Christian Krumm

Antimicrobial Poly(2‐methyloxazoline)s with Bioswitchable Activity through Satellite Group Modification

Biocides are widely used for preventing the spread of microbial infections and fouling of materials. Since their use can build up microbial resistance and cause unpredictable long-term environmental problems, new biocidal agents are required. In this study, we demonstrate a concept in which an antimicrobial polymer is deactivated by the cleavage of a single group. Following the satellite group approach, a biocidal quaternary ammonium group was linked through a poly(2-methyloxazoline) to an ester satellite group. The polymer with an octyl-3-propionoate satellite group shows very good antimicrobial activity against Gram-positive bacterial strains. The biocidal polymer was also found to have low hemotoxicity, resulting in a high HC50 /MIC value of 120 for S. aureus. Cleaving the ester satellite group resulted in a 30-fold decrease in antimicrobial activity, proving the concept valid. The satellite group could also be cleaved by lipase showing that the antimicrobial activity of the new biocidal polymers is indeed bioswitchable.

Impact of Functional Satellite Groups on the Antimicrobial Activity and Hemocompatibility of Telechelic Poly(2-methyloxazoline)s

Polyoxazolines with a biocidal quarternary ammonium end-group are potent biocides. Interestingly, the antimicrobial activity of the whole macromolecule is controlled by the nature of the group at the distal end. These nonreactive groups are usually introduced via the initiator. Here we present a study with a series of polymethyloxazolines with varying satellite groups introduced upon termination of the polymerization reaction. This allowed us to introduce a series of functional satellites, including hydroxy, primary amino, and double-bond-containing groups. The resulting telechelic polyoxazolines were explored regarding their antimicrobial activity and toxicity. It was found that the functional satellite groups greatly controlled the minimal inhibitory concentrations against the bacteria Staphylococcus aureus and Escherichia coli in a range of 10 to 2500 ppm. Surprisingly, the satellite groups also controlled the hemotoxicity but in a different way than the antimicrobial efficiency.

Conjugation of Ciprofloxacin with Poly(2-oxazoline)s and Polyethylene Glycol via End Groups

The antibiotic ciprofloxacin (CIP) was covalently attached to the chain end of poly(2-methyloxazoline) (PMOx), poly(2-ethyloxazoline) (PEtOx), and polyethylene glycol (PEG), and the antimicrobial activity of these conjugates was tested for Staphylococcus aureus, Streptococcus mutans, Escherichia coli, Pseudomonas aeruginosa, and Kleisella pneumoniae. Chemical structures of the conjugates were proven by (1)H NMR and electron spray ionization mass spectrometry. The direct coupling of PMOx and CIP resulted in low antimicrobial activity. The coupling via a spacer afforded molecular weight dependent activity with a molar minimal inhibitory concentration that is even higher than that of the pristine CIP. The antimicrobial activity of the conjugates increases in the order of PMOx < PEtOx < PEG. Conjugation of CIP and a quaternary ammonium compound via PMOx did not result in higher activity, indicating no satellite group or synergistic effect of the different biocidal end groups.

Well-defined amphiphilic poly(2-oxazoline) ABA-triblock copolymers and their aggregation behavior in aqueous solution.

Self-organization of block copolymers in solution is a way to obtain advanced functional superstructures. The synthesis of well-defined polymethyloxazoline-block-polyphenyloxazoline-block-polymethyloxazoline triblock copolymers is described and proven by (1) H NMR spectroscopy, SEC, and ESI-MS. The surprisingly water- soluble block copolymers do self-organize in aqueous solutions uniquely forming three coexisting well-defined structures: unimolecular micelles, micellar aggregates, and very form-stable polymersomes. This is the first example of a polymersome forming ABA-triblock copolymer with a glassy middle block. The spherical vesicles are analysed by scanning electron microscopy and transmission electron microscopy. It could be shown that these vesicles are indeed hollow spheres.

Investigations on the activity of poly(2-oxazoline) enzyme conjugates dissolved in organic solvents.

The use of enzymes in organic solvents offers a great opportunity for the highly selective synthesis of complex organic compounds. In this study we investigate the POXylation of several enzymes with different polyoxazolines ranging from the hydrophilic poly(2-methyl-oxazoline) (PMOx) to the hydrophobic poly(2-heptyl-oxazoline) (PHeptOx). As reported previously on the examples of model enzymes POXylation mediated by pyromellitic acid dianhydride results in highly modified, organosoluble protein conjugates. This procedure is here extended to a larger number of proteins and optimized for the different polyoxazolines. The resulting polymer-enzyme conjugates (PEC) became soluble in different organic solvents ranging from hydrophilic DMF to even toluene. These conjugates were characterized regarding their solubility and especially their activity in organic solvents and in some cases the PECs showed significantly (up to 153,000 fold) higher activities than the respective native enzymes.

Poly(2-oxazoline)–Antibiotic Conjugates with Penicillins

The conjugation of antibiotics with polymers is rarely done, but it might be a promising alternative to low-molecular-weight derivatization. The two penicillins penicillin G (PenG) and penicillin V (PenV) were attached to the end groups of different water-soluble poly(2-oxazoline)s (POx) via their carboxylic acid function. This ester group was shown to be more stable against hydrolysis than the β-lactam ring of the penicillins. The conjugates are still antimicrobially active and up to 20 times more stable against penicillinase catalyzed hydrolysis. The antibiotic activity of the conjugates against Staphylococcus aureus in the presence of penicillinase is up to 350 times higher compared with the free antibiotics. Conjugates with a second antimicrobial function, a dodecyltrimethylammonium group (DDA-X), at the starting end of the PenG and PenV POx conjugates are more antimicrobially active than the conjugates without DDA-X and show high activity in the presence of penicillinase. For example, the conjugates DDA-X-PEtOx-PenG and DDA-X-PEtOx-PenV are 200 to 350 times more active against S. aureus in the presence of penicillinase and almost as effective as the penicillinase stable cloxacollin (Clox) under these conditions. These conjugates show even greater activity compared to cloxacollin without this enzyme present. Further, both conjugates kill Escherichia coli more effectively than PenG and Clox.

Chapter 15:Antimicrobial Polymers and Surfaces – Natural Mimics or Surpassing Nature?

Fighting pathogenic microbes is one of the great current challenges of mankind. Nature has developed several techniques to counteract microbial attacks. Science has also yielded several technologies, including antimicrobial polymers as biocides and polymers used for microbe killing and repelling surfaces. Recent scientific antimicrobial approaches are mimicking natural concepts. In this chapter, current developments in antimicrobial and antifouling polymers and surfaces are reviewed and discussed regarding the question whether they mimic nature or surpass it.

Telechelic, Antimicrobial Hydrophilic Polycations with Two Modes of Action

Telechelic antimicrobial poly(2-oxazoline)s with quaternary ammonium (quat) end groups are shown to be potent antimicrobial polymers against Gram-positive bacterial strains. In this study, the activity against the Gram-negative bacterium Escherichia coli is additionally implemented by hydrolyzing the poly(2-methyl-2-oxazoline) with two quart end groups to poly(ethylene imine) (PEI). The resulting telechelic polycations are active against Staphylococcus aureus and E. coli. The contribution of the PEI backbone is determined by measuring the antimicrobial activity in the presence of calcium ions. The influence of PEI on the overall activity strongly depends on the molecular weight and increases with higher mass. The PEI dominates the activity against E. coli at lower masses than against S. aureus. The quart end groups require an alkyl substituent of dodecyl or longer to dominate the antimicrobial activity. Additionally, PEI and quart end groups act synergistically.

Entropically driven Polymeric Enzyme Inhibitors by End‐Group directed Conjugation

A new generic concept for polymeric enzyme inhibitors is presented using the example of poly(2-methyl-2-oxazoline) (PMOx) terminated with an iminodiacetate (IDA) function. These polymers are shown to be non-competitive inhibitors for horseradish peroxidase (HRP). Mechanistic investigations revealed that the polymer is directed to the protein by its end group and collapses at the surface in an entropy-driven process as shown by isothermal titration calorimetry. The dissociation constant of the complex was determined as the inhibition constant Ki using HRP kinetic activity measurements. Additional experiments suggest that the polymer does not form a diffusion layer around the protein, but might inhibit by inducing minor conformational changes in the protein. This kind of inhibitor offers new avenues towards designing bioactive compounds.