Diverse Properties of Guanidiniocarbonyl Pyrrole-Based Molecules

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Cite This: Acc. Chem. Res. XXXX, XXX, XXX−XXX

Diverse Properties of Guanidiniocarbonyl Pyrrole-Based Molecules: Artificial Analogues of Arginine Joydev Hatai and Carsten Schmuck*

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Institut für Organische Chemie, Universität Duisburg-Essen, Universitätsstrasse 7, 45141 Essen, Germany CONSPECTUS: The guanidinium moiety, which is present in active sites of many enzymes, plays an important role in the binding of anionic substrates. In addition, it was also found to be an excellent binding motif for supramolecular chemistry. Inspired by Nature, scientists have developed artificial receptors containing guanidinium scaffolds that bind to a variety of oxoanions through hydrogen bonding and charge pairing interactions. However, the majority of binding studies is restricted to organic solvents. Polyguanidinium based molecules can form efficient complexes in aqueous solvents due to strong electrostatic interactions. However, they only have moderate association constants, which are significantly decreased in the presence of competing anions and salts. Hence, to improve the binding affinity of the guanidinium moiety, our group developed the cationic guanidiniocarbonyl pyrrole (GCP) moiety. This rigid planar analogue binds efficiently to oxoanions, like carboxylates even in aqueous solvents. The lower pKa value (7−8) of GCP compared to guanidinium derivatives (pKa 13) favors the formation of strong, hydrogen bonded ion pairs. In addition, carboxylate binding is further enhanced by additional amide hydrogen bond donors located at the five position of the pyrrole core. Moreover, the design has allowed for introducing secondary interactions between receptor side chains and guest molecules, which allows for optimizing binding specificity and selectivity. The spectroscopic data confirmed stabilization of guanidiniocarbonyl pyrrole/oxoanion complexes through a combination of ion pairing and multiple hydrogen bonding interactions. The key role of the ionic interaction in a polar solvent, is demonstrated by a zwitterion derivative of the guanidiniocarbonyl pyrrole, which self-assembles in both dimethyl sulfoxide and pure water with association constants of K > 1010 M−1 and K = 170 M−1, respectively. In this Account, we discuss strategies for making GCP functionalized compounds, in order to boost their ability to bind oxoanions. Then we explore how these building blocks have been incorporated into different synthetic molecules and peptide sequences, highlighting examples that demonstrated the versatility of this binding scaffold. For instance, the high oxoanion binding property of GCP-based compounds was exploited to generate a detectable signal for sensing applications, thus improving selectivity and sensitivity in aqueous solution. Moreover, peptides and molecules containing GCP have shown excellent gene transfections properties. Furthermore, the self-assembly and zwitterionic behavior of zwitterionic GCP analogues was used to develop variety of supramolecular architectures such as stable supramolecular β-helix structure, linear supramolecular oligomers, one-dimensional rods or two-dimension sheets, fibers, vesicles, soft nanospheres, as well as stimuli responsive supramolecular gels.

1. INTRODUCTION In the biological environment, the guanidinium moiety is a widespread mediator of specific noncovalent binding in various catalytic processes. The active sites of many enzymes contain guanidinium groups participating in the binding of anionic substrates.1 In addition to ground state recognition events, the guanidinium group also plays a decisive role in transition state binding in many catalytic processes as well, like the hydrolysis of phosphate diesters.2 Guanidine-containing therapeutic agents are used for the treatment of a wide spectrum of diseases.3 Besides these important roles, the guanidinium group was also found to be an excellent binding motif in the field of supramolecular chemistry. Guanidinium containing compounds have the ability to bind anions such as carboxylates, phosphates, sulfates, and nitrates through hydrogen bonding and charge pairing interactions.4 Many artificial receptors based on this principle are restricted to organic solvents, where guanidinium cations and oxoanions form stable ion pair interactions. However, in aqueous solutions, the © XXXX American Chemical Society

competitive solvation of both donor and acceptor sites by individual solvent molecules significantly reduces hydrogen bonding strength and ion pair interactions. In Nature, biological recognition processes mostly occur in the rather hydrophobic interior of proteins. The overall hydrophobic character of the binding pocket significantly reduces solvation of the binding sites by water molecules. Furthermore, molecular recognition relying only on hydrogen bonding (Hbond) interactions alone usually also does not work in aqueous media. To overcome such limitations and to enhance the binding affinity, improved abiotic guanidinium systems were developed that surpass the parent guanidinium cation in their binding properties.5 Such modifications can rely on different modes of action such as hydrophobic interactions, ion−pair interactions, cation−π interactions, multiple complementary H-bonds, or metal coordination. Thus, although the individual Received: March 18, 2019

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DOI: 10.1021/acs.accounts.9b00142 Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Accounts of Chemical Research

Figure 1. Early examples of mono- and polyguanidinium receptors (1−6).

interaction between host and substrate is weak, the combined effect can lead to a high association constant. 1.1. Oxoanion Binding by Simple Guanidinium Cations

Inspired by natural systems, researchers have designed various excellent abiotic hosts based on alkylguanidinium cations for oxoanion binding mostly in organic solvents. To date, many excellent reviews have been published in the field of arginine and guanidine based synthetic receptors; thus, discussion of this topic will be brief.5−7 In their pioneering work, McKay and Kresling reported bicyclic scaffold 1 (Figure 1) in which the guanidinium group is constrained in such a way that only one face is available to the formation of hydrogen bonds.8 Using this scaffold as a substructure Schmidtchen and co-workers reported various compounds of type 2 and investigated their binding properties with monocarboxylates, dicarboxylates and biologically relevant phosphates ions.9 Different derivatives of bicyclic guanidinium scaffold 3 containing naphthyl, uracil, or Kemp’s acid substituents were reported to form complexes with p-nitrobenzoate, 3′-AMP, or dideoxyadenine dinucleotide, respectively, as reported by deMendoza and co-workers.10 Lehn and co-workers developed a method for incorporating guanidinium groups into macrocyclic molecules (4) and explored the anion binding properties in organic solvents.11 Later, Hamilton and Anslyn introduced receptors 5 and 6, respectively. Others research groups designed polyguanidinium receptors that function in more polar environments such as aqueous dimethyl sulfoxide (DMSO) or methanol. Despite the very strong electrostatic interaction of these polyguanidinium complexes, they have only moderate association constants in water. Moreover, the presence of competing anions and salts significantly decreases their binding affinity. For example, receptor 6 showed strong binding affinity for citrate 7 with an association constant of K = 6.9 × 103 M−1 in pure water. However, in buffer solution, the affinity drops by more than 2 orders of magnitude (K ∼ 102 M−1) due to the increased salt concentrations.12 Based on theoretical calculation, we therefore introduced a new and easily accessible cationic guanidiniocarbonyl pyrrole (GCP) moiety 8 for efficient binding of carboxylates even in aqueous solvents (Figure 2).13 Acyl guanidiniums have lower

Figure 2. Guanidiniocarbonyl pyrrole cation 8 efficiently binds carboxylates due to a combination of ion pair formation and additional H-bonds.

pKa values (7−8) than simple guanidiniums (pKa 13), which favors formation of strong H-bonded ion pairs. Furthermore, binding is significantly increased by additional H-bond donors such as the amide and pyrrole NH. The motif is planar, rigid, and perfectly preorientated for binding of planar anions such as carboxylates. In addition, secondary interactions between receptor side chains and guest molecules can be easily introduced in order to achieve selectivity and specificity. Our group has reported the design and synthesis of various GCPcontaining ligands and their unique chemical properties. The strong oxoanion binding properties of GCP were exploited for sensing biomolecules, and the self-complementary binding agent GCP-zwitterion was developed for advanced supramolecular materials, like stable β-helix structures, one-dimensional rods or two-dimension sheets, as well as stimuli responsive supramolecular gels. Moreover, peptides and molecules containing GCP have shown excellent gene transfections properties. In this Account, we describe the strategies behind the rational design of GCP-derivatives for efficient binding of oxoanions in water as well as selected examples illustrating the usefulness of this scaffold both in vitro and in vivo. B


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Figure 3. (a) Structure of receptor 6 and isotherm of the binding of citrate with receptor 6 in D2O with added phosphate. (b) Structure and citrate binding isotherm of receptor 9 in bis-tris buffer and 1000-fold excess of chloride. The dotted line indicates changes in absorption due to simple dilution during titration. Panel (a) reprinted with permission from ref 12. Copyright 1997 Wiley-VCH. Panel (b) adapted with permission from ref 14. Copyright 2005 American Chemical Society.

Figure 4. “Knock out” analogues and NMR-titration curves of receptors (10, 13, 14, 17, picrate salts) with N-acetyl alanyl carboxylate (NMe4+ salt, 1 mM) in H2O/DMSO-d6, 2:3 (v/v) and binding constants (M−1) calculated thereof. Adapted with permission from ref 13. Copyright 2000 WileyVCH.

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2. GUANIDINIOCARBONYL PYRROLE: AN IMPROVED BINDING MOTIF FOR OXOANIONS The binding interaction of GCP based receptors with carboxylates in aqueous media was investigated by NMR, UV, CD, fluorescence titrations, and molecular mechanics calculations. The complex is stabilized through a combination of ion pairing and multiple hydrogen bonding interactions. For example, receptor 10 binds carboxylates (K ≈ 103 M−1) at least 1−2 orders of magnitude more efficiently than simple alkyl guanidinium cations in 50% water in DMSO. The GCP cation was found to be the most efficient of the monocationic binding sites for oxoanions reported so far. The improved binding affinity of GCP can be seen directly in a modified version of Anslyn’s citrate receptor 6, in which all three dihydroimidazole moieties were replaced by GCP groups. Receptor 9 binds to citrate (K = 105 M−1) in the presence of a 1000-fold excess of chloride anions even in aqueous buffer (Figure 3).14 The question arises: Which of the multiple binding interactions present are responsible for the superior anion binding by the GCP motif? Plausible candidates are the higher acidity of the acyl guanidinium cation or the additional Hbonds, as well as a combination of both. Therefore, a series of “knock out” analogues were synthesized and studied (10−17) (Figure 4a).13 The receptors were designed in such a way that each receptor lacked one or more potential binding feature from the parent GCP motif. Comparison of their binding interaction with suitable substrates allowed for estimating the energetic contribution of each specific interaction. The energetic contributions of the individual noncovalent interactions within our binding motif were found to be significantly different. Most interestingly, it turned out that the amide NH in position five of the pyrrole ring is crucial for effective binding of a carboxylate substrate. Its effect even exceeds that of the central pyrrole NH. In addition, the pyrrole system was found to be superior to benzene, pyridine or furan derivatives (Figure 5).

Furthermore, the ionic interaction plays an important role in stabilizing the complexation between carboxylates and GCP cations in polar solvents. For example, compound 18 selfassembles both in DMSO and pure water with association constants of K > 1010 M−1 and K = 170 M−1 respectively, whereas the neutral “knock-out” analogue 19 only selfassembles in chloroform with K > 104 M−1, although it contains an identical hydrogen bonding pattern (Figure 6).15 Thus, self-assembly of 19 strongly depends on the solvent compositions. For example, addition of >5% DMSO leads to complete dissociation of these dimers due to the competitive formation of H-bonds to solvent molecules. To investigate the origin of the high association constant of 18, we performed high-level theoretical density functional theory analyses taking into account several “knock-out” analogues in which hydrogen donor groups were replaced with either a methylene group or an ether bridge. The calculated results showed that the high stability of GCP associates like 18 is due to both the tailored hydrogen bond network and the electrostatic interaction, as well as their mutual influence on each other. For example, the dimerization energy of 20 (ΔE = 13.6 kcal/mol) is only half that of 18 (ΔE = 23 kcal/mol) (Figure 6). The above results demonstrated that charge interactions alone are also not sufficient and hydrogen bonding interactions also play a crucial role. After the detailed structural investigation, we have designed several GCP-based artificial receptors and in the following section their potential application will be discussed.

3. RATIONAL DESIGN AND POTENTIAL APPLICATION OF GCP-BASED MOLECULES 3.1. Artificial Receptors for Amino Acids

The design of artificial receptors, which efficiently bind amino acids under physiologically relevant conditions, still remains a challenging task.16 Receptors for amino acids often need hydrophobic effects and/or strong metal ligand interactions to achieve substrate binding in water. For example, efficient binding of amino acids by cyclodextrin or calixarene based receptors is often facilitated by hydrophobic interaction with an aromatic amino acid side chain. The superior binding properties of the GCP cation allows for oxoanion binding in water solely based on electrostatic interactions. Receptor 10 strongly binds carboxylates with binding constants up to K ≈ 2800 M−1 in H2O/DMSO-d6, 2:3 (v/v), which is much more effective than the parent N-acetyl guanidinium cation.13 Moreover, the complex stability also depends on the nature of the amino acid side chain.17 For example, the association constants for binding of D/L-phenylalanine, D/L-alanine, and D/ −1 −1 L-lysine by receptors 10 are K = 1700 M , K = 770 M , and −1 K = 360 M , respectively. The above results clearly show that in addition to electrostatic interactions the cation−π interaction between the aromatic ring of phenylalanine and

Figure 5. Repulsion between the lone pairs of the pyridine nitrogen and the anionic carboxylate oxygen is responsible for the less efficient binding of pyridine based acylguanidinium cations compared to the pyrrole systems.

Figure 6. Dimerization of different guanidiniocarbonyl pyrrole zwitterions. D


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rial chemistry. However, the realistic theoretical calculation for supramolecular complexes of this size is very difficult. Thus, in most cases, the de novo design approach on its own is not suitable. However, we used simple molecular mechanics calculations for rather small substrates to check for structural complementarity between host and guest. Following this principle, the receptor 23 was designed to bind dipeptides with a free carboxylate (Figure 9a).23 An additional hydrogen bonding motif was introduced that stabilized the complex and should improve the selectivity for dipeptides over simple anionic receptors. The binding of receptor 23 to anionic dipeptides strongly depends on the amino acids and decreases in the order of Gly < Ala < Val. This unexpected order of binding interactions between 23 and amino acids could be explained as follows: (1) The interactions within the complex with 23 are similar to those observed in a β-sheet, and valine is known to induce β-sheet rich peptide conformations.24 (2) The isopropyl side chains of valine effectively shield the H-bonds between the backbone amides from the surrounding solvents which provided additional strength. However, the receptor 23 fails to distinguish between different peptides. It could be that the interaction limited to the amide backbone of the dipeptide and complexation did not invoke any direct interaction with the amino acid side chain. Thus, receptor 24 was designed, where the GCP cation was linked to a novel cyclotribenzylene moiety (Figure 9b). It provides a hydrophobic, bowl-shaped cavity that can accommodate a methyl group but not any larger alkyl chain.25 Indeed, the solution studies and molecular mechanics calculations confirmed that receptor 24 strongly binds (1:1 stoichiometry, K = 33 100 M−1) alanine containing peptides such as D-Ala-D-Ala or its enantiomer L-Ala-L-Ala over dipeptides with other side chains.

acyl guanidinium unit of receptor 10 stabilizes the complex (Figure 7). However, the weak association constant of lysine is

Figure 7. Calculated structure of receptor 10 with N-acetyl phenylalanine carboxylate in water derived from molecular modeling (intermolecular H-bonds are shown as broken lines). The aromatic ring forms an additional attractive cation−π interaction with the guanidiniocarbonyl pyrrole moiety. Adapted with permission from ref 17. Copyright 1999 Royal Society of Chemistry.

most probably due to unfavorable electrostatic interactions between the positively charged ω-ammonium group in lysine and the guanidinium group. This finding inspired the introduction of additional binding interactions to the GCP N′ (21, Figure 8) in order to enhance selectivity as well as increase the recognition events with respect to the bound carboxylate.18 Based on this strategy, 22 binds to valine (K = 1750 M−1) and preferably to alanine (K = 1000 M−1), while the unsubstituted GCP cation 14 fails to discriminate between these amino acids. Molecular mechanics calculations confirmed the stronger binding of valine associated with favorable hydrophobic interactions between the two isopropyl side chains in water. In addition, the carboxylate forms an ion pair with the guanidinium moiety which is further stabilized by additional H-bonds shown in Figure 8.

3.3. Efficient Gene Delivery into Cells

Efficient delivery of nucleic acids into cells is crucial for many medical applications like gene therapy.26 Due to fundamental problems associated with viral gene carriers, nonviral cationic lipids or polymer vectors, like polyethylenimine (PEI),27 or lysine- and arginine-rich oligopeptides (often called cell penetrating peptides, CPPs)28 have been developed. Moreover, several modifications of arginine-rich CPPs such as, addition of a hydrophobic part to the peptide sequence, have been described in the literature to improve the translocation and transportation ability.29 However, at least 6−10 arginine residues were necessary for decent gene transfection by simple arginine based polypeptides.30 In comparison to guanidinium groups, GCP had shown superior binding affinity to carboxylates and it was likely that superior binding to phosphates present in the DNA backbone would also occur

3.2. Artificial Receptors for Peptides

Developing artificial receptors that target biologically important oligopeptides is of key importance to the design of various sensors19 and new therapeutics.20 In Nature, site selective (e.g., C-terminal) molecular recognition of oligopeptides plays a vital role in a variety of processes, like the mode of action of the antibiotic vancomycin21 or in Ras-protein induced oncogenesis.22 In principle, one can follow two distinct strategies: (i) de novo design of a complete receptor based on theoretical calculations or (ii) development of a motive with combinato-

Figure 8. Introduction of a substituent to the GCP N′ enhanced selectivity due to additional hydrophobic interactions with the side chain of the amino acid. E


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Figure 9. (a) Binding of various dipeptide carboxylates by receptor 23. (b) Receptor 24 and energy-minimized structure for the complex formed between receptor 24 (green) and the dipeptide D-Ala-D-Ala (gray) from the top, highlighting the hydrogen bonds (red dotted lines), and from the side, showing the interaction between the methyl group and the cyclotribenzylene cavity. Adapted with permission from ref 25. Copyright 2006 Wiley-VCH.

very efficient in both DNA-binding and transfection (Figure 11). The efficiency of 29 as a transfection agent was comparable to that of Lipofecatmine or PEI. But, in contrast to these commercial vectors, 29 showed only minimal toxicity. Later, we found that side chain functionalization of a tetra cationic cyclic peptide 30 with a single GCP moiety led to the formation of cationic nanofibers of micrometer length (Figure 12). This fiber formation led to efficient gene transfection without a need for any helping agents.34 The plasmid seemingly attached to the surface of the cationic nanorods. Further investigation showed that gene transfection occurs through a nonendocytic cellular uptake pathway.

(Figure 10a). Thus, we expected that replacement of the guanidinium group in arginine by GCP would improve gene transfection efficiency. Based on these considerations, we designed a library of three-armed molecules 25 composed of the dipeptide Lys-Phe as well as a terminal GCP moiety (Figure 10b).31 As other nonviral vectors, the receptor has the ability to bind both linear and circular DNA (K of ca. 107 M−1) and condense it into small polyplex-like structures. These can be taken up by cells via an endosomal pathway (Figure 10c). Nevertheless, often DNA binding ligands form polyplexes with DNA and show no or only very low efficiencies as transfection vectors. This could be due to the degradation of trapped DNA by lysosomal enzymes before transfection can occur. Often additional helper molecules, such as lipids or chloroquine, need to be added to facilitate endosomal release. This, however, was not necessary in the case of 25. On the other hand, receptors 26 and 27, which carry a higher number of charges and have a different binding mode (26: K of ca. 106 M−1; 27: K of ca. 107 M−1) also bind to DNA with high affinity and show a similar concentration-dependent DNA condensation. However, they did not facilitate transfection. Hence, we investigated structure−activity relationships of a set of similar molecules. It was concluded that for efficient gene transfection we need a sensitive balance between all effects32 such as (a) the number of positive charges, (b) the binding affinity, and (c) the buffering capacity. As an example, the best DNA binding ligand 28 identified by screening a library of 259 molecular tweezers composed of two identical peptide arms (amino acids AA1−AA3) and GCP (Figure 11) showed only minimal gene transfection efficiency.33 However, introduction of two lipophilic C18 hydrocarbon chains led to 29, which was

3.4. Inhibition of Beta Tryptase Activity

In recent years, development of multivalent ligands for biological recognition, particularly the inhibition of protein− protein interactions, has come into the focus of current research.35 Therefore, a wide variety of receptors has been developed that bind on protein surfaces. However, the recognition of protein surfaces decorated with distinct and unique patterns of hydrophobic and charged residues with high affinity and specificity is still a challenging field. We chose βtryptase as a target, which is the predominant serine protease of human mast cells. The inhibitors of β-tryptase reported in the literature were mainly based on either classical active site inhibition36 or heparin antagonists.37 Even though these compounds inhibit tryptase in nanomolar concentrations, they have poor selectivity relative to other serine proteases such as trypsin or chymotrypsin.38 β-Tryptase (PI 5.0−6.0) has some highly acidic “hot spots” which consist of clusters of negatively charged acidic amino acids (Glu, Asp). Furthermore, a new docking study suggested that the enzyme has at least two different binding sites for the cationic F


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Figure 10. (a) Binding interaction of phosphate present in the DNA backbone with a GCP group. (b) Artificial three-armed receptors composed of dipeptide Lys-Phe and GCP molecules. (c) Mediated DNA uptake mechanism: ligands bind to the plasmid, which are channeled into the cell via endosomal uptake. But only ligand 25 can act as a proton sponge in the endosomes, leading to their rupture and ultimately to DNA release and GFP expression.

ligands (Figure 13). Hence, we thought ligands with two different types of arms (31) could be a potent inhibitor (Figure 14a). The results of the screening clearly showed that the GCP containing inhibitor 31e is nearly 2 orders of magnitude more potent than inhibitors which consist of similar peptide sequences but lack the GCP group 31a.39 A closer look at the kinetic data revealed that peptide 31e showed two modes of inhibition (Ki = 0.056 ± 0.026 μM and Ki = 19.59 ± 2.63 μM) (Figure 14b). This biphasic inhibition profile, which was only observed for the GCP containing peptide, together with a docking study (Figure 14c) indicated that most likely two different sets of binding sites are present on the surface of βtryptase. Thus, the terminal GCP group containing peptide can not only significantly improve enzyme inhibition, it can also alter the mode of binding compared to analogous peptides containing only proteinogenic amino acids. As described above, the use of combinatorial chemistry has several advantages; however, solid phase synthesis and screening of a focused library still require significant effort. Dynamic combinatorial chemistry (DCC) is a method that generates a mixture of all possible combinations using reversibly forming covalent or noncovalent bonds between a set of molecular buildings blocks.40 In particular, disulfide41 and imine exchange42 reaction have been used successfully under physiological conditions. Considering the inherent limitations of imines (stability, isolation, pH), Lehn et al.43 introduced pre-equilibrated DCLs using reversible hydrazone formation in order to find efficient binders for given

Figure 11. Addition of a lipophilic tail to receptor 28 that had nanomolar DNA binding affinity and little transfection efficiency transformed into efficient and nontoxic transfection vectors 29. Adapted with permission from ref 33. Copyright 2013 Wiley-VCH.

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biomolecules (acetylcholinesterase, HPr kinase/phosphatase) based on a deconvolution strategy. Hence, we employed this alternative approach for identifying a potent inhibitor for β-tryptase through the screening and deconvolution of pre-equilibrated dynamic combinatorial libraries (DCLs). These were generated by a reversible hydrazone formation between tetrapeptidic hydrazides and di/trialdehydes (Figure 15a).44 Hydrazide 32C containing a GCP group at the terminus of the peptide sequences (GKWK(GCP)) was found as an active inhibitor (Ki= 22 nM) in the library. Removal of 32C restores the enzyme activity by 47% relative to the inhibition by the full library. In contrast, hydrazides 32A and 32B containing terminal arginine and lysine residues (ca. 20−25% recovery) are less active. Molecular mechanics calculations indicated that ligands bind to the active site of β-tryptase, blocking access to its central pore (Figure 15b). 3.5. Receptors for DNA and RNA Sensing

Small molecules that specifically interact with DNA and RNA have attracted significant attention, due to the relevance of such recognition events in medicinal, biochemical, and biological processes.45 In supramolecular chemistry the interaction with double-stranded (ds) DNA/RNA is often dominated by noncovalent binding modes (e.g., intercalation, minor or major groove binding, external electrostatic binding).46 Thus, we designed fluorescent probes by connecting a planar aromatic fluorophore (for intercalation) and a GCP unit with a flexible linker (for ion pairing interaction with the sugar backbone).47 This should allow for multiple noncovalent interactions with the DNA/RNA. For instance, the novel pyrene-guanidiniocarbonyl-pyrrole cation 33 was developed that efficiently differentiates between ds-DNA and ds-RNA by two different mechanisms.48 The mixture of ds-DNA and 33 exhibits a strong CD signal at about λ = 300 nm, while under the same conditions titration with dsRNA an excimer fluorescence maximum was observed at λ = 480 nm. The planar pyrene molecule likely intercalated into the double helix of DNA, while the GCP cation interacts with the DNA minor groove through selective H-bonds and ion pair interactions at pH 5. In contrast, most likely more than one molecule of 33 interacts with the major groove of ds-RNA thus forming a π-stacked excimer (Figure 16). Furthermore, we have seen that binding of GCP containing peptides to DNA is enthalpy driven compared to commonly observed entropy-controlled stabilization. We therefore hypothesized that more GCP moieties should increase binding events between peptides and DNA. Thus, the binding affinity of a series of tetrapeptides 34 (a−e) (Figure 17a) consisting of a different number of GCP and lysine residues was tested using calf thymus DNA (ctDNA). The results showed that binding became more enthalpy favored with an increasing number of GCP groups, compared to the free lysine residues (Figure 17b).49 Therefore, the introduction of GCP moieties into the peptide could effectively alter the thermodynamic signature of peptide/DNA binding from an entropy driven process to an enthalpy driven one.

Figure 12. Peptide 30 self-assembles into cationic nanorods which are able to shuttle DNA into cells. Adapted with permission from ref 34. Copyright 2016 Wiley-VCH.

Figure 13. Potential binding sites of β-tryptase derived from a computational docking study with the tripeptide KWR indicates two types of binding sites one with higher affinity at the two monomer− monomer interfaces (green), and one each nearby the four catalytic centers (red). Adapted with permission from ref 39. Copyright 2013 The Royal Society of Chemistry.

3.6. Self -Assembly of GCP derived Zwitterions

The self-association properties of 5-(guanidiniocarbonyl)-1Hpyrrole-2-carboxylate prompted us to design efficient building blocks for the construction of larger, self-assembled structures in polar solvents. For example, we have investigated that depending upon the structure and the experimental conditions H


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Figure 14. (a) General structure of inhibitor 31. (b) Biphasic inhibition curve of β-tryptase by inhibitor 31e. (c) (i) Calculated structure of a possible binding mode of inhibitor 31e, (ii) close-up of inhibitor 31e (shown as sticks with carbon atoms colored green), and (iii) schematic representation of the interactions between the ligand and the protein residues. Only two different arms of the inhibitor are shown. Adapted with permission from ref 39. Copyright 2013 Royal Society of Chemistry.

Figure 15. (a) Structures of acyl Hydrazones. (b) Calculated binding modes of (i) inhibitor 32(C)3 and (ii) inhibitor 32(D)3 to the surface (shown as mesh) of β-tryptase. Close-ups of the interactions of (iii) 32(C)3 and (iv) 32(D)3 (shown as balls and sticks) with the enzyme surface. The red, blue, and gray colors on the enzyme surface indicate the negative, positive, and neutral electrostatic potentials, respectively. Catalytic serine residues are shown using the space-filling model. Adapted with permission from ref 44. Copyright 2015 Royal Society of Chemistry.

Figure 16. Structure of receptor 33 and one of the low energy complexes of a dimer of 33 (stick representation) bound into the dsRNA major groove. H-bonds are broken lines; RNA atoms (oxygens) participating in H-bonds are shown in CPK representation (gray balls). Adapted with permission from ref 48. Copyright 2008 Elsevier.

GCP containing molecules can fold intramolecularly into loops50 or form linear supramolecular oligomers,51 fibers,52 vesicles,53 as well as soft nanospheres.54 I


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Figure 17. (a) Chemical structure of 34 (a−e). (b) Shift of the thermodynamic profile of peptide−DNA binding from entropy to enthalpy by increasing the number of GCP modifications. Adapted with permission from ref 49. Copyright 2016 Royal Society of Chemistry.

The long flexible linker allows for the formation of intramolecular ion pairs, thus leading to dimers. In contrast, molecular rigidity, which prevents any intramolecular ion pair formation is responsible for the oligomer structures. Surprisingly, the self-assembly behavior was also affected by the nature of the parent pyrroles derivative. After introduction of a sec-amide substituent at the rearward periphery (3position, 36c), the discoid dimers aggregated into a onedimensional rod following a nucleation elongation mechanism (Figure 19).55 Molecules such as 36 (a, b) were unable to form a rodlike structure. Therefore, it was obvious that π−π interaction between the discoid dimeric zwitterion dimers alone was insufficient for this aggregation. The formation of intermolecular hydrogen bonds between the peripheral amide groups was an important additional factor. Nevertheless, destruction of the discoid dimers by change of pH, and thus the protonation state of the zwitterion, converted the dimers into typically cationic amphiphiles that formed vesicles, tubes, and flat sheets, instead of linear rods. Importantly this change in assembly behavior could be switched back and forth by adjusting the pH accordingly.

For example, depending on alkyl chain length, zwitterion 35a forms large aggregates at low concentrations, whereas the more flexible zwitterions only form small oligomers (35b) or dimers (35c−e) at much larger concentrations (Figure 18).52

Figure 18. Self-assembly of flexible zwitterions such as 35 can in principle lead to various aggregates depending on the length and flexibility of the spacer in between the two opposite charges. Adapted with permission from ref 51a. Copyright 2007 American Chemical Society.

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CONCLUSIONS AND FUTURE OUTLOOK In this Account, we have summarized design strategies and potential applications of GCP in the field of supramolecular and bioorganic chemistry. We have shown that ion pair formation by guanidinium cations in aqueous solution can be

Figure 19. (I) Chemical structure of zwitterion 36 (a−d). (II) (A−C) Schematic self-assembly of the zwitterion 36c starting at infinite diluted solution (A) via dimerization (B) and nucleation (C) to formation of one-dimensional rodlike structures (D). Adapted with permission from ref 55. Copyright 2013 American Chemical Society. J


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significantly improved by acylation and by incorporating additional H-bond donor sites into the recognition motif. Both experimental and theoretical studies of several knock-out analogues revealed that each single factor such as hydrogen bond counting or charge interaction on its own fails to explain the dimer stabilities. Rather it depends on the combination of various factors, e.g., the acidity of the NH hydrogen atoms, the individual H-bond pattern, and the secondary interactions within the recognition motif. The GCP containing receptors strongly bound to amino acids and peptides, and inhibited protein activities. Moreover, peptides and molecules containing GCP have shown excellent gene transfection properties. Furthermore, the GCP derived zwitterion is a self-complementary agent, that can form dimers and is reversibly switchable by either acid or base. It was used to develop a variety of supramolecular architectures such as stable supramolecular β-helix structures, linear supramolecular oligomers, one-dimensional rods or two-dimensional sheets, fibers, vesicles, soft nanospheres, as well as stimuli responsive supramolecular gels. Currently, extensive research is being dedicated in our lab using both de novo and combinatorial approaches toward the development of water-soluble GCPbased artificial receptors that have the capability to inhibit the function of different proteins responsible for a variety of diseases. Also, more challenging in vivo applications of GCP containing molecules are currently being investigated.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies Joydev Hatai completed his B.Sc., M.Sc. at Calcutta University, and Ph.D. studies at IISER-Kolkata. In 2017, after completing his postdoctoral studies at Weizmann Institute of Science, he joined Prof. Dr. Carsten Schmuck research group as a postdoctoral fellow. His areas of research include exploration of the GCP containing building blocks for inhibition of protein activity and supramolecular assembly. Carsten Schmuck studied chemistry at the Ruhr University Bochum, where he also received his Ph.D. After a postdoctoral stay at Columbia University (New York), he started his independent career at the University of Cologne, before accepting a Professorship in Würzburg in 2002. Since 2008 he holds a chair in Organic Chemistry at the University of Duisburg and Essen. His main research interests lie in supramolecular and biomolecular chemistry, with a special focus on the application of taylor made binding motives in aqueous media.

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ACKNOWLEDGMENTS Fellowship from University of Duisburg Essen to J.H. is gratefully acknowledged.

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REFERENCES

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