidine and arginine monomers reported in this review. The synthesis of the different guanidinium monomers is well-detailed, most of them easily accessible synthetically, and can be found in the related references.

Also worth mentioning are neutral urea [65] and thiourea [66,67] host monomers for stoichiometric molecular imprinting of oxoanions. Urea and thiourea monomers possess two parallel NH hydrogen bond donor groups to interact with oxoanionic substrates. A strong interaction with the oxoanion can be obtained if the hydrogen is deprotonated. A series of urea monomers was synthesized by Hall et al. [65], among which the most performant was N-3,5-bis-(trifluoromethyl)-phenyl-N-4-vinylphenyl urea. Due to the electron withdrawing $-CF_3$ group on the aryl urea, the NH groups possessed higher acidities and thus a high association constant of $8.8 \times 10^3 M^{-1}$ was observed with tetrabutylammonium benzoate in DMSO-d6. However, it is known that H bonds are substantially weaker in polar protic organic solvents as well as in aqueous buffers. Effectively, selective oxoanions recognition in water by these monomers, has been rarely demonstrated.

The amidinium-based functional monomers designed for phosphate derivatives and carboxylate recognition could possibly target another class of oxoanions such as derivatives of sulfates and sulfonates, among which are sulfated glycosaminoglycans namely heparan, dermatan, chondroitin and keratan sulfates, which are interesting physiological targets as they are abundant on the cell surface. These stoichiometric monomers pave the way to the fabrication of a novel generation of MIPs with improved selectivities to operate in aqueous real-world situation, and should be applicable to various biotechnological and biomedical areas.

### CRediT authorship contribution statement

**Bernadette Tse Sum Bui:** Writing – review & editing, Writing – original draft, Conceptualization.
**Karsten Haupt:** Writing – review & editing, Supervision, Funding acquisition, Conceptualization.

### Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Karsten Haupt reports financial support was provided by French Universities Institute. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgement

K.H. acknowledges financial support from Institut Universitaire de

France.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.trac.2025.118509.

### Data availability

No data was used for the research described in the article.

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