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Cationic Antimicrobial Peptides (AMPs): Thermodynamic Characterization of Peptide–Lipid Interactions and Biological Efficacy of Surface-Tethered Peptides

The worldwide emergence of bacterial superbugs, and the limited number of effective antibiotic against them, has become an unresolved public catastrophe. According to a report from the World Health Organization in 2014, the resistance rate in a common bacterial pathogen such as Escherichia coli is high enough to limit the efficacy of currently available oral treatment options for patients with urinary tract infections; if cases where bacteria adsorb and form biofilms on implant materials, the treatment options are even more limited. Thus, the development of novel antibiotics for oral administration and as coatings for biomaterial surfaces represents an important and pressing research goal. 
 
A wide variety of organisms produce antimicrobial peptides (AMPs) as part of their defense system. The main site of action of the mostly positively charged and amphipathic AMPs is the membrane of the target cells. AMPs accumulate on the surface of the bacterial cell membrane and insert into the lipid matrix. This leads to disruption of the barrier function of the membranes and cell death. The AMPs bacterial selectivity is highly dependent upon the peptide structural motifs as well as the differences in organization of the cell membranes. Unlike the cholesterol-rich electrically neutral lipid membrane of eukaryotic cells, responsible for low peptide affinity and insertion, the bacterial cytoplasmic membrane possesses negatively charged phospholipids that, together with additional cellular envelope features such as the lipopolysaccharide (LPS) and peptidoglycans in Gram-negative and Gram-positive bacteria, respectively, represent ideal targets for cationic AMPs binding. This mode of action of AMPs could represent a basis for developing a novel class of antibiotics to counteract the issue of bacterial resistance. 
 
In this doctoral research, the structural basis of activity of short cyclic AMPs rich in arginine and tryptophan residues, as promising anti-E. coli candidates, was studied to understand membrane constituents and the peptide structural motif important for selectivity against Gram-negative bacteria. In addition, optimized chemical strategies were developed for tethering AMPs with different modes of action on polyethylene glycolylated (PEGylated) resin beads as a model solid surface. The biological activity of the surface-bound AMPs against bacteria was investigated and compared with the activity of the corresponding free peptides in solution. In particular, we sought to understand how a) the physical properties of the resin, such as the spacer length between the solid matrix and the AMP, as well as the capacity of the functional groups on the surface, and b) the tethering at different positions (peptide termini and side chain) affect the activities of the tethered AMPs.


CAPs
CAPs have been found in almost all species of eukaryotic organisms and are recognized as the evolutionarily conserved components of their innate immune system that defend the host against microbes through membrane or metabolic disruption [1]. A distinguishable advantage of CAPs over conventional antibiotics is that they do not provoke immune responses [2]. Regardless of their origin and biological efficacy, CAPs share common features. They are short peptides (usually between 350 amino acid residues), which include a large content of basic amino acids and a global distribution of hydrophobic and hydrophilic residues. Due to the high frequency of amino acid residues, such as R and K in their sequences, they carry an overall positive charge in the physiological pH range, which is of great importance for their interactions with the negative charges of bacterial cell membranes [1]. CAPs adapt an amphipathic conformation at polar-nonpolar interfaces with a hydrophobic domain consisting of nonpolar amino acid residues on one side and polar or charged residues on the opposite (Fig. 1). Because of these physicochemical characteristics, CAPs have a tendency to accumulate on the negatively charged microbial surfaces and membranes. Each CAP has a unique pattern of activity against a variety of Gram-positive and Gram-negative bacteria, yeasts, fungi and viruses [4]. While the antimicrobial peptide cecropin A is only active against Gram-positive bacteria [5], MG 2 and dermaseptin show activity against both types of bacteria as well as fungi [6,7], and the membrane-lytic peptide MEL attacks both prokaryotic and mammalian cells [8].

Structure diversity and basis of activity
In spite of their common mechanism of action, CAPs show remarkable structural diversity. The relationship between their structure and the spectrum of antimicrobial activity is a complex subject, because even CAPs of the same structure may have different effects upon microorganisms and mammalian cells [9,10]. Due to the high structural diversity of the large number of peptides discovered so far, it is difficult to classify CAPs into generally accepted groups. Nevertheless, CAPs can be roughly classified based upon their secondary structure into four major categories: -helical, -sheet, loop, and extended peptides [11] ( Fig. 2). CAPs with -helical and -sheet secondary structures are among the most ubiquitous peptides in nature. Besides this classification, there are a few groups of CAPs, which are classified based on frequent occurrence of one or more amino acid residues, e.g., Bac5 [13] and PR-39 [14] are PR-rich antimicrobial peptides; indolicidin has a high content of W residues, and histatin 5 [15] contains numbers of H residues. The positively charged residues are presented in black, the hyrdrophilic residues in gray, and the hydrophobic residues in white colors. Q, H, , and  represent the total positive charge of the peptide, peptide hydrophobicity, the hydrophobic moment, and the angle subtended by cationic residues, respectively. Adapted from ref. [3]. The strutures from top to bottom are: (a) -sheet structure; e.g., human -defensin 1, which forms a triple--sheet structure stabilized by three disulfide bonds. The N-terminal region of -defensin 1 also contributes to an -helix segment. (b) -helix structure, e.g., MG 2, (c) extended structure, e.g., indolicidin, which does not contain structural elements of helices or -sheets. Instead, it has a stretched spiral structure; (d) loop, which is restrained by disulfide bridge, e.g., tachyplesin. It adopts a -hairpin fold. Taken from ref. [12].
The knowledge and understanding of activity-modulating structural parameters responsible for the antibacterial activity and specificity of CAPs may provide insight into both the relationship between structure and activity as well as the mechanism of action. Because of their wide dissemination, -helical peptides are the best-studied CAPs. Dathe et al. demonstrated that the physicochemical parameters of CAPs, such as (i) the helicity and amphipathicity, (ii) the hydrophobicity, (iii) the hydrophobic moment, (iv) the magnitude of permeabilizing activity of the peptides. The reinforcement of the electrostatic interactions between positively charged peptides and negatively charged membrane components had a higher influence upon the bactericidal acivity than on the hemolytic effect.

Cellular basis of activity and bacterial selectivity 1.2.1 Membrane composition
The majority of CAPs work differently compared to conventional antibiotics, which generally block an enzymatic activity essential for a microorganism's reproduction through a specific interaction with a protein or a protein-nucleic acid complex [21]. Because CAPs often act via permeabilization of the bacterial cell membranes; one should consider the differences in structure of the outer layer of bacterial species, their cell profiles, and the variable composition of lipids in the plasma membrane in order to understand the basis of their different spectra of activity.
Bacterial cell membranes are usually rich in anionic lipids [1]. A schematic representation of the cell wall of Gram-positive and Gram-negative bacteria is shown in Fig. 3. The cell enevelope of Gram-negative bacteria consists of an outer and an inner (cytoplasmic) membrane. The outer membrane is highly asymmetric, with the outer leaflet covered by highly negatively charged LPS ( 90%) and the inner leaflet mainly composed of POPE. A thin layer of peptidoglycan, which constitutes the largest part of the preplasmic space, covers the distance between the outer and cytoplasmic membranes. The cytoplasmic membrane, which is considered as the target for CAPs, is composed of: POPE, negatively charged POPG phospholipid and cardiolipin. In contrast, Gram-positive bacteria have no outer membrane, but a thick peptidoglycan layer which protects the cytoplasmic membrane.
The main difference arises from the ratio of POPE and POPG phospholipids in the cytoplasmic membrane of Gram-positive and Gram-negative bacteria (Table 1). Whereas the  Table 1. Phospholipid composition of the plasma membranes of selected microorganisms. Adapted from ref. [22,23].

Phospholipid Composition (%)Wt Species/cell POPG POPS POPC POPE CL Other
Cholesterol LPS The bacterial negatively charged outer surface provides the basis for accumulation of cationic peptides, which is driven by electrostatic interactions. Compared to bacterial membranes, the outer layer of the mammalian cell membrane contains exclusively zwitterionic POPC, POPE, and neutral SM, and the inner layer contains POPE and POPS [24]. In addition, the plasma membrane of mammalian cells is characterized by the presence of cholesterol in both layers.
This endows the membrane with different mechanical properties responsible for reduced binding of amphipathic molecules, such as CAPs to these membranes (Fig. 4) [25].

Bacterial LPS
LPS is quite different from the membrane phospholipids and plays an important role as an efficient barrier to certain hydrophobic antibiotics [26]. As a result of destroying Gramnegative bacteria, LPS can be released from the cell wall into the blood stream and induce septic shock [27]. Thus, the interaction of peptides with LPS for both the antibacterial, as well as the anti-inflammatory, effects of the peptides is of crucial importance. LPS is an amphiphilic heteropolymer and consists of a conserved fatty acyl chain backbone known as LA and a large polysaccharide chain [28]. LA is the toxic moiety of LPS. The backbone of LA consists of a diglucosaminecarbohydrate backbone in a beta-(1'→6)-linkage, which is phosphorylated at positions 1 and 4' (Fig. 5) [28]. A total of four 3-hydroxy-fatty acyl chains, generally having a length of 12 to 14 carbon atoms and known as "fatty acids primary", are attached directly to the carbohydrate backbone by either ester-or amide-linkages (Fig. 5). The primary 3-hydroxy fatty acids can be further esterified by a total of 4−7 fatty acids. These fatty acid chains are so-called "fatty acids secondary", which are not directly bound to LA. The polysaccharide structure of LPS consits of an oligosaccharide core with a limited number of sugars, and highly variable polysaccharide chains composed of one or more oligosaccharide repeating units, the so-called O-antigen (Fig. 5). The O-antigen has several biological activities; it serves as a receptor for bacteriophages, modulates the activation of the alternative complement pathway, and inhibits the attachment of membrane attack molecules to the bacterial outer membrane [26,30]. When an LPS contains the O-antigen chain, it is described as s-form LPS (smooth) because the bacterial colonies have a smooth morphology ( Fig. 6). However, not all Gram-negative bacteria possess an O-antigen. Such bacteria are termed rough (r) because they form rigid, incomplete colonies on solid agar and autoagglutinate in saline. Several rough (r)-mutants were shown to have truncated polysaccharide cores due to defects in genes that code for glycosyl or phosphoryl transferases.
Depending upon the length of the core region of the polysaccharides, they are described as Re-, Rd-, Rc-, Rb-, and Ra-LPS (Fig. 6) [28]. The rough mutant containing the complete core is termed Ra-LPS. LPS which lack the terminal sugar are known as Rb-LPS, and Re-LPS is the shortest LPS mutant which occurs in nature. Re-LPS contains only two singly-negatively charged Kdo. The diversity of sugar moieties in the core region and the composition of LA are conserved in different E. coli strains. However; the O-antigen region varies strongly among different E. coli strains. Charged residues are located preferentially in the core oligosaccharide region. Ra-Re denote the different rough chemotypes of LPS. Native, complete LPS is called smooth LPS (s-LPS). Adapted from ref. [28].
LPS possess binding sites for calcium and magnesium ions are responsible for the structural stability of the outer membrane of Gram-negative bacteria [31]. The susceptibility of Gram-negative bacteria to CAPs has been proposed to be associated with factors that facilitate the transport of the peptide across the outer membrane, such as the magnitude and the location of the LPS charge, the concentration of LPS in the membrane and the absence of O-antigen chains. CAPs are either trapped in the outer barrier [32] or increase their permeability by causing disordering of the LPS organization [12]. A mechanism of "selfpromoted uptake" has been proposed for the transport of CAPs across the complex outer membrane, thus facilitating peptide accessibility to the inner target membrane [12].

Membrane permeabilization
Most of the soluble CAPs penetrate into the cell membrane and enhance the permeability. Electrostatic interactions cause the accumulation of the cationic peptides at the negatively charged bacterial membrane, and hydrophobic interactions drive their insertion into the lipid bilayer [33]. Additionally, counter ion exchange initiating a "self-promoted uptake" across the outer LPS-rich membrane modulates the peptide accessibility to the inner target membrane of Gram-negative bacteria [12]. Several models have been proposed to explain the modes of action of CAPs, which are presented in Fig. 7. However, the evidence for the anticipated modes of action is based on experiments carried out on model lipid systems [34]. Peptides bind to the membrane by assuming an amphipathic helix with the polar domain exposed to the membrane surface and the hydrophobic helix face buried in the lipid acyl chain region. Changes in the lipid bilayer structure and reorientation of the bound peptides lead to membrane permeabilization.

(A) barrel-stave model (transmembrane helix bundles)
This model describes a pore as an aggregated set of membrane-bound CAPs. The axes of the -helical sequences are oriented perpendicular to the membrane surface (Fig. 7A). The peptide sequences should be long enough to span a 30 Å thick lipid membrane, and thus this model is not appropriate for short CAPs. A typical example for this model is alamethicin, which forms stable ion channels [35]. The pore formation is initiated with alamethicin N terminus insertion into the membrane interior where the peptide hydrophobic region binds to the hydrophobic chains of lipids and the peptide hydrophilic region forms a central lumen.
The channel diameters range from 0.2 and 2 nm [36] and the number of helices which form the alamethicin pore varies between 3−11 [37,38].

(B) toroidal model (wormhole)
The toroidal pore model is comparable to the barrel-stave model; however, the pores are instable and poorly reproducible (Fig. 7B) [33]. In this model, the peptides associate with the polar lipid headgroups and penetrate into the membrane. The bound peptides cause lipid chain disorder and induce an enhanced lipid surface expansion and curvature modulation, which lead to an unfavorable strain between the outer and inner lipid layers. At high peptide concentration, the peptides are lined perpendicular to the membrane surface. Then, both peptides and lipids form a transmembrane pore which connects the inner and outer lipid layers [39]. The membrane activity of MG 2 is associated with this type of pore [40−42]. The pore diameter reaches 3 nm, about twice as large as the alamethicin channel [43]. Carpet-like model (e.g., model KLAL peptide). Taken from ref. [3].

Alternative mechanisms of action
In spite of the membrane-permeabilizing activities, a different view on the mechanism of action of CAPs involves their penetration across the membranes of pathogens without pore formation [4]. This process is followed by inhibition of intracellular processes, such as protein synthesis [45], DNA replication [46], or by targeting mitochondria and causing efflux of ATP [5]. Support for these mechanisms of action comes from investigations of BUF [46], histatins [47], the P-rich antimicrobial peptide indolicidin [48], and others [4].
Besides these modes of antimicrobial activity, lipid segregation induced by CAPs is another alternative model which leads to the formation of domains in bacterial membranes.
This model was proposed to explain the membrane permeabilizing action of cyclic RW-rich hexapeptides [49] (Fig. 8).

Significance of short CAPs
Over the last few decades, thousands of CAPs of various length, amino acid composition and conformation have been described [50]. Most of them belong to the class of -helical peptides. However, unresolved problems of toxicity against eukaryotic cells, the limited stability against proteolytic digestion in vivo and high production costs prevent using such CAPs orally and systemically. There is therefore a requirement for the development of new classes of CAPs with improved activity profiles [2].
Small CAPs rich in particular amino acid residues, such as R, W, and P have gained high interest as lead compounds [51−55]. They are usually found as small antimicrobial motifs of much larger natural compounds [56]. The mechanisms of interaction of small and conformationally constrained peptides with cellular membranes, as well as the key factors that provide bacterial specificity for these peptides, are much less well understood [57]. Thus, among others, small RW-rich peptides with improved toxicity against bacteria are interesting candidates to study the structural motifs and forces responsible for selectivity and may pave the way to develop new therapeutics with potent activity against multi-resistant bacteria.
The role of R and W residues for biological and bilayer permeabilizing activities has been described for several sequences. Examples are: the hexapeptide lactoferricin [58], indolicidin [59], TP [60], Pac-525 [61], the short bovine bactenecins analogs [54], and synthetic linear RW-rich hexapeptides indentified from combinatorial library studies [62]. On the level of the lipid membrane, the W residue has a high propensity to insert into the membrane and partition near the membrane-water interface [63], while positively charged R residues, with hydrogen bonding properties, provide the basis for peptide interaction with the anionic components of bacterial membranes. The electric dipole moment and hydrogen bonding ability of W with both anionic components of lipid membrane, e.g., phosphate groups and water molecules in the membrane interface, as well as intramolecular cation- electron interactions between R and W have been suggested to be responsible for favoured partitioning and the interfacial location of the peptides [64]. These unique properties make peptides, even if shortened to three amino acid residues, highly active [52].
Furthermore, cationic and bulky hydrophobic amino acids proved to be the best mimics of the amphipathic topology of the active-site -strands of LPS-binding proteins [65].
W, as a component in many LPS-binding motifs, also points to a significant role of peptidesugar interactions for selective toxicity against Gram-negative bacteria. Studies with lactoferricin-derived peptides and LPS mutant E. coli strains underlined the importance of an appropriate location of R and W residues for antimicrobial activity [66]. It has been suggested that the peptides first interact with the negative charges present in the inner core. LPS-disorganization finally results in facilitated approach of W residues to the LA as the preferred hydrophobic binding site [66].

Particular properties of RW-rich hexapeptides
RW-rich hexapeptides derived by structural modifications of the synthetic hexapeptide Ac-RW ( Fig. 9) [53] create one group of small CAPs. The parent sequence, Ac-RW, was identified by screening of a synthetic combinatorial library [62]. The peptide adopts an amphipathic structure in a membrane-mimetic environment [67]. Recently, it has been shown that head-to-tail cyclization of Ac-RW ( Fig. 9) distinctly enhances the peptide's activity against Gram-negative E. coli [53]. However, in contrast to its high antimicrobial activity, the cyclic peptide only weakly permeabilizes lipid bilayers [53,68]. Single amino acid substitutions or replacement of L-amino acid residues by Denantiomers were demonstrated to enhance or abolish the antimicrobial activity [53,68,69].
The peptides are able to permeabilize the outer and inner membranes of E. coli [70], but, interestingly, their activity decreases against wall-deficient L-forms of E. coli. Moreover, studies with mutant E. coli strains demonstrated that the activity of sequences with three adjacent aromatic residues, for instance, c-WFW, is reduced upon removal of the O-antigen and shortening of the core region of the outer-membrane LPS [70]. This points to a particular role of LPS in peptide activity and selectivity against E. coli.
Using solution NMR spectroscopy, it was shown that the c-RW consists of two -turn motifs [71]. On interaction with detergents and lipids, c-RW develops pronounced amphipathicity compared to its rather flexible structure in water [73]. Molecular dynamic simulations led to the suggestion that the peptide backbone lies parallel to the lipid bilayer surface, the positively charged R residues interact with the phosphate groups of the lipids, and insertion of the aromatic residues into the acyl chain region reduces the permeability barrier of a DPPC bilayer to water [74]. Furthermore, the peptide induces lipid demixing and formation of peptide-rich domains in DPPG/DPPE bilayers (Fig. 8) [49].
The observation that many cyclic hexapeptides are similar in conformation, but differ in the sequence [73] and their antimicrobial activities raises the question of whether specific amino acid residues are responsible for the cyclization-induced pronounced activity and selectivity of the RW-rich hexapeptides against Gram-negative bacteria. Studies using trimesic acid as a template mimicking the peptide backbone have demonstrated an essential role of the guanidino moiety for bacterial selectivity [74]. However, it seems that not only charge-driven peptide accumulation at negatively charged E. coli membranes, but also the exact nature of the interaction of W residues with LPS domains is important to be understood.
Only a few studies have investigated the influence of the aromatic W residues on the bacterial selectivity of the peptides. So far, c-RW analogs with a lipophilic Nal or a bulky non-aromatic amino acid residue, i.e., bicyclo[1.1.1]pentane, instead of W were demonstrated to increase the bactericidal activity of the peptides, whereas introduction of Y or F residues abolished the activity [53,68,69].
non-specific long-and short-range forces [77]. The most common invading pathogens that cause implant-associated infections include Gram-negative E. coli and Pseudomonas species, Gram-positive Staphylococci and Candida species [78]. Biofilm is rarely resolved by host defense mechanisms [75]. In addition, antibiotic therapy typically reverses the symptoms caused by planktonic cells released from the biofilm, but fails to destroy the biofilm itself [79]. The factors considered to be responsible for biofilm resistance include: inability of antimicrobial penetration in all areas of the biofilm, reduced growth rate in biofilm, and possible expression of resistance genes [80]. Due to their intrinsic resistance to conventional antibiotic therapy, biofilm formation leads to a significant increase in costs for removing them. Industrial companies spend billions of dollars a year to control them. To combat this threat, research must focus on antimicrobial coatings in order to reduce the initiation of microorganism aggregation on such materials and thus prevent biofilm formation.
Various approaches have been designed in ordrer to develop such biomedical surfaces including: (i) surfaces covered with bacteria-repellent or anti-adhesive agents using highly hydrated and close-packed, chain-like molecules, such as PEG [81] or bearing negative charges [82]; (ii) polymer matrices with incorporated antibiotics (non-covalently), which are released into the surrounding medium in a controlled manner [83,84]: and (iii) antimicrobial polymers, which are either prepared by polymerization of constitutive monomers with therapeutic moieties [85−87] or covalently bound to antimicrobial agents [88−93] (Fig. 11).
However, these strategies suffer from several disadvantages, such as limited affinity of biomaterials for antibiotics, modification of the mechanical properties of the materials, and a limited spectrum of therapeutics and active monomers with polymerization-compatible chemistry. The major obstacle is their hemolytic activity [90,94,95], particularly the toxicity of surfaces modified with quaternary ammonium, pyridinium and related compounds [88,96,97] to human cells [98].
The production of surfaces covalently covered with antibiotics is another important approach to overcome the problem of biofilm formation. Examples of these surfaces are ampicillin and penicillin attached to expanded poly(tetrafluoroethylene) [99,100].   immobilized cecropin-MEL hybrid peptide on a variety of different types of substrates such as amidated polymer brushes, hydrogel and beads [104]; nisin-tethered on block copolymers made of ethylene oxide and propylene oxide monomers [105]; cellulose membranes covered with short bactenecins analogs (Bac2A) [106]; a synthetic -helical CAP, namely E14LKK immobilized with poly(ethylene) [107]; resin beads covered with a -sheet CAP [108]; silicaand titania-cationic decapeptide (KSL) nanoparticles [109]; titanium surfaces coated with vancomycin [110], human host defense peptide LL-37 [111] or antimicrobial peptoids [112]; and TP derivatized amphiphilic block copolymers [113]. Inhibition of a biofilm culture of Pseudomonas aeruginosa, or other oral pathogens, by the human host defense peptide LL-37 or a peptide mimictic based on the structure of MG 2 are examples of this application [114,115]. The enzymatic degradation of a tethered peptide is significantly slower than the corresponding soluble peptide [101] and thus the antimicrobial activity mainly originates from immobilized, as opposed to leached peptides [103,104]. Moreover, the activity of these antimicrobial coatings may remain conserved even after heating up to 200 C and over a broad pH range [116].

Peptide-based biofilm
Recent strategies for the immobilization of CAPs on solid surfaces used either: (i) covalent linkage via SPPS [101,106,108,116], thioalkylation [102,104,111], EDC/NHS activation on COOH-enriched surfaces [103,107], disulfide exchange reaction on polymers using thiolated-peptides [105], or (ii) non-covalent methods using highly specific interactions, such as the biotin-streptavidin system [106]. CAPs may be tethered randomly on the solid surfaces or specifically at the C terminus, N terminus or side-chains of the peptides via the amino group of selected K residues present within the peptide sequences. However, due to the random immobilization process there is no control over the orientation of the tethered peptides.
The pioneer studies by Haynie et al. demonstrated the relationship between peptide structure and activity of soluble and resin-tethered peptides [101]. It was shown that Cterminal immobilization distinctly reduced the activity of potent antimicrobial sequences and that the relationship between the antimicrobial activity and structural properties, such as amphipathicity, was retained. Furthermore, the structure-related activity profile of the investigated peptides did not change with immobilization [101]. Studies with a -sheet antimicrobial peptide attached to PEG-modified beads or directly to the hydrophobic MBHA resin demonstrated that peptide immobilization via a long spacer stays bactericidal even after extensive washing [108]. These reports let to the expectation that the activity of immobilized peptides strongly depends upon the length and kind of spacer between the active sequences and the solid matrices. However, the flexibility of the peptides, the peptide density on the surface and the position of immobilization will also influence activity.
The biocidal activity of immobilized peptides may be position-dependent. This means that the tethered peptides should be tethered on surfaces at a position, which allows effective interaction between the critical domains of the CAP and the bacterial cell membrane. Support comes from tethered MG 1 [103], a cecropin-MEL hybrid peptide [104], and an -helical peptide E14LKK [107]. Randomly immobilized MG 1 on gold surfaces [103], as well as the peptide C terminally tethered on MEO 2 MA-co-HOEGMA brushes, showed good activity against Gram-positive bacteria [102]: the tethered peptide reduced surface adhesion of bacteria by more than 50%. In contrast, the antimicrobial activities of a tethered cecropin-MEL hybrid peptide [104] and an -helical peptide E14LKK [107] are orientation-dependent with randomly immobilized peptides being almost inactive. The highest specific biological activities were achieved with the cecropin-MEL hybrid peptide and E14LKK when they were tethered at the C terminus and N terminus, respectively [104,107]. The screening of antimicrobial activities of a library of tethered peptides derived from Bac2A analogs against P. aeruginosa demonstrated that the antimicrobial activity of the immobilized peptides is influenced by the positioning of hydrophobic and cationic amino acid residues within a sequence in respect to the site of linkage to the solid surface [106]. For optimal activity, the

Preparation and perspectives of surface-tethered peptides
It is expected that the antimicrobial activity of tethered peptides is influenced by both the peptide sequence and the solid surface. The aim of second part of the study was to know: 1. How does immobilization influence the biological and membrane permeabilizing activities of membrane active peptides?  Table 9). The immobilized peptides included: (i) -helical membrane-active peptides, such as a synthetic model (KLAL) peptide and MK5E (a peptide derived from natural MG 2), which act according to the carpet-like and toroidal pore mode respectively [44,118]; (ii) MEL, a membrane-active peptide with the ability to form discrete ion channels [8]; (iii) BUF, a highly cationic peptide without membrane-permeabilizing activity, which targets intracellular moieties [46]; and (iv) TP, an RW-rich peptide with a typical turn-turn structure which has ambiguous modes of action ranging from membrane lysis to interaction with intracellular targets [119,120]. Standard SPPS, thioalkylation, and oxime-forming ligation strategies were used to immobilize the peptides at the C and N termini and via different side-chain positions.
In order to demonstrate the suitability of the peptides for preparation of bioactive surfaces, were prepared to assess the lipid bilayer-permeabilizing activities of soluble and tethered peptides. These results were correlated to their biological activities in order to study the influence of tethering upon the peptides mode of action.

W-substituted c-WFW analogs
In order to investigate the influence of the physicochemical properties of W residues upon the bacterial selectivity of c-WFW and to analyze the interaction with lipid bilayers mimicking the inner and outer membranes of bacteria, W residues were replaced in the sequence with unnatural amino acids, such as Dht, Igl, 1MeW, Bal, 5MeW, 5MeoW, 5fW and b3-hW (Fig. 13). Each modification endows the cyclic peptide with a unique property ( Table 2). The biological activities of the peptides were correlated with their affinity for lipid bilayers doped with LPS mutants in order to indentify structural motifs of the peptides and interaction partners on cellular level, which are responsible for the recognition of E. coli.

Description and physicochemical properties of W-analogs
The aromatic side chain W is regarded as a highly hydrophobic residue based on the Liu-Deber hydrophobicity scale [121], characterized by a dipole moment of ~ 2.1 D in magnitude, which is directed from N-1 in the five-membered ring to C-5 in the six-membered ring of indole [122] and able to form hydrogen bonds. Dht is the product of the reduction of W, is no longer planar in character and introduces steric, as well as charge distribution changes. With a changed distribution of electrons, it has neither the aromaticity nor the dipole moment of W, but hydrogen bonding ability is conserved. As the aromaticity of W has been proposed as the reason for its waterlipid interfacial preference [63], Dht does not seem to position at this region. Igl also bears a fused-ring aromatic side chain, which is; however, closer to the backbone compared to Dht and incapable of hydrogen bonding. Similar to Dht, Igl is not expected to lie at the lipid-water interfacial region; however, it is more hydrophobic than Dht (Fig. 14, Table 3). The hydrogen bonding ability of W is blocked in the case of 1MeW by the more hydrophobic and bulky methyl group. However, 1MeW has the amphipaticity of W and a dipole moment (~ 2.2 D) with similar direction and magnitude [122]. Bal has the same size as the indole ring, but because of the lower electronegativity of the sulfur atom in the five-membered ring, its dipole moment is reduced compared to W ( Table 2). It has no considerable amphipathic structure and does not have the hydrogen bonding characteristics of W [123]. Both 1MeW and Bal residues are more hydrophobic than W. This appeared in the higher t R -values of c-  Bal Bal 5fW * The classification is based on the chemical structures of the residues. Their influence on peptide hydrophobicity and amphipathicity as reflected in the RP-HPLC retention behavior is shown in Table 3.
Unlike 1Metrp, 5Metrp is potentially able to form hydrogen bonding with water or anionic phosphate and carboxylate groups of lipid membranes. However, the quadrupole moment (aromaticity) and the dipole moment of 5MeW are similar to that of W and 1MeW in magnitude and direction [122,124]. In contrast, the methoxy group and fluorine atom with their electron-withdrawing effect at C-5 will enhance the dipole moment of indole. However, the density of negative charges above and below the plane of the indole ring is weaker in the case of 5fW, which will lead to a lower quadrupole moment compared to W. On the other hand, the methoxy group will push electrons into the indole ring plane resulting in strengthening the -electron system and quadrupole moment compared to W [124].
Substitution of the hydrogen at C-5 by the methoxy group and fluorine atom will also enhance the hydrogen bonding character of the NH moiety. In addition, the methoxy group and fluorine atom can form hydrogen bonds with water molecules at the lipid bilayer interface.
Incorporation of b3-hW with the same side chain as W enhances the size and likely the flexibility of the backbone ring ( Fig. 13). Therefore, the lipid-bound peptide might adopt conformations different from the parent cyclic peptide (c-WFW) with a different degree of amphipathicity.

Cyclic peptide synthesis and their HPLC characterization
The sequences synthesized for this study (Table 3) are based on the previously described hexapeptide c-WFW with three adjacent aromatic and charged residues [53,68].
Key features, such as the number and distribution of the three R and non-charged residues were maintained.
The hydrophobicity/amphipathcity of c-WFW is characterized by t R = 18.83 min in RP-HPLC (Fig. 14, Table 3). Because of the protonation of NH at physiological pH, the peptide with two Dht residues is much more hydrophilic than c-WFW. Also, c-(Igl)F(Igl) is highly hydrophilic and characterized by t R = 15.91 min in RP-HPLC. c-(b3-hW)F(b3-hW) is slightly more hydrophobic than c-WFW as the result of two extra methylene groups. The Balcontaining peptide is the most hydrophobic one in this series. Introduction of 5MeoW, 5fW, 1Metrp, and 5MeW only slightly enhances the hydrophocitiy compared to the parent peptide (19.08 min < t R < 20.41 min). * The purities of the products were more than 95% (Fig. 14). † These unnatural amino acids are not enantiomerically pure. See section for Abbreviations.

Characterization of cyclic peptides by CD
To get structural information on representative cyclic peptides in comparison to the linear Ac-WFW, CD spectra of the peptides containing N-1 and C-5 substituted W analogs, c-(b3-hW)F(b3-hW), and c-(Bal)F(Bal), as well as the parent peptide, were recorded in phosphate buffer, a mixture of buffer and TFE and in the SDS micelle-and POPG liposomebound state (Fig. 15). While studies in buffer provide information on the structural flexibility of peptides in aqueous solution, TFE represents a solvent which induces intramoleular and intermolecular hydrogen bonding and thus favours helix formation of linear peptides [125].
TFE solvent conditions were taken to monitor the ability / propensity of peptides to adapt a secondary structure [17]. The anisotropic nature of SDS micelles and POPG bilayers represent more suitable models of cell membranes and the high negative charge provides the basis for high peptide binding [126,127].
The spectra of c-WFW dissolved in buffer shows a negative band and a shoulder at   structure of c-RW and Ac RW in micelle-and lipid-bound states [67,71] indicates that the amphipathic structures of the cyclic peptides were little modified with sequence modifications [73]. Thus, the CD spectra of c-WFW is assumed to reflect the structures derived from NMR measurements: a hydrophobic cluster of the aromatic residues in contact to the hydrophobic chains of SDS and POPG and the backbone and cationic side chains exposed to the negative charges of SDS and POPG. Introduction of 5MeoW, 5fW, 5MeW and 1MeW had little influence upon the spectral characteristics of the cycles. However, following introduction of Bal, the contribution of the aromatic side chain to the spectrum was lost. The pronounced band intensity of the SDS-bound peptide at lower wavelengths (210 nm), where the contribution of the backbone amid bonds predominates suggests that binding also reduces the number of backbone conformers of c-(Bal)F(Bal).
Introduction of b3-hW enlarges the ring size and is expected to enhance the backbone flexibility of the hexapeptide. The b3-hW-containing peptide shows a sharp negative band at ~ 225−230 nm, both in aqueous environment and when bound to POPG membranes (Fig. 15).
This pronounced conformational change might be due to the disturption of the -turn motifs in c-WFW [73] and induction of an amphipathic conformation different from that of c-WFW.
Interestingly, in the SDS-bound state, spectral characeristics comparable to the other cyclic peptides, with a pronounced band in the 200 nm region, was observed for c-(b3-hW)F(b3-hW). This suggests that this peptide is also able to assume an amphipathic structure as suggested for the cyclic parent sequence and other analogs. Recent studies with analogs of Gramicidin S, with ring sizes ranging from 10 to 16 amino acid residues, confirmed the importance of amphipathicity induced by the -turn/-sheet structure in interaction with membranes [128]. Disruption of the structure by increasing the size of the cyclic peptide weakened the peptides' interactions with POPC vesicles and reduced their hemolytic, antimicrobial, and antifungal activities.

Antibacterial and hemolytic activities
The antimicrobial activities of c-WFW, its analogs and the linear sequence (Ac-WFW) are summarized in Table 4. In general, the activity of the cyclic peptides was higher against B. subtilis than E. coli and correlated with the peptide hydrophobicity.   higher activity than the parent peptide against S. aureus and E. coli [123]. c-(5fW)F(5fW) had the same activity as c-WFW whereas c-(5MeW)F(5MeW) and c-(1MeW)F(1MeW), two peptides with comparable high hydrophobicity, were less active against E. coli.
Except for the most hydrophobic peptides, c-(Bal)F(Bal) and c-(1MeW)F(1-MeW), causing 70% and 30% hemoglobin release from human RBCs repectively at 200 M, all peptides showed no significant hemolytic activity ( Table 4). The different susceptibilities of cells can be explained on the basis of peptide accumulation at the lipid matrices of the target membranes driven by electrostatic interactions.

Lipid bilayers as model of biological membranes
The envelope of Gram-negative E. coli bacteria consists of an inner (cytoplasmic) membrane with a composition of 80% POPE, 15% POPG, and 5% cardiolipin [23], and a highly asymmetric outer membrane with LPS ( 90%) in the outer leaflet and POPE as the main components of the inner leaflet. To study peptide binding to lipid membranes, POPC, and outer membranes [127]. limited to the inner core oligosaccharide [28]. In this study, only the total negative charges of phosphates anions (nine charges) were considered for binding studies. r-LPS contains roughly nine negatively charged phosphate groups and thus peptide binding to POPC/r-LPS bilayers was expected to be higher than to POPC mixed with LA, which bears two phosphate groups. However, it was found that the increase in negative charge in r-LPS-doped bilayers compared to the POPC/LA system only slightly enhanced R b (Fig. 20). In contrast, in the presence of s-LPS, peptide binding distinctly increased and the R b values were doubled compared to r-LPS and LA with reduced size of the carbohydrate moiety ( Fig. 20). This binding behavior correlates with reduced antimicrobial activity against Oantigen-and outer core-deficient LPS mutant E. coli strains, which was observed for RW-rich cyclic hexapeptides with three adjacent aromatic residues [70].

Role of sequence composition upon binding
Low binding of the linear and the two most hydrophilic cyclic peptides to POPC is reflected by R b values (~ 1 × 10 -2 mol/mol at c P,f = 30 M; calculated according to the Eq. 4 shown in the experimental section) (Fig. 20). The R b values (at c P,f = 30 M) follow the order: Fig. 20). The negative charge in POPC/POPG bilayers enhanced the R b, but also reduced differences in R b between the individual peptides (Fig. 20). With respect to the magnitude and sequence dependency of the thermodynamic parameters, the binding of the cyclic peptides to POPC/LA was comparable to that of POPC/POPG lipid system. Interestingly, for POPC containing r-LPS and s-LPS, peptide sequence-related differences were reduced (Fig. 20). An exception is Ac-WFW; its binding to liposomes containing LA, r-LPS and s-LPS was as low as to POPC and POPC/POPG SUVs.
These differences might be due to favorable contributions of the sugar moieties of LPS to peptide binding. In a recent study, the interactions between carbohydrates and aromatic groups have been described in terms of the hydrophobic effect, dispersion forces and carbohydrate electron interaction [129]. Carbohydrates interact in a favorable manner with peptides and proteins via stacking involving aromatic side chains [129]. The -electron distribution, surface area, and flexibility of the aromatic systems affect the magnitude of the interaction. In recent studies on a -hairpin peptide containing a W residue, as well as a glucosyl or galactosyl analog of serine, strong intramolecular carbohydrate- electron interaction (2.13.3 kJ/mol; stronger than - or cation- interactions) were found to stabilize the peptide's secondary structure [129]. SUVs (T = 37 C). It is assumed that the cationic peptides cannot cross the bilayers and only 60% of the total lipid amount is accessible for binding. The data were calculated by combining a surface partitioning equilibrium with Gouy-Chapman theory. The fit parameters (K 0 , ΔH°, and z P ) are listed in Tables 5 and 6.  (Table 6). Based upon these observations, it

Influence of lipid composition upon binding
can be concluded that hydrophobic peptide-LPS interaction is essential for an efficient transport across the bacterial outer membrane, but that LA does not act as a specific activity modulating binding site of the hexapeptides. This is in accordance with observations with lactoferricin peptides suggesting that the tight fatty acid packing in LA is not the primary site of interaction [130].

Role of sequence composition upon binding
Binding to POPC followed the order: Table 5). The free energy of binding of Ac-WFW, c-(Igl)F(Igl) and c-(Dht)F(Dht) is low, while G° values for the more hydrophobic peptides (t R > 18 min) range between -31 and -35 kJ/mol and are almost identical. Among these, c-(b3-hW)F(b3-hW) shows lowest partitioning (G° ~ -31 kJ/mol), indicating the role of conformational constraints of the cyclic peptides in binding. As shown in Table 5 and  (Table 5), which is in agreement with the classical hydrophobic effect [131]. This effect was observed for the antimicrobial peptide dicynthaurin as well [132]. The authors considered water and counterion release from the peptide and a sodium binding equilibrium at the lipid headgroups as the major driving forces for peptide-membrane interactions. The binding reaction of the other peptides including the linear Ac-WFW to both POPC and POPC/POPG systems is driven by enthalpy with H° values varying between about ~ -40 to ~ -20 kJ/mol (Table 5, Fig. 21). The enthalpy contribution is highest (> -30 kJ/mol) for c-(5fW)F(5fW) and c-(5MeoW)F(5MeoW) and followed by c-WFW and Ac-WFW. The pronounced H° of c-(5MeoW)F(5MeoW) and c-(5fW)F(5fW) correlates with a slightly enhanced peptide hydrophobicity and an enhanced hydrogen bonding tendency in comparison to the cyclic parent peptide (Tables 3, 5). The high H° contribution is associated with a low positive and negative value of TS° (Fig. 21B). H° of c-(b3-hW)F(b3-hW), c-(5MeW)F(5MeW), and c-(1MeW)F(1MeW) binding ranges between ~ -20 and -27 kJ/mol and the contribution of entropy is about 6−12 kJ/mol. The common feature of these cyclic peptides is a mean hydrophobicity reflected by a t R of about 20 min (Table 3). Except the Dht, Igl-and Bal-containing cyclic peptides, peptide binding to POPC and POPC/POPG lipid bilayers is in good agreement with the nonclassical hydrophobic effect as described for other CAPs [55,133,134].
These results demonstrate that low peptide hydrophobicity and conformational flexibility of the peptides reduced binding whereas the hydrogen bonding ability of the W residue and its dipole and quadrupole moments did not make any distinguished contribution to bilayer binding, as reflected by studies with c-(1MeW)F(1MeW), and c-(5fW)F(5fW) / c-(5MeoW)F(5MeoW), respectively (Fig. 21A, Tables 3 and 5). Studies with a 1MeW-modified lactoferricin analog revealed enhanced membrane binding and showed 1MeW to be aligned at the membrane interface with an extent of motion similar to that of W [135]. Furthermore, the influence of dipole and quadrupole moments became apparent in intramolecular interactions between the -electrons of the indole ring of W with the positively charged guanidino moiety of R in RW-rich peptides, which were suggested to stabilize the structure of CAPs and enhance membrane binding [136,137]. Because of the higher quadrupole moment (aromaticity) of the indole ring in c-(5MeoW)F(5MeoW) compared with c-(5fW)F(5fW) [124], a higher affinity of the former peptide for lipid bilayers was expected [136,137]. However, the opposite was observed ( Fig. 21A and Table 5). This underlines the contribution of other driving forces, such as the dominant role of hydrophobicity for insertion of the RW-rich cyclic hexapeptides into a phospholipid membrane.  Lipid bilayers (SUVs)   Binding of the cyclic sequences to LA systems was observed to correlate with peptide hydrophobicity: G° values gradually decreased from ~ -28 to ~ -35 kJ/mol (Fig. 22A).
Interestingly, partitioning of c-(Dht)F(Dht) and c-(Igl)F(Igl) into POPC/LA became entropically driven ( Table 6). The G° values for interaction with r-LPS containing bilayers scatter around -32 kJ/mol while they seem to increase with increasing t R for POPC/s-LPS lipid bilayers. Compared to the POPC/LA system, r-LPS doped bilayers favored partitioning of the most hydrophilic peptides (Dht-and Igl-containing cycles). The interaction of the most hydrophobic, c-(Bal)F(Bal), with the three lipid systems is least differentiated, as is reflected by comparable G° and K 0 values (Fig. 22A, Table 6). Furthermore, the ring size has little effect on G°.  (Table 6). In contrast, the contribution of enthalpy to binding of the hydrophobic peptides increases whereas the entropic effect decreases.
Unlike the expectation of the differentiated activity pattern against E. coli [70], no dependency of the G° to s-LPS-doped bilayers was observed upon the composition of the cyclic peptide, the only exception was c-(5fW)F(5fW). The aromatic residue is characterized by a reduced quadrupole and enhanced dipole moment and an increased hydrogen bonding ability compared to W (Table 2). However, enhanced binding does not improve the activity (Table 4). The peptide is as active as the parent peptide c-WFW against E. coli.

Effect of ionic strength upon c-WFW binding to r-LPS and s-LPS lipid systems
To investigate whether the enhanced partitioning of the cyclic peptides into LPSdoped POPC bilayers is due to electrostatic interactions, titrations of c-WFW with POPC/r-  Fig. 23 and the thermodynamic parameters are presented in Table 7. The increasingly exothermic enthalpy change with enhanced ionic strength was compensated by an increasingly unfavorable entropic contribution.
As observed previously, binding was strongest to bilayers containing O-antigen-  (Table 7, Fig. 23). This is related to a low antimicrobial activity observed for cyclic RW-peptides against E. coli mutant strains with outer core-and O-antigen deficient-LPS [70].

Heat capacity change on membrane partitioning of c-WFW
A common feature of both classical and nonclassical hydrophobic effects is their strong temperature dependency, which is characterized by negative C p° values [138]. To determine the heat capacity of peptide-lipid interactions, C p°, the partitioning of c-WFW into SUVs composed of POPC or POPC/POPG (3/1 mol/mol) as a function of temperature was measured.
The results are summarized in Fig. 24 and respectively. This signature of the hydrophobic effect results from the release of ordered water molecules from hydrophobic surfaces upon partitioning of the peptide into the lipid bilayer [138]. Another contribution to C p° may come from an increase in lipid acyl chain motion induced by membrane expansion on peptide binding [131]. These results are in contrast to large positive C p° values found for membrane partitioning of magainins [139].

Summary
Induction of conformational constraints in small RW-rich peptides by cyclization has been found to distinctly enhance antimicrobial activity, in particular against Gram-negative E. coli [53,68]. RW-rich clusters seem to be responsible for the selectivity increase as well as for the high affinity to lipid bilayers [53], and LPS moieties have been suggested to exert a strong activity-modulating effect [70].
In this study, W residues in c-WFW were replaced by various unnatural amino acids, such as Dht, Igl, 5MeoW, 5fW, b3-hW, 5MeW, 1MeW, and Bal (Fig. 13). This study showed that: i. Peptide hydrophobicity and backbone constraints are the crucial parameters for the biological activity (Hydrophilic and the linear and large cyclic sequences are least active) ( Table 4).
ii. Compared to B. subtilis, the activity profile against the E. coli strain is much more differentiated (Table 4).
iii. E. coli is slightly less susceptible than B. subtilis to the cyclic peptides and the hemolytic activity of the cyclic peptides is low (Table 4).
iv. The activity profile against bacteria correlates with the profile of free energy of partitioning into phospholipid bilayers ( Fig. 21 and Table 4). The negative charge in POPC/POPG bilayers enhances K app. but K 0 is independent upon the presence of the anionic lipid ( Fig. 20 and Table 5).
v. Low partitioning into POPC and mixed POPC/POPG bilayers correlates with low hydrophobicity of the cyclic peptides, the large cycle containing -amino acid, and the linear sequence ( Fig. 21 and Table 5).
vii. Sugar moieties of r-LPS and s-LPS modify the partitioning behavior. Lipid interactions are strongest for POPC doped with s-LPS. The difference between the linear and cyclic peptides is conserved; however, partitioning does no longer depend on the structural properties of the cyclic sequences. The results clearly demonstrate a strong supporting role of the outer core and O-antigen in peptide binding to LPS rich bilayers ( Fig. 22 and Table 6).
Membrane permeabilization by many cationic peptides depends upon both electrostatic accumulation by negatively charged membrane constituents and insertion into the bilayer, which finally results in a breakdown of the barrier function of the lipid matrix [44].
This idea is supported by the correlation of ITC binding data with hemolytic and antibacterial activities of c-WFW and its analogs. Accordingly, low peptide concentrations in the vicinity of neutral lipid bilayers are responsible for low hemolytic activities, whereas electrostatic attraction to negatively charged membranes favors partitioning into POPC/POPG bilayers and POPG-rich (~70 mol%) B. subtilis membranes [23]. In addition to the effect of the outer membrane, the slightly reduced susceptibility of E. coli could be due to lower peptide accumulation at the inner target membrane, which contains only 15 mol% negatively charged lipids [23]. However, the hydrophobic contributions to binding (as expressed by K 0 ; see Table 5) were identical. Peptide hydrophobicity and backbone constraints were found to be the crucial determinants of biological activity.
A balance between electrostatic and hydrophobic contributions to peptide-lipid interactions has been suggested to determine the positioning of RW-rich hexapeptides in the bilayer interface [68]. The preferential location of W residues at the membrane interface has been attributed to their aromaticity and ability to form hydrogen bonds with both water and polar lipid headgroup moieties [58,63,136,137,140,141]. In this study, hydrophobicity and

Site-specific immobilization of CAPs
In order to investigate the suitability of CAPs for the generation of antimicrobial surfaces, and to analyze parameters such as: the distance between the active sequences and the solid matrix; the density of peptide-loaded surface and the surface area available for peptide loading, which may influence the activities of tethered peptides upon bacterial cells and lipid bilayers; synthesis resins, i.e., TentaGel S NH 2 , TentaGel M NH 2 , TentaGel MB NH 2 , HypoGel 400 NH 2 , and HypoGel 200 NH 2 were used as models of solid surfaces. The variability of resin beads in size, capacity and spacer length represent a set of critical parameters to be analyzed. The model KLAL peptide and the MAG 2-derived MK5E, two highly membrane active -helical CAPs, were used for this purpose. The model KLAL peptide has both antimicrobial and hemolytic activity [44], while MK5E is selectively active against bacteria [118]. KLAL peptide was suggested to act via a carpet-like mode of action [44] whereas MAG 2, the parent peptide of MK5E, was described to form toroidal pores on interaction with bacterial cells and model membranes [118].
The surface tethering of CAPs is known to reduce the flexibility of peptides and may limit the range of peptide penetration to bacteria cytoplasmic membrane. Due to different mechanisms being responsible for the antimicrobial effect of the peptides, site-specific tethering is expected to be used as a tool to get information on the mode of action of CAPs ( Fig. 7). To examine this hypothesis, KLAL and MK5E peptides, as well as three natural CAPs (i.e., MEL, TP, and BUF), were tethered at C terminus, N terminus, and side chains via an -amino group of K residues. MEL, BUF, and TP form amphipathic structures at peptidelipid interfaces. MEL was suggested to act on bacterial cells through pore formation [8], whereas BUF targets intracellular processes after translocation across the cytoplasmic membrane [46]. TP has ambiguous mechanisms of action: membrane depolarization coupled to secondary intracellular targeting [119,120].
The antimicrobial activities of tethered and soluble peptides were correlated to their lipid bilayers' permeabilizing activities, not only in order to identify critical parameters in desigining effective biocidal surfaces, but also to examine their applications in order to shed light on to the modes of action of CAPs.

Physical and chemical properties of PEGylated resins as model solid surfaces
Synthesized resins, with PEG spacers of different length, were used to couple the peptides at different chain positions. PEG has been described to provide interfacial protective coating [142] and thus to improve the applicability (solubility and stability against enzymatic degradation) of proteins [143] and peptides [144].
TentaGel S NH 2 , TentaGel M NH 2 , TentaGel MB NH 2 , HypoGel 400 NH 2 , and HypoGel 200 NH 2 belong to the classes of divinyl benzene cross-linked polystyrene containing PEG grafts (Fig. 25). They are highly porous. The size of the pores for a TentaGel S NH 2 resin bead ranged between 0.10.2 m, which is smaller than the size of a bacterium [145]. The PEG grafts have different spacer lengths and represent the majority of the mass of these polymers. Thus, the properties of these hybrid resin beads closely resemble those of PEG [146]. Furthermore, the reactive centers are located at the terminus of the glycol spacers. These properties provide the opportunities for synthesizing combinatorial libraries using organic solvents followed by bioassays in aqueous media [147,148]. These resins have various size distributions ranging from TentaGel M NH 2 (diameter  10 m) to TentaGel MB NH 2 (diameter ~ 300 m) ( Table 9). Whereas TentaGel S NH 2 is characterized by a long spacer (3 kDa) and has the lowest capacity, HypoGel 400 NH 2 , and HypoGel 200 NH 2 have much shorter PEG spacers and comparably high capacities.

Characterization of KLAL and MK5E peptides by HPLC and CD
N-terminally acetylated KLAL and MK5E peptides, as well as sequences bearing PEG 2 chains at the intended positions of immobilization, were prepared to assess the influence of immobilization related charge modification and introduction of the PEG moiety on the peptide structure and biological effect. Table 10 shows the PEGylated and acetylated KLAL and MK5E peptides, their helicity and the t R -HPLC values as a measure of hydrophobicity.
Introduction of the hydrophilic PEG 2 chain did not substantially influence the retention behavior. This might be due to the low molecular mass (163 Da) of PEG 2. In contrast, N-terminal acetylation caused reduction of positive peptide charge and this increase in hydrophobicity also enhanced the t R .
CD spectroscopic investigations of KLAL, MK5E and the acetylated and PEGylated analogs confirmed an unordered peptide conformation in phosphate buffer (Fig. 26). TFE induced -helical conformation, as reflected by negative ellipticities at 207 nm and 222 nm and a positive CD band below 200 nm (Fig. 26). Although, the helical content of MK5E and Ac-MK5E was quite similar (~ 50%), the helicity of the most hydrophobic Ac-KLAL was enhanced by more than 30% compared with KLAL (Table 10). PEGylation decreased the peptide helicity independent of the position of PEG introduction (Table 10). Steric hindrance might be responsible for this effect.

Preparation and characterization of tethered KLAL and MK5E
peptides C-terminally immobilized KLAL and MK5E on TentaGel S NH 2 , HypoGel 200 NH 2 , and HypoGel 400 NH 2 were prepared by standard SPPS using Fmoc-amino acids [149]. It is known from the literature [101] that the major sources of impurity in SPPS are deletion sequences and chain termination that might occur at each coupling step due to nonquantitative coupling reactions. Thus, the activities of C-terminally immobilized peptides might result not only from the right sequences, but also from minor contributions of deficient sequences. N terminus and side chain immobilization of KLAL and MK5E on TentaGel S NH 2 using the thioalkylation [150] and oxime-forming ligation strategies [151] were performed with HPLC-purified sequences (Fig. 27). Thus, the antimicrobial activities of these immobilized peptides result only from the correct sequences. The formation of tethered peptides was confirmed after cleavage of the sequences immobilized on TentaGel S RAM resin (Fig. 28). TentaGel S RAM resin is similar in the chemical structure to TentaGel S NH 2 resin, but has a TFA cleavable linker. The molecular masses of the peptides coupled via the thioalkylation and oxime formation were 57 Da. and 69 Da. larger than those values for the Cys-and AOA-modified peptides, respectively ( Fig. 29).  A comparison of the resin capacity and the density of immobilized peptides shows that about 1/3 of the reactive groups of resins was occupied with C-terminally immobilized peptides (Tables 12, 13). Furthermore, under the applied coupling conditions, the density of C-terminally TentaGel S NH 2 -bound peptides (0.099 and 0.133 mol/mg for KLAL and MK5E peptide respectively) was about three times higher than the density of the N-terminally and side chain-tethered sequences. This might be partially due to the different synthesis procedures and limited accessibility of the reactive functional groups. It has been reported that at most 15% of the total amount of functional groups of typical TentaGel beads are located on the surface of the bead [152]. Moreover, protein immobilization onto TentaGel was shown to be limited to the bead surface [153]. Whereas SPPS on the porous resin will result in a substantial amount of peptides, which might be not accessible for interaction with biological membranes, immobilization of the complete peptide sequences via thioalkylation and ligation strategies will be restricted to the surface of the beads. The enhanced capacity of the HypoGels (Table 9) leads to an enhanced (Table 13) but, compared to TentaGel S NH 2 , the coupling efficiency slightly decreased as reflected by an increase in the ratio of resin capacity to peptide density.

Biological activities of free and tethered KLAL and MK5E peptides
Antimicrobial activity of the free peptides All KLAL and MK5E peptides showed activity against B. subtilis and E. coli in the micromolar range (Table 11).
To model the loss of charge connected with immobilization via the N-terminal amino group, acetylated and N-terminally free KLAL and MK5E sequences were compared.  (Table 10). Likely, the t R value of Ac-KLAL was further enhanced by an increased amphipathicity, which is based on the enhanced helicity at interfaces as has been described for other peptides [154], and found here for Ac-KLAL as compared with KLAL under structure inducing solvent conditions.

Although the introduction of PEG 2 at different positions of both KLAL and MK5E
caused the antimicrobial activities and the helicity of the N-terminally free and acetylated parent sequences to decease slightly (differences of one dilution step among the antimicrobial activities of PEGylated KLAL and MK5E peptides), changes in the activity spectrum were not observed (Tables 10, 11). One might speculate that these small changes are due to the low molecular weight of the attached PEG 2 moiety. However, recently it has been reported that even coupling of much larger PEG units did not influence the basic mechanism of membrane permeabilization of amphipathic peptides [155,156]. Thus, modification of MG 2 with PEG (5 kDa) resulted only in slightly reduced antimicrobial activity and did not change the interaction patterns with lipid bilayers [155]. Similar observations were made with β-sheet tachyplesin [156]. In contrast, the loss of the antimicrobial activity of PEG-nisin [144] could only be explained by the disturbance of the peculiar mechanism of action, which is based on selective lipid II binding and consecutive migration of the peptide's C terminus through the cell membrane.
These results show that the loss of the N-terminal charge may result in a drastic reduction of the antimicrobial activity, in particular against Gram-negative bacteria, whereas introduction of PEG chains at different chain positions might be expected to have little influence. Therefore, it is concluded that the conservation of the cationic charge with peptide immobilization is particularly important for maintaining the peptide activity against Gramnegative bacteria.

Antimicrobial activity of the tethered peptides
Peptides immobilized by the thioalkylation and ligation strategies inhibited the growth of both bacteria at similar MIC regardless of the position of immobilization (Table 12).
Concentrations between 0.1−0.2 mM, and 0.6−0.8 mM of KLAL peptides immobilized at the N terminus and side chains were required to inhibit bacterial growth and to act bactericidally against B. subtilis and E. coli, respectively (Table 12). The activity of randomly immobilized KLAL was comparable to other side chain-specific immobilized sequences.
The activity profile of TentaGel S NH 2 -bound MK5E peptides was found to be slightly different from KLAL (Table 12). The side chain and C-terminally immobilized MK5E sequences showed comparable activities, but a charged N-terminal -amino group seems to be necessary for maximal bactericidal activity in particular againat E. coli, which diminish with either acetylation or N-terminal immobilization. The comparably high MIC values of C-terminally immobilized KLAL and MK5E are likely related to the fact that the majority of these sequences (~ 75%; see ref. [152]) were tethered in the interior of the porous TentaGel S NH 2 and might not be accessible for membrane interaction [152,153]. Indeed, the small pore size of TentaGel S NH 2 [145] does not allow for bacterial entry and penetration. In all cases, the MBC values of the immobilized peptides were identical to the MIC. * The amount of resin-immobilized peptides was determined from three independent experiments based on the absorption of the Fmoc-chromophore at 301 nm ( = 6000 M -1 cm -1 ). The standard deviations ranged between 2 and 10 %. † Each MIC of peptide-covered resin was determined in one experiment using a serial dilution of the peptideloaded resin. The values were confirmed in two independent experiments using the determined MIC and two resin concentrations below and above the MIC. No changes were found (Fig. 40). ‡ The MIC of the immobilized peptides was calculated on the basis of the concentration of peptide-covered resin causing growth inhibition and taking into consideration the density of the peptides on the resin beads. Based on the standard deviation of the surface density, variations in the MIC are less than 10 %. § The numbers give the chain position of immobilization. K stands for the lysine residue. ¶ ND, not determined Consequently, tethering conserved the activity spectra of the peptides at reduced concentrations. The resin-bound peptides were antimicrobial against E. coli and B. subtilis in the millimolar range compared to the results seen with micromolar concentrations of the free peptides. Moreover, insertion of either KLAL or MK5E into the membrane as a mode of action is supported by the fact that the activity is independent of the site of immobilization. As the immobilization at TentaGel S NH 2 was performed at the chain termini and the K residues in the polar face of peptide helices, the immobilized peptides are assumed to arrange their amphipathic helix in the membrane surface.

Hemolytic activity of the free and tethered peptides
KLAL peptides were active towards RBCs with EC 25 values of about 10 M (Table 11). MK5E was not hemolytic up to concentrations of about 400 M. TentaGel S NH 2 showed a concentration dependent hemolytic effect. Hemolysis was less than 10% up to about 80 mg/ml, but rapidly increased at higher concentrations (Fig. 30)

MK5E peptides
The ability of peptides to permeabilize the lipid bilayer of liposomes and thus to induce the release of incorporated dye was measured in order to compare the membrane

The free peptides
The EC 50 values of initial calcein leakage (Fig. 31) showed that all acetylated and  (Table 11, Fig. 31).

The tethered peptides
The kinetics of the dye release from POPC, and mixed POPC/POPG LUVs induced by C-terminally TentaGel S NH 2 -bound KLAL and MK5E peptides is presented in Fig. 32.
Although different in the magnitude, each of the resin-tethered peptides induced disruption of the bilayers in a dose-dependent manner. The activity profile of the bound peptides correlated well with the effects of the corresponding free peptides. Non-modified TentaGel S NH 2 was not bilayer active. These results suggest that C-terminal immobilization via a large PEG chain conserved the bilayer permeabilizing ability of the free KLAL and MK5E peptides. The dye release was monitored as increase in the fluorescence intensity at 514 nm at c L  25 M.

Effect of spacer length
Because the thickness of the cell envelope for E. coli and B. subtilis has been reported to be 46 nm and 45−55 nm respectively [157−159], the role of the spacer length upon the antimicrobial activities of KLAL and MK5E peptides C-terminally immobilized on  [1]. The observation suggests that a long spacer-related high flexibility of the immobilized peptide is of advantage for antimicrobial activity.

Surface density of tethered peptides
The bacterial membrane possesses in the order of 10 5 anionic charges [160]. Taking into consideration that a Gram-negative E. coli has an average surface area of 0.5 × 2.0 m 2 , the membrane charge density is approximately 10 13 charges/cm 2 . The capacity of TentaGel S NH 2 is 280−330 pmol/bead (Table 9). Taking KLAL and MK5E peptides, the density of positive surface charges per TentaGel S NH 2 bead is much higher than the surface charge density of an E. coli bacterium, even if only one part of the total charge of the resin (at least ~ 2 × 10 17 charges/cm 2 ) is considered. Therefore, the peptide density on the surface of TentaGel S NH 2 beads is sufficient to promote resin-cell interaction as demonstrated by the fact that higher loading of the HypoGels did not enhance the activity (Tables 12, 13).
Moreover, the peptides loading on HypoGel 200 NH 2 , which is about twice of the peptides density on HypoGel 400 NH 2 ( Table 13), showed that increasing the peptide loading did not improve the biocidal activity if the flexibility of the immobilized peptide is limited by a short spacer. These results lead to the suggestion that with increased constraints induced by a reduced spacer length, the ability of peptides to bind to the bacterial membrane was conserved; however, the membrane permeabilization efficiency, e.g. the ability of the peptides to insert into the target membrane was reduced. Nevertheless, the antimicrobial activity of HypoGel-bound peptides suggests that interactions with the outer layer of bacteria provide a substantial contribution to the effect. For cationic biocidal polymers an exchange of structurally essential divalent cations in the bacterial membrane leading to the disturbance of the permeability barrier has been suggested [88,97,161−165]. The high cationic charge density of peptide-loaded HypoGel resin beads might have the same effect.

Effect of particle size
To investigate the effect of the surface area of the peptide-coated solid matrix upon biological activity, the model KLAL peptide was immobilized on resin beads of different size: TentaGel MB NH 2 and TentaGel M NH 2 ( Table 9). The surface area of TentaGel M NH 2 is closer to the size of a bacterium ( 0.5 × 2.0 m; along the short and long axes of the ellipsoidal body) than that of TentaGel MB NH 2 . However, both resin beads have an identical overall loading capacity (0.20.3 mmol/g). Taking the diameters of spherical beads and the number of beads in 1 g resin (Table 9), the surface area of TentaGel M NH 2 is calculated to be almost 35 times larger than that of TentaGel MB NH 2 .
The density of C-terminally immobilized KLAL on TentaGel MB NH 2 and TentaGel M NH 2 was 0.167 and 0.102 mol/mg, respectively (Table 14). Inverse to the density, KLAL immobilized on TentaGel M NH 2 was more active than the TentaGel MB NH 2 -tethered peptide against Gram-positive and Gram-negative bacteria (Table 14). According to the suggested mode of action, the peptide accumulates onto the membrane of bacteria until it reaches a threshold concentration followed by lysis of the membrane in a carpet-like mode [44].  . 33).  Taken together, these results suggest that enlargement of the surface area available for peptide tethering may lead to enhanced peptide aggregation onto the bacteria membranes and thus, enhanced the antibacterial activity of CAPs with a membrane-lytic mode of action.

Characterization of MEL, BUF, and TP peptides
MEL (26 amino acids) shows antibacterial and hemolytic activity. It has five basic residues with two -helical segments, which have been connected together with a helixbreaking P residue. Basic and hydrophobic residues are located mainly at the C termius and N terminus, respectively (Table 15). The hinge allows the N terminus and C terminus helices to localize independently upon interaction with the bacteria membrane. The hydrophobic flexible "GIG" hinge sequence at the N terminus has been proposed to be important for insertion into the lipid bilayer [10]. Membrane insertion and association of the peptides into ion-permeable pores, as proven by discrete conductivity levels, has been suggested to lead to permeabilization of the bacteria cell membrane by toroidal pore formation (see Fig. 7) [166].
BUF (21 amino acids) is selectively active against bacteria. It is highly basic with charged residues (+7 including one H residue) dispersed throughout the sequence (Table 15).
BUF is not membrane active and the peptide appears to target intracellular nucleic acids after translocation across lipid bilayers without significant permeabilizing activity [167]. A hinge induced by a P residue within the sequence was found to be responsible for translocation across the cell membranes [46]. TP (13 amino acids) is a cathelicidin-derived CAP, which is rich in R, W, and P residues (Table 15) [168] with membrane permeabilizing activity as well as the ability to translocate across the cytoplasmic membrane [119]. The peptide has four R residues localized at the peptide termini with three W residues in the center of the sequence. The presence of two P residues induces a unique amphipathic -turn structure in interaction with detergent micelles [64].  (Fig. 39).
The peptide sequences, the AOA-modified analogs and the t R -HPLC values are presented in Table 15. AOA modification influenced the peptide hydrophobicity. Whereas, modification of MEL at the C terminus resulted in a more hydrophilic peptide (t R  22.5 min compared with t R  23.2 min for the parent peptide), no difference in the t R values was seen between MEL and AOA-MEL (Table 15). In contrast, AOA modification at peptide termini had no effect upon the t R values for both BUF and TP. However, TP-AOA and AOA-TP were more hydrophilic than TP (Table 15).

Characterization of tethered MEL, BUF, and TP peptides
MEL, BUF, and TP were immobilized at the C terminus and N terminus on TentaGel S NH 2 resin beads using the oxime-forming ligation strategy (Fig. 27). HPLCpurified AOA-modified peptides were used. Linkage of peptides at the N terminus results in reduction of one positive charge compared with the C-terminally modified counterparts. The tethering was confirmed by the immobilization of the peptides on TentaGel S RAM as described before (Fig. 34). The derived molecular masses of the immobilized MEL, BUF, and TP correspond to the theoretical values.
The density of MEL and TP tethered at the C terminus and N terminus on TentaGel S NH 2 was almost identical (~ 0.02 mol/mg for MEL, ~ 0.15 mol/mg for TP) whereas for BUF, the density of tethered peptide at N terminus was higher (Table 16).

Antimicrobial activity of free peptides
The peptides showed antimicrobial activities at micromolar concentrations (

Inner and outer membrane-permeabilizing activities of MEL, BUF, and TP peptides
To further analyze the membrane activity of MEL, BUF, TP and their AOA-modified analogs, we examined the effect of the peptides on the integrity of the inner and outer membrane of E. coli (strain ML-35p). The results are summarized in Fig. 35. Membraneactive KLAL peptide was used as the positive control. The peptide concentration was around the MIC values for E. coli (strain DH 5). BUF and its AOA-modified analogs do not show significant permeabilizing activities at concentrations of 10 M and 30 M respectively, the activity patterns were comparable to the buffer control. The good antimicrobial activity (Table 15), but inability to permeabilize the bacterial membranes, is consistant with a membrane-independent mechanism of action as previous studies have suggested [46,167,169]. are much lower than the MICs of the N-terminally tethered MEL (up to a factor 5). This position-dependent activity indicates that the N terminus of MEL should be presented for optimal interaction with bacteria cell membrane and supports the suggested mode of MEL action: Insertion of the peptide's hydrophobic N-terminal domain into the membrane while the charged C terminus is located in the membrane surface. A similar membrane localization has been suggested for the highly antimicrobial and hemolytic cupiennin 1 peptide [170], which has a structural similarity to MEL [166]. The polar C terminus modulates the peptide accumulation at negatively charged cell surfaces via electrostatic interactions [171]. The hydrophobic N terminus inserts into the hydrophobic core of the lipid membrane and acts as a driving force for pore formation and membrane disruption. Membrane insertion of both peptides results in formation of an ion-conducting pathway.

Antimicrobial activity of tethered peptides
No differences in the activities of TP immobilized at the C terminus or N terminus exist against B. subtilis (MIC = 0.74 mM) or E. coli (MIC = 2.94 mM). With TP insertion into the membrane, the three consecutive W residues form a large hydrophobic domain and the charged termini remain exposed to the surface [120]. Lipid displacement and pore formation are the consequences. Tethering of the peptide at both termini has obviously little influence upon membrane localization and organization, thus excluding a mechanism, which is related to deep penetration or translocation across the membrane. So far, no convincing results were obtained for immobilized BUF (Table 16). Because BUF has interacellular targets [46,167,169], it has to translocate across the bacterial membrane. It is expected that immobilization excludes the peptide from the cytoplasm and thus abolishes the biological effect (studies will be continued). For nisin, Joshi et al. showed that the covalent immobilization results in the loss of antimicrobial activity [105]. Nisin belongs to the group of lantibiotics [172] and targets the lipid II, which is located on the cytoplasmic side. The authors immobilized thiolated nisin on poly[ethylene oxide]poly[propylene oxide]-poly[ethylene oxide] triblocks via formation of a disulfide bridge, but the peptide was active against bacteria only after reduction of the disulfide bridge, which means after peptide release. The peptide had to be free for migration of its C terminus across the cell membrane and the formation of nisin-lipid II complexes [173].

Bilayer permeabilizing activities of free and tethered MEL, BUF, and TP peptides
The bilayer-permeabilizing activities of the peptides against negatively charged POPC/POPG (3/1 [mol/mol]) LUVs mimicking the charge properties of the lipid matrix of the inner membrane of Gram-negative bacteria (Fig. 36) correlate well with the activity profile derived for peptide-induced inner membrane permeabilization (Fig. 35). The activity of the peptides decreases according to the following order: MEL peptides > TP peptides >> BUF peptides. As MEL, TP and BUF showed comparable antimicrobial activity against E. coli (Table 15), the results suggest different bactericidal modes of action for the peptides. Unlike the channel-forming MEL, Yang et al. suggested that the effect of TP is due to depolarization of the bacterial cell membrane, coupled to targeting intracellular components [119]. In contrast, the BUF peptides showed no permeabilizing activity even at the concentration of 400 M, which is consistent with the results of Kobayashi et al. [167,169] and the idea of the peptide translocation across the cytoplasmic membrane to reach intracellular targets. MEL, TP and BUF immobilized at the C terminus and N terminus confirmed the activity pattern (Fig. 37). BUF and TP are both inactive and the kinetics of peptide induced bilayer permeabilization is slow at about 1 mM concentration. In contrast to the free peptides, tethered BUF peptides cause higher fluorescence intensity compared to the tethered TP peptides. Considering the density of loaded peptides (Table 16), this might be due to the light scattering induced by higher amount of resin used for BUF peptides (Fig. 37). On the other hand, C-terminally tethered MEL induced dye release more efficiently than the peptide bound at the N terminus at much lower concentration (0.1 mM). The dependency of activity upon the site of MEL immobilization again underlines the role of the hydrophobic N terminus domain for membrane insertion. In summary, the distribution of the hydrophobic and charged amino acid residues within a peptide sequence is an important issue, which has to be taken into account for preparation of tethered peptides. Because the free hydrophobic region within a peptide sequence has to insert into the cell membranes in order to disturb their integrity, this domain should always be far away from the site of immobilization. Whereas, for peptides, such as KLAL and MK5E with almost identical distribution of positively charged amino acid residues along the sequenece, and TP with its charges at the sequence termini, the activities of the immobilized sequences are not position dependent (Tables 12 and 16), MEL should be tetherd specifically at C terminus for maximal activity. Moreover, not all CAPs may be active against bacteria when they are tethered. The primary support comes from the resin bead-tethered BUF, which causes neither cell death nor permeabilization of lipid bilayers.

Summary
Contact-active cationic antimicrobial surfaces have been proposed to exert their bactericidal effect by penetrating the bacterial cell wall if their biologically active site is far enough away from the surface of the solid matrix (Fig. 38). Autolysis, initiated by the exchange of membrane stabilizing cations, has been suggested as the mode of action [88,97,161−165]. Because of the toxicity of these surfaces to human cells, in recent years increasing attention has been focused upon surfaces covered with CAPs. Among the various methods of immobilization of CAPs, covalent attachment offers several advantages such as long-term stability and lower toxicity of the biomolecules compared to their incorporation into release-based systems [101]. However, the application of the appropriate chemistry for peptide tethering on materials, which induces little modification of the peptide structural parameters, such as conformation, charge, hydrophobicity and amphipathicity is of particular importance [16]. Chemical strategies including SPPS, thioalkylation and oxime-forming ligation were used in order to link CAPs with different activity spectra and modes of action at the C terminus, N terminus, and side chains on PEGylated resin beads as model solid surfaces (Fig. 27). The CAPs include membrane-active peptides, such as model KLAL peptide [44], MG 2-derived MK5E [118], MEL [8], BUF [46,167,169], and TP [119,120,168].
Reasonable antimicrobial activity of surface-bound peptides requires the optimization of the coupling parameters such as the length of the spacer, the surface area of solid matrices, and the amount of target-accessible peptide. Studies with the tethered membrane active peptides, e.g., KLAL, and MK5E, on TentaGel S NH 2 resin (the resin, which is characterized with the PEG spacers (3 kDa) ( Table 9) long enough to span the thickness of bacteria cell membrane) demonstrated that tethering conserved the activity spectrum of the peptides at reduced concentrations (Table 12). The antimicrobial activity distinctly decreased with reduction of the spacer length and even an increase in the loading capacity of the resin, e.g., HypoGel 200 NH 2 , was not sufficient to compensate for the spacer length-related activity decrease (Tables 12,16). Furthermore, an increase in surface area of the solid matrices for peptide tethering improved the antimicrobial activity. The analyzed parameters are relevant for the establishment of a more general approach to obtain efficient biocidal solid matrices loaded with CAPs.
Furthermore, the studies demonstrate that immobilization can be used as a powerful tool to gain insight into the modes of peptide interaction with biological and model membranes. Differences in the activity of N-and C-terminally tethered MEL confirmed the important role of the MEL N terminus for insertion and peptide orientation within the lipid matrix to form pores, whereas the independency of KLAL activity upon the site of immobilization confirmed an orientation of the helix parallel to the lipid bilayer and a carpetlike mode of action. Hopefully, ongoing studies on immobilized BUF would enable us to distinguish between the peptides with a membrane-perturbing mode of action and those with intracellular targets.

Summary
CAPs are components of the innate immune system of mammalians and play an important role in the defense of all organisms, including plants, against invading pathogens.
Their mode of action, based on interaction with the cell membrane and their properties, such as rapid action and a low tendency to stimulate the bacterial resistance have been considered as a promising basis for the development of a new class of antibiotics, particularly for topical application. In recent years, much effort has been focused upon modifying such peptides with respect to antimicrobial activity. However, only identification of the structural motifs and optimization of peptide interactions with the different classes of pathogens, such as Grampositive or Gram-negative bacteria will provide the basis for the development of highly selective antimicrobial compounds. Furthermore, increasing efforts have been focused upon the development of antimicrobial surfaces, which inhibit the growth of bacterial adhesion and subsequent biofilm formation.

W-substituted c-WFW analogs
In the first part of this work, it was planned to identify interaction moieties on the cellular level and the driving forces responsible for the pronounced activity increase in RWrich hexapeptides after induction of conformational constraints by peptide cyclization, using systematic modification of the hydrophobic cluster. For this, a small library of peptides based upon the sequence of cyclo-RRRWFW was prepared, in which W residues were replaced by unnatural analogs such as Dht, Igl, 5MeoW, 5fW, 5MeW, 1MeW, Bal and the -amino acid b3-hW with altered hydrophobicity, dipole and quadrupole moments, ability of hydrogen The reason why distinct differences in hydrogen bonding ability, dipole moment, and aromaticity of the W residues are not reflected in the thermodynamic characteristics of the peptide interactions with LPS-containing lipid bilayers remains to be elucidated.

Site-specific immobilization of CAPs
In the second part of this work, the influence of immobilization upon the activity profile of CAPs was analyzed. This study was to analyze a number of coupling parameters

Synthesis of peptides 6.2.1.1 Synthesis of linear peptides (automated synthesis)
The linear peptides were synthesized automatically by SPPS using a standard Fmoc/t-Bu protocol [149]. Syntheses were carried out on TentaGel S RAM resin (0.26 mmol/g) using presence of 10 equiv. DIEA in DMF. Double couplings for 20 min were allowed. N terminus deblocking was carried out twice with 20% piperidine in DMF for 5 min. Washes were made with DMF. Acetylation was carried out twice and for 10 min using Ac 2 O/DIEA/DMF (0.5/1/3.5 v/v/v). For acetylation of the PEGylated peptides the same methodology was used. The strategy of SPPS was also used to introduce PEG 2. For side chain PEGylation of K residues, Fmoc-Lys(Dde)-OH was used instead of Fmoc-Lys(Boc)-OH. The Dde side chain was removed by exposure to 2% hydrazine in DMF twice for 5 and 10 min. After washing, PEG 2 was coupled to the free amino group using HBTU activation.
Final cleavage from the resin and deprotection of the side chain functionalities were achieved by exposure to a mixture of 5% phenol, 2% TIPS, and 5% water in TFA for 2 h. After cleavage of the protecting groups, peptides were precipitated by addition of cold diethyl ether.

Synthesis of cyclic peptides (manuel synthesis)
The peptide acids, as precursors for the preparation of cyclic peptides, were synthesized manually by SPPS using the standard Fmoc/t-Bu protocol. Syntheses were carried out on ClTrt-Cl resin (1.03 mmol/g) using Fmoc-protected amino acids. Before the synthesis, the resin was placed in a reaction vessel equipped with a porous frit and swollen in DCM for

HPLC purification of crude peptides
The crude peptides were dissolved in a mixture of water/ACN (1/1 v/v) and purified by RP-HPLC on a Shimadzu LC-10A system (Japan) using a PolyEncap C-18 column (250 × 20 mm, 10.0 m, 300 Å) (Bischoff Analysentechnik, Germany) operating at 220 nm and 10 mL / min to produce final products that were more than 95% pure.

Characterization of peptides based upon t R -HPLC.
Measurement of the t R value of CAPs using RP-HPLC is a convenient method to compare peptide hydrophobicity. This method is based on the ability of the hydrophobic residues in a peptide to interact with the hydrophobic surface of the HPLC reversed stationary phase. Thus, the differences due to increases in hydrophobicity are reflected in the retention time t R .
Chromatographic characterization was performed on a Jasco analytical HPLC system (Jasco, Japan) with a diode array detector operating at 220 nm. Runs were carried out using a

Thioalkylation: N terminus and side chain immobilization of KLAL
An additional C residue was introduced at the N terminus or an -amino group of the K residues of the peptides. BrAcOH (Fluka, Germany) was introduced at the aminofunctionalized resin using its anhydride, which was prepared in situ by mixing

Oxime-forming ligation: N terminus and side chain immobilization of MK5E, MEL, BUF, and TP
AOA was introduced at the C terminus, N terminus or an -amino group of K residues.
For the C terminus immobilization of the peptides, we introduced an extra K residue at the first position of the C terminus of the sequences. Fmoc-Lys(Dde)-OH was used for this purpose. To overcome the problem of over-acetylation of nitrogen atoms in the ─NH─O─ moiety, which would lead to AOA(AOA)-peptides during the synthesis of an aminooxy-peptide for chemical ligation [174], the aminooxy-peptides were prepared by using 3 equiv. DIC, 3 equiv. HOBt as coupling reagents and 3 equiv. AOA in DCM for 1 h. The ketone-containing resin was prepared as followed: Three equiv. of pyruvic acid were added to the reaction vessel containing TentaGel S NH 2 , 3 equiv. DIC and 3 equiv. HOBt in an appropriate amount of DCM. After 2 h, the reaction was followed with the Kaiser test.
Finally, the resin was washed several times with DMF and DCM and subsequently dried under vacuum overnight for further use. Peptides modified with AOA (~ 15 mM in acetate buffer, 6 M GnHCl, pH 4.6) were added to the dry ketone-functionalized resin and allowed to react at room temperature overnight. After washing with DMF followed by DCM, the resins were dried under vacuum and stored at 4 °C (Fig. 27).

Fmoc-chromophore
The density of immobilized peptides was determined by measuring the absorption of the cleaved Fmoc-chromophore upon treatment of the peptide-loaded resins with 20% piperidine in DMF. The resin-bound peptides were exposed to a 5-fold excess of Fmoc-Cl, and DIEA in DCM (calculated on the basis resin capacity and the number of available amino groups in the sequence of the individual peptide). After 1 h, the resin was washed in DCM and subjected to 20% piperidine in DMF for 20 min. Afterwards an aliquot of the supernatant was added to a cuvette containing piperidine and the absorption was measured at 301 nm ( = 6000 M -1 .cm -1 ) using a Lambda 9 spectrophotometer (Perking-Elmer, Germany).

Bacterial culture preparation and determination of MIC and MBC
Bacteria were grown at 37 °C, shaking at 180 rpm, in LB to the mid-log phase as   Appropriate amounts of peptide-bearing resin were added to the culture tubes containing 1 mL of LB medium. An aliquot of the cell suspension was then added resulting in a cell concentration of ~ 1.6 × 10 6 cells/mL. Non-modified resins were used as a control.
Depending upon the cell line, the tested concentrations ranged between 0.5 and 80 mg/mL. concentration at which no colonies were detected (Fig. 40B). The experiments were performed in triplicates.

Hemolytic activity
The hemolytic activity of the peptides was determined using human RBCs (Charité-

MEL, BUF and TP and their AOA-modified analogs
Most CAPs act via permeabilization of the bacterial cell membrane. In order to assess the permeabilizing activity of the peptides in vitro, Lehrer et al. developed a protocol, which allows the study of the Gram-negative bacterial inner and outer membrane permeabilizing activity of CAPs [175]. To perform this assay, the E. coli (strain ML-35p) was used. The cells are characterized by a lack of lactose permease. The strain expresses periplasmic -lactamase and cytoplasmic -galactosidase. Due to the lack of lactose permease, NCF (-lactamase substrate) and ONPG (-Galactosidase substrate) are blocked from being transported across the outer and inner membrane of the bacterial cell respectively. However, as the result of peptide-induced outer membrane permeabilization, -lactamase can cleave NCF, which is detected by a color change from yellow to red. The inner membrane permeabilization allows ONPG to reach the cytoplasm, where -galactosidase metabolizes its substrate, which can be followed by a color change from colorless to yellow (Fig. 41). To make the POPC/LPS (12/1 [mol/mol]) lipid film, we employed a protocol called "dry method" [176]. According to this protocol, a certain amount of dry LPS was weighed and added into a 10 mL round-bottomed glass tube, prefilled with specific amount of dried POPC lipid film, such that the molar ratio of POPC/LPS reaches 12/1. Based on the structure of LPS shown in Fig. 16, we assumed that Rd-LPS and s-LPS have molecular masses of approximately 2.5 and 10 kDa., respectively. Because LPS is a heterogenous molecule and has the tendency to aggregate in varying sizes, the exact value for the molecular mass of LPS cannot be determined [177]. The assumed molecular masses for Rd-LPS and s-LPS used in these studies fit well with the masses reported for Rd-LPS from Salmonella Minnesota and s-LPS from E. coli (strain O8:K127) [177]. However; considering the overall heterogeneity, it has been reported that the molecular mass for s-LPS from E. coli K-235 varies between 10−80 kDa. [178]. The dried POPC/LPS lipid was then suspended in pyrogen-free water (Milli-Q, Element System, Millipore, France) and extensively vortexed. It was then heated up to 60 C followed by sonication in a bath sonicator for 10 min. The suspension was vortexed again and the cycle repeated twice. This turbid mixture was next lyophilized and made ready for further application. This protocol leads to the maximum amount of LPS incorporated into POPC phospholipid membrane [179]. To prevent any contamination of the POPC/LPS vesicles, no further attempt was done to separate non-incorporated LPS.

LUVs
Appropriate amounts of POPC alone, and its mixture with POPG at molar ratios of 3:1

Lipid bilayer permeabizing activities
In the dye release assay, aliquots of the LUV suspensions, loaded with calcein at selfquenching concentration were injected into cuvettes containing the dissolved peptides or

Isothermal titration calorimetry
ITC is a useful technique to study chemical reactions quantitatively and describe them thermodynamically. Each reaction is connected with an enthalpy change ( H°), which means that heat is either released into (exothermic) or taken in from (endothermic) the surroundings.
This universal property is independent of molecule size and so that all chemical and biochemical reactions can be studied [180]. This includes the formation of complexes between proteins, DNA, lipids or low molecular weight biomolecules, which are mostly based on non-covalent bonds. A particular example is the interaction between peptides (P) and lipid (L) bilayers mimicking bacterial membranes [181]. The complex formation (L.P) can be described by the simple equilibrium shown below: L + P L.P This equilibrium is defined by a binding constant (K) as given by: (1).
In Fig. 43, the operation of an isothermal titration calorimeter is schematically shown.
It consists of a reference and a sample cell. The temperature difference between two cells can be very precisely measured. The ITC syringe is filled with a solution of the interaction partner L (liposomes) and a solution of binding partner P (peptide) is present in the sample cell. The reference cell is filled with buffer. It serves as the reference for the temperature change, which takes place in the sample cell. Both cells have a higher temperature than the carefully thermostated environment (hold isothermal) and are supplied with controlled and measurable heating to keep the temperature constant. Each injection from the syringe leads to the formation of the L.P complex and this process is accompanied by heat absorption/release from/to the sample cell. The calorimeter responds to the endothermic or exothermic reaction with a corresponding increase or decrease of the electrical heating power to keep the temperature in the sample cell constant. These responses appear as differential heating power, as shown e.g. for heat release in the upper part of Fig. 43B. By integrating the area under each signal, the free-set quantity of heat versus the increasing ratio of L and P is obtained (lower part of Fig. 43B). By adjusting the theoretical curve to these data, K is obtained. . The smallest changes in temperature (∆T) between the two cells are detected and compensated by changing the heating power. In the right panel, the change in heating power for each injection into the sample cell is presented as a trace of negative peaks, which become smaller by increasing the number of injection because less substance is available for binding.
ITC can provide the comprehensive thermodynamic characterization of binding of antimicrobial peptides to different lipid vesicles [181]. The thermodynamic parameters in terms of ∆H°, ∆S°, and ∆G° are useful to understand the mechanism of action of CAPs and their selectivity towards different lipid membranes.

Theory and description of surface partitioning equilibrium model
The interaction between a peptide and a lipid membrane surface can be described by a two-step model. The interaction is initiated by electrostatic contributions resulting in surface accumulation. This is followed by hydrophobic interactions causing peptide insertion [181].
In order to describe the peptide-lipid binding, a surface partitioning equilibrium model was used to fit all ITC experimental data and draw the thermodynamic parameters [182,183]. where R b is: .
γ was taken as 0.6 for membrane-impermeant peptides interacting with SUVs [181]. R b was assumed to be given by: where z i is the valence of ith species, and c i is the concentration of the i th dissociated electrolyte in the solution. σ is obtained according to the theoretical calculation described by Seelig [184], which is corrected for the effect of counterion binding due to the association of Na + with the POPG headgroups as given by: where A L is 68 nm 2 and A P is assumed zero when the peptide adsorbs superficially. K Na+ is the binding constant of the sodium to the negatively charged lipid headgroup (0.6 M -1 ) with a concentration of c Na+ .
In an ITC experiment, the normalized heat produced or consumed on peptide-lipid interactions (Q) is given by (see ref. 182 for details): where ∆H° stands for the molar transfer enthalpy of the peptide from the aqueous into the bilayer phase, and Q dil , for the heat of dilution normalized with respect to the molar amount of lipid injected, ∆n L . ć P,b and c P,b are considered as the equilibrium concentrations of bound peptide before and after an injection of volume ∆V cell , respectively, and c P,s that in the syringe.
On the basis of this model, we fitted the ITC data to obtain K 0 , ∆H°, and z e for each peptide. The ∆G° was then taken as:

Instrument setup and measurement
High-sensitivity ITC was performed on a VP-ITC (GE Healthcare, Sweden). All experiments were run at 37 °C to study peptide binding to POPC doped with LA and LPSs above the gel-to-liquid-crystalline phase transition temperature. At lower temperatures, the binding process is endothermic [186]. To determine peptide-lipid interactions, a 40 µM peptide solution in the calorimeter cell was titrated with an SUV suspension ranging from 5 to 40 mM. All peptide solutions were degassed before ITC experiments. In a typical experiment, 3−10 µL aliquots of the SUV suspension were titrated into the peptide solution during each injection and the content of the sample cell was stirred continuously at 310 rpm. Because of a small loss of titrant during the mounting of the syringe and the equilibration stage preceding the actual titration, the first injection volume was usually set to 3−5 µL. The first injection peak was excluded during data analysis. Time spacings of 5 min were long enough to allow the ITC signal to return to the baseline value. The measured heat of binding decreased with consecutive lipid injections because less free peptide was available. Control experiments, injecting lipid vesicles into buffer without peptide, showed that the heats of dilution were small and constant.

ITC data analysis and curve fitting
The raw ITC data were collected based on the program supplied by the manufacturer.
Baseline correction, peak integration and its adjustment for data acquisition and analysis were conducted as described by the manufacturer. Nonlinear least-squares data fitting was performed in an Excel (Microsoft, U.S.A.) spreadsheet using the Solver add-in (Frontline Systems, U.S.A.) [187].

CD spectroscopy
CD spectroscopy is a technique by which conformational properties of peptides can be quickly measured in solution. CD occurs due to the difference in absorption of left or right circularly polarized light in the UV−VIS wavelength range (100−1000 nm) by optically active chromophores. In addition to inherent chiral absorbing systems, achiral chromophores also show a CD when they are disturbed by the dissymmetric field of an optically active group. In the absence of aromatic amino acid residues, only the peptide backbone contributes significantly in the far UV region. Thus, the CD spectra of peptides are determined by transition dipole moments of the amide groups, which are influenced by the chiral C α -atoms.
Absorption occurs mainly in the range between 180−240 nm: π−π * transitions (λ < 200 nm) and n−π * transitions (λ = ~ 218 nm) with less intensity. The superposition of contributions from aromatic residues in the 220 to 240 nm region influences the spectral characteristics. The induction of a secondary structure confers a structural asymmetry upon peptide sequences, which is reflected by characteristic CD patterns. There are three main classes of secondary structures: the α-helix, the β-sheet, and the random coil. The α-helix produces the most distinctive CD spectrum: a very strong positive band near 192 nm, which corresponds to π−π * ⊥ transitions and two negative maxima of approximately equal intensity near 222 nm (n−π * ) and 208 nm (π−π * || ). The β-sheet exhibits a single negative band near 217 nm, representing n−π * . The random coil exhibits a strong negative band at 197 nm (π−π * || ) and a small positive band in the 220 nm (n−π * ) region. In the spectra of complex proteins, the contributions of the individual structures superimpose. The spectra provide less information about the conformation of certain parts of the molecule, but rather provide an integrated picture of a peptide's structure. The value of the method is in the sensitivity of the CD signal with respect to structural changes induced by variations in the peptide concentration, pH, change in temperature, solvent polarity or ionic additives [188]. However, based upon the helicity of an ideal helix, or the correlation of CD spectra of proteins and their known crystal structure, also the content of helix or amount of other structures of peptides and proteins of interest can be calculated. However, these methods are only slightly suitable for small peptides because small sequences form poor helices and the data evaluation is based on the analysis of proteins with secondary and tertiary structures [189]. In general, the CD spectrum of a peptide represents the Θ mr versus λ. being the amount of helix [190].