Cyclic peptides in nature
Ion Binding
Cation Binding
Challenges associated with anion binding
Anion binding methods
Electrostatic Binding
Metal Ligand Binding
Hydrogen Bonding
Peptides as Ion receptors
Ion receptors and Transport
Lugdunin: A novel Cyclic peptide
Introduction
Antimicrobial resistance (AMR) is a growing challenge facing mankind. It describes what happens when microorganisms, such as bacteria and viruses, can no longer be combatted with using standard treatments# http://www.who.int/antimicrobial-resistance/en/. This means that infections that were formerly easily treatable become harder to fight and are more likely to spread. New antimicrobials, with novel modes of actions, are required to ensure this new challenge can be met.
One avenue to tackling these infections relies on exploiting differences in ion concentrations and functions. Ions are ubiquitous in nature and the selective and controlled transport of ions is critical for cellular function.1 If this natural equilibrium could be disrupted through ion transporters or receptors in a selective fashion this could provide a new source of antimicrobials.
Thiocarlide (#) and trichlorocarbanalide (#) are antibiotics which have shown modest transporting ability for anions.2 Potent small molecule anion transporters, with anion binding abilities, can induce apoptosis in cancer cells through transport mechanisms.3-6 This has been linked to their ability to disrupt cell homeostasis6 and more specific antimicrobial mechanisms.7
Cyclic peptides possess innate features that make them interesting scaffolds for ion receptors. Naturally occurring cyclic peptides have been shown to be biologically active against #, #, #. 8-9 This biological activity could be linked to ion binding and transport. If so then they could provide a new avenue for drug discovery.10
1. Cyclic peptides are important in nature
Peptides play an important biological role. They are integral to protein synthesis and cell functions. They also have interesting applications in a variety of fields including #blah, #blah and #blah.
Peptides have several advantages over traditional small molecule drugs, including low levels of tissue accumulation and potentially lower toxicity.11 A potential therapeutic agent must also be stable under biological conditions. However, linear peptides are susceptible to degradation by endo and exo proteases.12-14 Cyclising these peptides can help stabilise them, improving their therapeutic chemical and physical properties. 15 The synthesis of linear peptides is well established, however, the cyclisation of small linear peptides has historically been more challenging .
2. Ion binding
Ion binding molecules have been developed for several applications including anion sensors, anion responsive gels, the extraction and separation of anions from liquids and mixtures,16 in the field of catalysis.17 Another application of ion binders is as transmembrane ion transporters,18 this will be discussed in more detail later.
Generally, a number of factors impact a receptor’s binding ability including the type and number of binding sites, the relative size and geometry of the ion and receptor, the solvent in which binding takes place and the strength of ion solvation and the charge on the ion.19
2.1 Cation Binding
The field of cation binding is a well established field. The 1987 Nobel Prize in Chemistry was awarded for the discovery of crown ethers (#) which demonstrated ability to bind cations.20 Crown ethers and their derivatives demonstrate the ability to selectively bind cations through neutral binding motifs.19
Two cation binders of interest are valinomycin and monensin due to their role in membrane transport studies#reference.
Figure # Valinomycin (#) and monensin (#)
Valinomycin is a natural product with biological activity.21 It is a selective cationophore for potassium with a binding affinity of "5×" 〖"10" 〗^"4" 〖" M" 〗^"-1" . 22 It is a depsipeptide, where some of the amide peptide bonds are replaced with esters, and can encapsulate the potassium ion within the cyclic structure.23
Monensin is an ionophore with a quasilinear arrangement of heterocyclic rings.24 It is a selective sodium ionophore.25 In its deprotonated form it forms a pseudo cyclic structure stabilised by head to tail hydrogen bonding.24 In this form it can complex to a cation 25 and displays a ten fold selectivity for sodium.26
2.2 Challenges Associated with Anion Binding
In comparison to cations anion binding presents many challenges.27-28 Anions are larger than their equivalent isoelectric cation counterparts (Table #, extract data from Shannon Acta Cryst. ). Due to this they have lower charge to radius ratios than cations leads to weaker binding interactions.27-29
Table # the
Cation Radius/Diameter/Size? Anion
Na+ F-
K+ Cl-
Rb+ Br-
Cs+ I-
Anionic species may also be pH sensitive, which means different charged forms cay be present at different pHs.27-28 Therefore, receptors must function in the same pH range in which the target anion is unprotonated. Anions also have differing geometries (Figure # #Make figure) which need to be considered when designing receptors for a specific target.27-28
#Figure of different geometries#
Different geometries can provide an opportunity for receptor selectivity30 by positioning binding sites in a way which favours some geometries over others. Solvents also impact anion binding. Protic solvents, such as water or methanol, can form hydrogen bonds to anions. Anions have higher free energies of solvation that isoelectric cations of comparable size.30 An anionic receptor must therefore interact more strongly with the anionic species to compete with neighbouring molecules in the solvation sphere.30
#Something on the hoffmeister series?#
2.3 Anion Binding Methods
Anion receptors utilising different bonding interactions have been developed.
2.3.1 Electrostatic Binding
Electrostatic interactions can be utilised through the inclusion of a charged moiety within the receptor #Figure to explain this#.
In 1967 Park and Simmons reported the encapsulation of halides, including chloride, by macrocyclic polyammonium receptors (#).31 They demonstrated that the ammoniums’ protons pointed towards the encapsulated ion. A later crystal structure of this complex confirmed this.32
For example, a tren-based amine, N,N’,N’’-tris(2benzylaminoethyl)amine, reported by Bowman-James et. al can form a triprotonated structure # with selectivity for dihydrogen phosphate and hydrogen sulfate anions over other anions.33 This selectivity was explained through proton exchange with the oxo acids whilst the general affinity was explained the by charge and basicity of the receptor.33
Figure 2 # triprotonated tren- based amine, N,N’,N’’-tris(2benzylaminoethyl)amine
A number of other receptors have been synthesised, employing various moieties such as #, #, #. However, the inclusion of charged species in a receptor has several disadvantages. The positive charge necessitates a counterion which may compete for the binding site.27 Additionally, binding moieties that rely on protonation, such as the ammonium moieties employed in # and #, are subject to pH constraints. This adds an additional restriction as the target anion must be unprotonated at the pH required to operate the receptor.
2.3.2 Metal-Ligand Binding
The ability of metal ions -particularly d-block metals- to bind anions, can be exploited in the design of anion sensors.29 In particular, metals are useful for sensing in a competitive solvent mixture,34 because of the strength of metal ligand interactions. The energetic binding interaction between a metal centre and an anion relies only on the electrical charge on the metal ion, its electron configuration, and the change to the ligand field stabilisation energy induced by coordination.29 However, despite these advantages, metal ions by themselves are imperfect receptors. They have many possible binding sites, which can result in poor for charged anions and binding with uncontrolled stoichiometry.29 A simple way of increasing the anion selectivity of metal centres is attaching a multidentate ligand, which can limit the available binding sites and provide a secondary function such as signalling.29
Dipicolylamino (DPA) moieties are often used to sense anions by metal-lingand binding of complexed metal cations.34 The three nitrogen donor atoms complex to zinc(II) with selectivity over other common biological cations such as potassium, sodium, magnesium and calcium.34
An example of a receptor utilising metal-ligand bonding interactions was synthesised by Jolliffe et al. and utilised DPA moieties complexed to zinc(II) on a single face of the backbone #.35 It was shown to selectively bind pyrophosphate.35 A cyclic peptide backbone containing oxazole units was used to give the structure rigidity and to compliment the target anions size and geometry.35 Nuclear magnetic resonance (NMR) data displayed shifts in the oxazole and α-protons suggesting a change in the peptide’s geometry or that the amide protons were also involved in the binding.35
Figure 3 # redraw this image without pyrophosphate
However, these positively charged receptors may not always be suitable for applications. Charges can limit solubility in organic solvents#reference lei said this but didn’t ref and positively charged macromolecular species are often cytotoxic.36 Additionally, charged receptors require a counterion, such as nitrate in the case of #, which may interfere with potential applications. The metal used for complexation could also be toxic upon break-down of the complex (###or something).
2.3.3 Hydrogen Bonding
Considering the aforementioned limitations and challenges associated with electrostatic and ligand-metal anion binding, hydrogen bonding addresses some of these issues. Hydrogen bonds are non-charged, allowing for use in a more diverse range of applications. Additionally, hydrogen bonds are directional and could be utilised to design selective receptors based on the geometry of the target ion.28
Whilst hydrogen bonding interactions are comparatively weak (#Table of relative strength of binding interactions#) anions may still be bound using a convergent set of hydrogen bonds arranged to compliment the target anion.1
Table #
Type of Interaction Electrostatic Bonding Metal-Ligand
Bonding Hydrogen Bonding
Typical Strength
The sulfate binding protein (SBP) is found in Salmonella typhimurium and facilitates active transport of sulfate anions.1, 27-28 When bound to the protein its is desolvated and bound by neutral hydrogen bonding residues (Figure #).27 Of these hydrogen bonds, 5 are donated from the peptide backbone.37
#Figure of sulfate binding protein#
The arrangement of the binding groups matches the geometry of the tetrahedral dianion and is selective for sulfate over other anions.1 The charge of the dianion is stabilised by the cumulative contribution of dipole interactions particularly with the donated hydrogen bonds.38
More generally, proteins can utilise these same elements of multiple directional hydrogen bonds to bind and stabilise anions. Macrocyclic structures can preorganise these hydrogen bonding elements and reduce conformational flexibility.1
3. Peptides as Ion Receptors
In nature, large peptides and proteins are used to selectively bind anions.39 Amino acids provide opportunities for hydrogen bonding through amide protons on the peptide backbone. Side chains of amino acids including threonine and tryptophan also have potential for hydrogen bonds from the OH and NH indole groups respectively.
Molecular mechanics, molecular dynamics and ab initio calculations have been used to show the propensity of simple cyclic peptides to bind to both cations and anions in solution.40 In the modelled system by Cho et al. a cyclic hexapeptide of repeating alanine units (#) was shown to change conformations to accommodate different guest ions. Cations, in this case sodium, interacted with the carbonyl oxygen atoms which reorientated to point inwards whilst anions, such as fluoride, interacted with the NH groups which pointed to the guest anion.
Figure 4: # A cyclic hexapeptide of repeating alanine units and # a cyclic peptidic molecule with alternating Aba and Ala units
Ishida and coworkers demonstrated the ability of cyclic peptidic receptors to bind to phosphoesters via hydrogen bonds to the amide protons of the peptide backbone.41 They used alternating alanine and 3-aminobenzoic acid units, to promote a rigid structure, in a hexapeptide type structure (#) and demonstrated binding upon addition of a phosphate salt.
Other unnatural amino acids can be incorporated into a peptide backbone structure to improve rigidity. Oxazoles#, thiazoles#, azoles#, and rigid aromatic units can limit available conformations to promote selective binding.39 Cyclic peptide based receptors also provide opportunities to tune cavity size to induce selectivity. This can make cyclic peptide scaffolds appealing starting points for anion receptors. The modularity of peptide synthesis allows additional binding moieties to be incorporated as side chain functionalities (as in # zndpa one) such as ureas#, thioureas# and squaramides#.
4. Ion binding and transport
Nature employs many various proteins to facilitate the transport of ions across biological membranes.42-44 Anions such as chloride, bicarbonate and phosphate play important physiological roles and exist in high concentrations in cells.43 However, this concentration is not uniform and can vary across membranes (# reference needed). Phospholipid membranes are hydrophobic, and semi-permeable4 and allow the diffusion of small neutral molecules but prevent the transport of ions.44 This allows large concentration differences on either side of the membrane, for example the concentration of chloride anions ranges from 5-15 mM intracellularly, to 110 nM in extracellular regions, with even greater variance between different organelles.43
Controlling these differing concentrations, and the movement of ions between different regions in the cell is integral to cellular health.1, 45 The movement of ions across membranes affects a variety of functions in cells from maintaining pH# and regulating volume# to immune responses# and signalling.45 Mutations of native channels proteins are involved in many diseases – known as “channelopathies”.46 Cystic fibrosis is one such disease, caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR).47 This channel transports chloride anions across epithelial cell membranes.48
Biological transporters can operate through channel, relay, and mobile carrier mechanisms (Figure #, of the different mechanisms).42-44
Figure # REDRAW FIGURE# have better labelling
Channel proteins differ in the stoichiometry and direction of transport.44 (Figure # of symport, antiport and uniport).
Figure # REDRAW FIGURE Maybe combine with previous as two sideways panels
Cotransporters transport move two solutes either in opposite directions, antiport, or in the same direction, symport.44 This transport can be passive, down an electrochemical gradient, or active, against an electrochemical gradient and requiring an input of energy.44-45
An important feature of ion transporters is there ability to bind to the target ion particularly when operating through a carrier mechanism (Figure#). The mobile carrier needs to bind to the target anion, and this bound complex then diffuses across the membrane. Hence, the ability to bind anions is an important feature of transporters.16
As mentioned previously, ion transporters have shown potential as therapeutic agents.
#Should there be an example of a transporter with therapeutic activity here?#
5. Lugdunin: A Novel Cyclic Peptide
Natural peptidic products often provide scaffolds with potential biological activity that can be further explored and improved upon.8
A recent investigation of the metabolism of staphylococcus aureus in the human nose involved the collection of 87 different strains of staphylococci.49 These strains were then screened for antimicrobial activity against S. aureus.9 One strain, S. lugdunensis IVK28, prevented growth of S. aureus under # conditions through the production of an antibacterial substance.9 Through comparison with an inactive mutant strain and structural determination methods including nuclear magnetic resonance, electrospray ionization and high resolution mass spectrometry, a thiazolidine containing cyclic peptide (Figure #, image of lugdunin) was identified as the cause of the antimicrobial activity.9
Figure 5 Cyclic peptide structure containing thiazolidine unit. Redrawn with thiazolidine unit in colour#.
This cyclic peptide contains many structural elements that could indicate and ability to bind ions. The carbonyl groups could coordinate to cations and the restricted size of the cycle could provide opportunities for selectivity. There are also many hydrogen bond donors, from the peptide backbone amides, thiazolidine unit and the indole proton of the tryptophan moiety. This could allow it to bind to anions. Interestingly, the hydrophobic amino acids, valine and leucine, might allow it to pass through phospholipid membranes to facilitate ion transport.
These structural features make lugdunin an ideal candidate for further investigation of the ion binding and transport abilities of cyclic peptides. This could potentially explain its antimicrobial activity and open up further research into a novel mode of antimicrobial action – Ion binding.
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