# Patent

## Title
Antimicrobial polymers

## Abstract

The invention generally relates to novel polymers (SMAMPs) and their syntheses and use. The polymers exhibit promising properties of AMPs. In particularly, for example, a ring-opening metathesis polymerization (ROMP) platform was developed that allows syntheses of SMAMPs that employ a minimum number of norbornene-based building blocks and/or enable easy and independent variation of hydrophobic and hydrophilic groups in the monomer units and/or along the polymeric backbone to finetune and select desirable properties of the polymers.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of priority from U.S. Provisional Application Ser. No. 61/185,035, filed Jun. 8, 2009, the entire content of which is incorporated herein by reference in its entirety.


FIELD OF THE INVENTION
The invention relates to novel antimicrobial polymers. More particularly, the invention relates to antimicrobial polymers that are synthetic mimics of antimicrobial peptides, and related preparation, methods and uses thereof.
BACKGROUND OF THE INVENTION
Natural antimicrobial peptides (AMPs) are among the first line of defense when organisms are under attack by bacterial pathogens. These host-defense peptides have shown broad-spectrum antimicrobial activity. (Brodgen, Nature Rev. Microbiol. 2005, 3, 238-250.) Because their production in the organism is much faster than that of specific antibodies, the AMPs are a vital component of innate immunity. AMPs are found in many species, including humans, animals, plants and invertebrates. Different from common antibiotics that target specific cell structures, AMPs use non-receptor interactions, including in many cases direct action against the bacteria's membranes. (Yang, et al, J. Am. Chem. Soc. 2007, 129, 12141.) The cells of the host organism are less affected. Thus, AMPs can selectively attack bacteria within a host organism. Bacteria could become immune to AMPs only when they change their entire membrane chemistry or other targets—thus resistance to AMPs is retarded as compared to other antibiotics. (Zasloff, Nature 2002, 415, 389-395.) Due to this promising feature, there has been increasing interest in synthetic mimics of antimicrobial peptides (SMAMPs). These include the SMAMPs made of α- and β-amino acids, peptoids, aromatic oligomers, and synthetic polymers. (Zasloff, Proc. Natl. Acad. Sci. USA 1987, 84, 5449; Castro, et al., Curr. Protein Peptide Sci. 2006, 7, 473; Chen, et al., J. Biol. Chem. 2005, 280, 12316; Won, et al., J. of Biol. Chem. 2004, 279, 14784; Hamuro, et al., J. Am. Chem. Soc. 1999, 121, 12200; Porter, et al., Nature 2000, 404, 565; Liu, et al., J. Am. Chem. Soc. 2001, 123, 7553; Epand, et al., Biochemistry 2004, 43, 9527; Patch, et al., J. Am. Chem. Soc. 2003, 125, 12092; Brouwer, et al., Peptides 2006, 27, 2585; Haynie, et al., Antimicrob. Agents Chemother. 1995, 39, 301; Tew, et al., Proc. Natl. Acad. Sci. USA 2002, 99, 5110; Liu, et al., Angew. Chem. Internat. Ed. 2004, 43, 1158; Tang, et al., Chem. Commun. 2005, 12, 1537; Kuroda, et al., J. Am. Chem. Soc. 2005, 127, 4128; Ilker, et al., J. Am. Chem. Soc. 2004, 126, 15870; Arnt, et al., J. Am. Chem. Soc. 2002, 124, 7664; Arnt, et al., Langmuir 2003, 19, 2404; Arnt, et al., J. Polymer Sci. A, Polymer Chem. 2004, 42, 3860; Mowery, et al. J. Am. Chem. Soc. 2007, 129, 15474.)
One common feature of most AMPs is their positive charge and facial amphiphilicity. Regardless of the secondary structures, these peptides generally display one hydrophobic and one hydrophilic face along their backbone. (Brodgen, Nature Rev. Microbiol. 2005, 3, 238-250; Boman, Immunol. Rev. 2000, 173, 5; Hancock, et al., Trends Biotech. 1998, 16, 82.) Due to positive charges in the hydrophilic part, AMPs bind preferentially to the anionic outer membranes of bacterial pathogens or other anionic targets including proteins and DNA. (Zasloff, Nature 2002, 415, 389-395; Yeaman, et al., Pharmacol. Rev. 2003, 55, 27.) In many cases, their facial amphiphilicity allows them to insert into the bacterial membrane and to locally change the membrane's lipid organization in such a way that trans-membrane pores are formed, although other mechanisms of action are also known. This interaction may lead to a breakdown of the membrane potential, the leaking of the cytoplasm and eventually the death of the pathogen cell. (Yount, et al., Biopolymers (Peptide Sci.) 2006, 84, 435.)
There is an unmet need for novel SMAMPs that possess desirable and tunable antomicrobial properties while at the same time are easy to prepare.
SUMMARY OF THE INVENTION
The invention is based in part on certain novel polymers (SMAMPs) obtained through advanced polymer design and synthetics methods, which polymers exhibit promising properties of AMPs. In particularly, for example, a ring-opening metathesis polymerization (ROMP) platform was developed that allow syntheses of SMAMPs. The platform employs a minimum number of norbornene-based building blocks and enables easy and independent variation of hydrophobic and hydrophilic groups in the monomer units and/or along the polymeric backbone, thereby allowing one to finetune and select desirable properties of the polymers, e.g., potency and selectivity.
In one aspect, the invention generally relates to a monomer unit having the structure of:




wherein one of R1 and R2 comprises a hydrophobic group and the other one of R1 and R2 comprises a hydrophilic group; and X is C—R3, O or S, wherein R3 is a hydrogen or a C1-C12 alkyl or alkoxy group (e.g., a C1-C6 alkyl or alkoxy group).

The hydrophobic group may include a substituted or unsubstituted alkry group, e.g., a substituted or unsubstituted C1-C10 alkyl group (e.g., a C1-C6 alkyl group). The hydrophilic group may include a group selected from ammonium ions, sulfonium ions, guanidinium ions, phosphonium ions, and mono- and di-alkyl substituted amino groups, for example.
In certain embodiments, X is O; R1 is a linear or branched C1-C12 alkyl group (e.g., a C1-C6 alkyl group); and R2 is




or a guanidinium ion





wherein p is an integer from 1-10 (i.e, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

In certain embodiments, X is C—R3; R1 is a linear or branched C1-C12 alkyl group (e.g., a C1-C6 alkyl group); and R2 is




or a guanidinium ion





wherein p is an integer from 1-10. In some embodiments, R3 is hydrogen.

In another aspect, the invention generally relates to a polymer comprising a monomer unit having the structure of:




wherein one of R1 and R2 comprises a hydrophobic group and the other one of R1 and R2 comprises a hydrophilic group; and X is C—R3, O or S, wherein R3 is a hydrogen or a C1-C12 alkyl or alkoxy group.

The hydrophobic group may include a substituted or unsubstituted alkry group, e.g., a substituted or unsubstituted C1-C10 alkyl group (e.g., a C1-C6 alkyl group). The hydrophilic group may include a group selected from ammonium ions, sulfonium ions, guanidinium ions, phosphonium ions, and mono- and di-alkyl substituted amino groups, for example.
In certain embodiments, X is O; R1 is a linear or branched C1-C12 alkyl group (e.g., a C1-C6 alkyl group); and R2 is




or a guanidinium ion





wherein p is an integer from 1-10 (i.e, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

In certain embodiments, X is C—R3; R1 is a linear or branched C1-C12 alkyl group (e.g., a C1-C6 alkyl group); and R2 is




or a guanidinium ion





wherein p is an integer from 1-10. In some embodiments, R3 is hydrogen.

For example, n is an integer from about 1 to about 1,000, for example, from about 1 to about 100; from about 1 to about 20; from about 1 to about 10; from about 1 to about 5; from about 3 to about 50; from about 3 to about 20; from about 3 to about 10; from about 3 to about 5.
In some applications, the polymer may preferably be a co-polymer. In certain embodiments, the co-polymer may include structural monomer units of:




wherein each of X′ and X′′ is C—R3, O or S, wherein R3 is a hydrogen or a C1-C12 alkyl or alkoxy group (e.g., a C1-C6 alkyl or alkoxy group); each of R1′ and R1′′ is a hydrophobic group and each of R2′ and R2′′ is a hydrophilic group, provided that R1′ and R1′′ are not the same, R2′ and R2′′ are not the same, or X′ and X′′ are not the same; and each of m and n is an integer from about 1 to about 1,000.

Each of m and n is an integer from about 1 to about 1,000, for example, from about 1 to about 100; from about 1 to about 20; from about 1 to about 10; from about 1 to about 5; from about 3 to about 50; from about 3 to about 20; from about 3 to about 10; from about 3 to about 5.
In some embodiments, each of R1′ and R1′′ is independently a linear or branched C1-C12 alkyl group (e.g., a C1-C6 alkyl group); and each of R2′ and R2′′ is independently




or a guanidinium ion





wherein p is an integer from 1-10 (i.e, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

In some embodiments, each of X′ and X′′ is O. In some other embodiments, each of X′ and X′′ is independently C—R3. In some other embodiments, one of X′ and X′′ is O and the other is C—R3.
In certain embodiments, one or more (e.g., all) of —C(═O)O— groups in (I), (II), (III) or (IV) occupy the exo-stereo position.
In another aspect, the invention generally relates to a polymer prepared by ring-opening metathesis polymerization, the polymer comprising hydrophobic and hydrophilic groups attached to the polymeric backbone such that, within a monomeric structural unit, the hydrophobic and hydrophilic groups are attached to the polymeric backbond at adjacent atoms. For example, in the polymer the hydrophobic and hydrophilic groups are attached to the polymeric backbond via ester linkages. The polymers of the invention may have a molecular weight of Mn between about 1,000 and about 100,000, between 1,000 and 50,000, between 1,000 and 10,000, for example.
The invention also relates to a product comprising a polymer of the invention and methods for preparing polymers of the invention, for example, by ring-opening metathesis polymerization.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates an embodiment of a ‘construction kit’ approach to obtain facially amphiphilic monomers and polymers.
FIG. 2 shows certain exemplary biological data.
FIG. 3 shows certain characterization data of exemplary propyl oligomers.
FIG. 4 shows certain exemplary biological data.
FIG. 5 shows certain exemplary biological data.
FIG. 6 shows certain characterization of propyl oligomers.
FIG. 7 shows exemplary highly active polymer.
FIG. 8 shows exemplary moderately active polymer and inactive polymer.
FIG. 9 shows exemplary HC50 curve of certain polymer.
FIG. 10 shows an illustrative representation of certain cell membrane and cell wall morphology.
FIG. 11 shows certain exemplary structure and antimicrobial properties for different molecular weights of the SMAMP.
FIG. 12 shows certain exemplary dye-leakage experiments on model membranes.
FIG. 13 schemetically illustrates certain rationalization of the results of the dye-leakage experiments in the presence of peptidoglycan.
FIG. 14: shows certain exemplary dye-leakage experiments on model membranes.
FIG. 15: shows certain exemplary GPC data for the SMAMP homopolymer series.
FIG. 16: shows certain exemplary dye-leakage curves.
FIG. 17: shows certain exemplary structural comparison.
FIG. 18: shows certain exemplary cartoon representation of local charge surplus.
FIG. 19: shows certain exemplary structure of model polymers.
FIG. 20. shows certain exemplary antimicrobial and hemolytic properties of certain copolymers.
FIG. 21 shows certain exemplary illustration of a dye-leakage experiment.
FIG. 22 shows certain exemplary dye-leakage data.
FIG. 23 shows certain exemplary representative cartoon and polymer structures.
FIG. 24 shows certain exemplary methods to endow polynorbornenes.
FIG. 25 shows certain exemplary 50/50 random copolymers.
FIG. 26 shows certain exemplary MICs of selected polymers.
FIG. 27 shows certain exemplary cartoon regarding polymer interactions.
FIG. 28 shows certain exemplary graph of polymer-induce dye leakage.
FIG. 29 shows certain exemplary fluorescence microscopy of stained E. coli in the absence and presence of polymer.
FIG. 30 shows certain exemplary synthetic scheme.
FIG. 31 shows certain exemplary molecular evolution of antibacterials.
FIG. 32 shows certain exemplary MIC and HC curves.
FIG. 33 shows certain exemplary monomer and polymer synthesis.
FIG. 34 shows certain exemplary library of ROMP-based SMAMP polymers.
FIG. 35 shows certain exemplary biological data for three exemplary series of SMAMP homopolymers.
FIG. 36 shows certain exemplary biological data for exemplary series of SMAMP copolymers.
FIG. 37 shows certain exemplary biological data for exemplary series of SMAMP copolymers, and illustration of SMAMP-membrane interactions.
FIG. 38 shows certain exemplary biological data for three exemplary series of SMAMP copolymers.
FIG. 39 shows certain exemplary biological data for exemplary imid-based SMAMP polymers.
FIG. 40 shows certain exemplary biological data for exemplary ester-based SMAMP copolymers.
FIG. 41 shows certain exemplary MIC90 data.
FIG. 42 shows certain exemplary molecular weight dependence of biological properties for various SMAMPs.
FIG. 43 shows certain exemplary dye-leakage percentage versus SMAMP concentration.
FIG. 44 shows certain exemplary fluorescence microscopy image.
FIG. 45 shows certain exemplary dynamic light scattering studies.
FIG. 46 shows certain exemplary dye-leakage curves and fluorescence microscopy images.
FIG. 47 shows certain exemplary schematic representation of modifications of polymers.
FIG. 48 shows certain exemplary NMR data.
FIG. 49 shows certain exemplary NMR data.
FIG. 50 shows certain exemplary GPC traces.
FIG. 51 shows certain exemplary MIC and HC50 data.
FIG. 52 shows certain exemplary MIC and HC50 data.
FIG. 53 shows certain exemplary MIC and HC50 data.
FIG. 54 shows certain exemplary GPC traces.
FIG. 55 shows certain exemplary chemical structures of norbornene monomers with multiple amine functionalities.
FIG. 56 shows certain exemplary antimicrobial polynorbornene derivatives.
FIG. 57 shows certain exemplary hemolytic activity (HC50).
FIG. 58 shows certain exemplary percentage of calcein dye leakage data.
FIG. 59 shows certain exemplary percentage of calcein dye leakage data.
FIG. 60 shows certain exemplary GPC analysis.
FIG. 61 shows certain exemplary NMR data.
FIG. 62 shows certain exemplary NMR data.
FIG. 63 shows certain exemplary NMR data.
FIG. 64 shows certain exemplary NMR data.
FIG. 65 shows certain exemplary NMR data.
FIG. 66 shows certain exemplary NMR data.
FIG. 67 shows certain exemplary NMR data.
FIG. 68 shows certain exemplary NMR data.
FIG. 69 shows certain exemplary NMR data.
FIG. 70 shows certain exemplary NMR data.
FIG. 71 shows certain exemplary NMR data.
FIG. 72 shows certain exemplary NMR data.
FIG. 73 shows certain exemplary NMR data.
FIG. 74 shows certain exemplary NMR data.
FIG. 75 shows certain exemplary NMR data.
FIG. 76 shows certain exemplary NMR data.



DEFINITIONS
Definitions of specific functional groups and chemical terms are described in more detail below. General principles of organic chemistry, as well as specific functional moieties and reactivity, are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999.
Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.
Given the benefit of this disclosure, one of ordinary skill in the art will appreciate that synthetic methods, as described herein, utilize a variety of protecting groups. By the term “protecting group”, as used herein, it is meant that a particular functional moiety, e.g., O, S, or N, is temporarily blocked so that a reaction can be carried out selectively at another reactive site in a multifunctional compound. In preferred embodiments, a protecting group reacts selectively in good yield to give a protected substrate that is stable to the projected reactions: the protecting group should be selectively removable in good yield by readily available, preferably non-toxic reagents that do not attack the other functional groups; the protecting group forms an easily separable derivative (more preferably without the generation of new stereogenic centers); and the protecting group has a minimum of additional functionality to avoid further sites of reaction. Oxygen, sulfur, nitrogen, and carbon protecting groups may be utilized. Examples of a variety of protecting groups can be found in Protective Groups in Organic Synthesis, Third Ed. Greene, T. W. and Wuts, P. G., Eds., John Wiley & Sons, New York: 1999.
It will be appreciated that the compounds, as described herein, may be substituted with any number of substituents or functional moieties.
The term “alkyl”, as used herein, refers to a saturated linear or branched (including cyclic) hydrocarbon free radical, unsubstituted (i.e., with corresponding number of carbon and hydrogen atoms), or optionally substituted with substituents known to those skilled in the art. For example, alkyl groups include (C1-C6) alkyl (or C1-C6 alkyl), which refers to a saturated linear or branched free radical consisting essentially of 1 to 6 carbon atoms (i.e., 1, 2, 3, 4, 5, or 6 carbon atoms) and a corresponding number of hydrogen atoms. Exemplary (C1-C6) alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, etc. Of course, other (C1-C6) alkyl groups will be readily apparent to those of skill in the art given the benefit of the present disclosure.
The terms “hydrophobic group” or “hydrophobic moiety”, as used herein, refer to a group having a property such that an affinity of the group for water is low (e.g., low polarity). Non-limiting examples of hydrophobic groups or moieties include substituted and unsubstituted alkyl groups (e.g., lower alkyl groups having 6, 7, 8, 9, 10, 11, 12, or 13-30 or more carbon atoms, linear, branched or cyclic), alkenyl groups, alkynyl groups, aryl groups, saturated or unsaturated cyclic hydrocarbons, heteroaryl, heteroarylalkyl, heterocyclic, and corresponding substituted groups. A hydrophobic group may contain some hydrophilic groups or substituents insofar as the hydrophobic character of the group is not outweighed. In further variations, a hydrophobic group may include substituted silicon atoms, and may include fluorine atoms. The hydrophobic moieties may be linear, branched, or cyclic.
The terms “hydrophilic group” or “hydrophilic moiety” as used herein, refer to a group having a property such that an affinity of the group for water is high (e.g., high polarity). Non-limiting examples of hydrophilic groups or moieties include hydroxyl, methoxy, phenyl, carboxylic acids and ions and salts thereof, methyl, ethyl, and vinyl esters of carboxylic acids, amides, amino, cyano, isocyano, nitrile, ammonium ions or salts, sulfonium ions or salts, phosphonium ions or salts, mono- and di-alkyl substituted amino groups, polypropyleneglycols, polyethylene glycols, glycosyl groups, sugars, epoxy groups, acrylates, sulfonamides, nitro, —OP(O)(OCH2CH2N+RRR)O−, guanidinium, aminate, acrylamide, pyridinium, piperidine, and combinations thereof, wherein each R is independently selected from H or alkyl. Further examples include polymethylene chains substituted with alcohol, carboxylate, acrylate, or methacrylate. Hydrophilic moieties may also include alkyl chains having internal amino or substituted amino groups, for example, internal —NH—, —NC(O)R—, or —NC(O)CH═CH2— groups, wherein R is H or alkyl. Hydrophilic moieties may also include polycaprolactones, polycaprolactone diols, poly(acetic acid)s, poly(vinyl acetates)s, poly(2-vinyl pyridine)s, cellulose esters, cellulose hydroxylethers, poly(L-lysine hydrobromide)s, poly(itaconic acid)s, poly(maleic acid)s, poly(styrenesulfonic acid)s, poly(aniline)s, or poly(vinyl phosphonic acid)s. A hydrophilic group may contain some hydrophobic groups or substituents insofar as the hydrophilic character of the group is not outweighed.
As used herein, MIC90=minimum inhibitory concentration that will reduce the growth of a certain bacteria by 90% as compared to an untreated control. Low MIC90 values indicate a high antibacterial activity. HC50=hemolytic concentration at which 50% of human red blood cells are lysed. Samples that yield high HC50 values have a low toxicity. Selectivity=HC50/MIC90. High selectivity values imply a good tolerance by the host organism, combined with a high toxicity towards the target organism.
DETAILED DESCRIPTION OF THE INVENTION
The invention generally relates to certain novel polymers (SMAMPs) developed through advanced polymer design and synthetics methods. In particularly, for example, a ring-opening metathesis polymerization (ROMP) platform was developed that allow syntheses of SMAMPs that employ a minimum number of norbornene-based building blocks and enable easy and independent variation of hydrophobic and hydrophilic groups in the monomer units and/or along the polymeric backbone to finetune and select desirable properties (e.g., potency and selectivity) of the polymers. The polymers exhibit promising properties of AMPs.
In one aspect, the invention generally relates to a monomer unit having the structure of:




wherein one of R1 and R2 comprises a hydrophobic group and the other one of R1 and R2 comprises a hydrophilic group; and X is C—R3, O or S, wherein R3 is a hydrogen or a C1-C12 alkyl or alkoxy group (e.g., a C1-C6 alkyl or alkoxy group).

The hydrophobic group may include a substituted or unsubstituted alkry group, e.g., a substituted or unsubstituted C1-C10 alkyl group (e.g., a C1-C6 alkyl group). The hydrophilic group may include a cationic group, which may be selected from ammonium ions, sulfonium ions, guanidinium ions, phosphonium ions, and mono- and di-alkyl substituted amino groups, for example.
In certain embodiments, X is O; R1 is a linear or branched C1-C12 alkyl group (e.g., a C1-C6 alkyl group); and R2 is




or a guanidinium ion





wherein p is an integer from 1-10 (i.e, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

In certain embodiments, X is C—R3; R1 is a linear or branched C1-C12 alkyl group (e.g., a C1-C6 alkyl group); and R2 is




or a guanidinium ion, wherein p is an integer from 1-10. In some embodiments, R3 is hydrogen.

In another aspect, the invention generally relates to a polymer comprising a monomer unit having the structure of:




wherein one of R1 and R2 comprises a hydrophobic group and the other one of R1 and R2 comprises a hydrophilic group; and X is C—R3, O or S, wherein R3 is a hydrogen or a C1-C12 alkyl or alkoxy group.

The hydrophobic group may include a substituted or unsubstituted alkry group, e.g., a substituted or unsubstituted C1-C10 alkyl group (e.g., a C1-C6 alkyl group). The hydrophilic group may include a cationic group, which may be selected from ammonium ions, sulfonium ions, guanidinium ions, phosphonium ions, and mono- and di-alkyl substituted amino groups, for example.
In certain embodiments, X is O; R1 is a linear or branched C1-C12 alkyl group (e.g., a C1-C6 alkyl group); and R2 is




or a guanidinium ion, wherein p is an integer from 1-10 (i.e, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

In certain embodiments, X is C—R3; R1 is a linear or branched C1-C12 alkyl group (e.g., a C1-C6 alkyl group): and R2 is





or a guanidinium ion, wherein p is an integer from 1-10. In some embodiments, R3 is hydrogen.

For example, n is an integer from about 1 to about 1,000, for example, from about 1 to about 100; from about 1 to about 20; from about 1 to about 10; from about 1 to about 5; from about 3 to about 50; from about 3 to about 20; from about 3 to about 10; from about 3 to about 5.
In some applications, the polymer may preferably be a co-polymer. In certain embodiments, the co-polymer may include structural monomer units of:




wherein each of X′ and X′′ is C—R3, O or S, wherein R3 is a hydrogen or a C1-C12 alkyl or alkoxy group (e.g., a C1-C6 alkyl or alkoxy group); each of R1′ and R1′′ is a hydrophobic group and each of R2′ and R2′′ is a hydrophilic group, provided that R1′ and R1′′ are not the same, R2′ and R2′′ are not the same, or X′ and X′′ are not the same; and each of m and n is an integer from about 1 to about 1,000.

Each of m and n is an integer from about 1 to about 1,000, for example, from about 1 to about 100; from about 1 to about 20; from about 1 to about 10; from about 1 to about 5; from about 3 to about 50; from about 3 to about 20; from about 3 to about 10; from about 3 to about 5.
In some embodiments, each of R1′ and R1′′ is independently a linear or branched C1-C12 alkyl group (e.g., a C1-C6 alkyl group); and each of R2′ and R2′′ is independently




or a guanidinium ion, wherein p is an integer from 1-10 (i.e, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

In some embodiments, each of X′ and X′′ is O. In some other embodiments, each of X′ and X′′ is independently C—R3. In some other embodiments, one of X′ and X′′ is O and the other is C—R3.
In certain embodiments, one or more (e.g., all) of —C(═O)O— groups in (I), (II), (III) or (IV) occupy the exo-stereo position.
In another aspect, the invention generally relates to a polymer prepared by ring-opening metathesis polymerization, the polymer comprising hydrophobic and hydrophilic groups attached to the polymeric backbone such that, within a monomeric structural unit, the hydrophobic and hydrophilic groups are attached to the polymeric backbond at adjacent atoms. For example, in the polymer the hydrophobic and hydrophilic groups are attached to the polymeric backbond via ester linkages.
The polymers of the invention may have a molecular weight of Mn between about 1,000 and about 100,000, between 1,000 and 50,000, between 1,000 and 10,000, for example
The invention also relates to a product comprising a polymer of the invention and methods for preparing polymers of the invention by ring-opening metathesis polymerization.
a. Highly Selective Antimicrobial Polymers
Access to synthetic AMP polymers may open up new applications, for example in the materials area, where bacterial infections from medical plastics are a current critical problem in our hospitals. Synthetic polymers can be obtained easily and in large quantities while still presenting facial amphiphilicity and positive charge, the important features of AMPs. Although there have been several recent reports of polymeric SMAMPs, their overall activities and selectivities remain far from optimal. Examples include: DeGrado and coworkers reported SMAMPs based on poly(ammonium methylmethacrylate) salts copolymerized with poly(butylmethacrylate) to tune the amphiphilicity; Klajnert et al. produced dendritic SMAMPs; Liu et al. synthesized SMAMPs from poly(maleic acid) linked to peptide tetramers; Makovitzki et al. recently made SMAMPs based on lipopeptides; and Gellman and coworkers presented a poly(amide) based polymer with good activities (12.5 μg/mL against E. coli and 3.1 μg/mL against S. aureus) and selectivities up to 32 for bacterial over mammalian cells. (Kuroda, et al., Polymer Prepr. 2004, 45, 610; Klajnert, et al., Int. J. Pharm. 2006, 309, 208; Liu, et al., J. Med. Chem. 2006, 49, 3436; Makovitzki, et al., Proc. Natl. Acad. Sci. USA 2006, 103, 15997; Mowery, et al. J. Am. Chem. Soc. 2007, 129, 15474.) Tew and coworkers synthesized facially amphiphilic antibacterial polymers based on arylamides, urea, and poly(phenylene ethynylene). (Tew, et al., Proc. Natl. Acad. Sci. USA 2002, 99, 5110; Tang, et al., Chem. Commun. 2005, 12, 1537; Arnt, et al., J. Am. Chem. Soc. 2002, 124, 7664; Arnt, et al., Langmuir 2003, 19, 2404.)
SMAMPs based on poly(norbornene) derivatives were previously described by Tew and Coughlin: they reported polymers with facially amphiphilic repeat units that had tunable antimicrobial activity depending on a defined ratio of hydrophobic and hydrophilic moieties in the repeat unit. Their most selective polymer had a hundred times higher activity towards bacteria than against human red blood cells. (Ilker, et al., J. Am. Chem. Soc. 2004, 126, 15870.) They also very recently reported poly(norbornenes) with quaternary pyridinium groups (selectivities up to 20 against E. coli). (Eren, et al., Macromol. Chem. Phys. 2008, 209, 516-524.)
Previously reported poly(norbornene) based SMAMPs required extensive synthetic effort to tune the amphiphilicity of the repeat units, or did not allow copolymer synthesis. Although previous work has shown that antimicrobial activity can also be achieved with random copolymers of hydrophilic and hydrophobic monomers, having facially amphiphilic monomers, i.e. monomers with a hydrophilic cationic and a hydrophobic part on the same polymerizable unit, allows for more precise tuning of the antibacterial activity. (Kuroda, et al., J. Am. Chem. Soc. 2005, 127, 4128; Mowery, et al. J. Am. Chem. Soc. 2007, 129, 15474.)
Disclosed herein is a novel and unique approach based on a ring-opening metathesis polymerization (ROMP) platform that (i) uses a minimum number of building blocks and (ii) allows the easy and independent variation of the hydrophobic and hydrophilic residues on the monomer. The important components of our molecular construction kit are highlighted in FIG. 1. The hydrophilic and hydrophobic components are attached to the polymerizable oxanorbornene group and can be varied independently.
The molecular construction kit approach described here allows the synthesis of a whole library of synthetic facially amphiphilic antibacterial homopolymers and copolymers with tunable activity and selectivity. The hydrophilic and hydrophobic components can be varied independently, allowing future synthesis of an even more extensive library of antibacterial polymers. Homopolymers of the 3k series thus obtained show a remarkable antibacterial activity against E. coli and S. aureus, which could be tuned over two orders of magnitude by variation of the hydrophobic residue R. Some of the homopolymers of the 10k series showed a double selectivity of E. coli over mammalian cells as well as over S. aureus. It was further demonstrated that molecular weight can be used as a parameter to tune the antibacterial activity. The optimum activity against S. aureus was found to be at 750 g/mol to 1,100 g/mol and against E. coli at 3,000 g/mol and 10,000 g/mol. Selectivities of S. aureus over red blood cells as high as >533 were obtained by copolymerizing an inactive/non-hemolytic monomer with an active/hemolytic monomer. These polymers were also doubly selective with >533 times higher selectivity of S. aureus over red blood cells, and with >53 times more selective for S. aureus over E. coli. Such a high selectivity against S. aureus is particularly exciting as the Methicillin-resistant form of S. aureus, MRSA, is an antibiotic-resistant ‘superbug’ and one of today's most serious health threats for hospital patients.
This is a clear example thus far of how the biological properties of cationic amphiphilic polymers can be tuned to first increase the selectivity of bacteria over mammalian cells, and second increase the selectivity towards one bacteria family over another. This discrimination is important for antimicrobial agents since many bacterial types are in fact beneficial to the body.
Examples
Monomer Synthesis. To obtain new synthetic antimicrobial polymers via ROMP, the first task was to design an easy and modular synthetic pathway towards facially amphiphilic monomers (FIG. 1). The three-step approach taken to obtain these monomers is presented in Scheme 1. In the first step, furan and maleic anhydride underwent a Diels-Alder reaction, yielding exclusively the exo-adduct in accordance with the literature. (Mantovani, et al., J. Am. Chem. Soc. 2005, 127, 2966.) This facile step provided compound 1 containing a polymerizable oxanorbornene group and a cyclic anhydride that allowed twofold and unsymmetrical functionalization. The anhydride 1 was ring-opened with an alcohol to introduce the desired hydrophobic moiety R, which was varied from methyl to hexyl, yielding a series of half-monomers 2a-f with different hydrophobicities. All compounds were crystallizable and thus easy to purify. In the last step, the designated cationic group was attached. As ROMP usually does not tolerate the presence of unprotected amines due to their ligating properties, the desired hydrophilic group (NH3+) was introduced in its protected tert-butyl carbamate (NHBoc) form. (Slugovc, et al., Macromol. Rapid Commun. 2004, 25, 1283.) The half-monomers 2a-f were reacted with the Boc-protected 2-amino ethanol by DCC coupling, yielding a series of masked amphiphilic monomers 3a-f (Scheme 1). This last step required purification by column chromatography to yield pure products. The overall yield of all three reaction steps was  ̃40%.








Homopolymer Synthesis and Molecular Weight Characterization. From the monomers 3a-f, two series of homopolymers with molecular weights of about 3,000 g/mol (the “3k” series) and abut 10,000 g/mol (the “10 k” series) were synthesized. The polymerization of the monomers shown in Scheme 2 was carried out using the third generation Grubbs catalyst (G3) and yielded the precursor polymers 4a-f. (Dichloro-di(3-bromopyridino)-N,N′-Dimesitylenoimidazolino-Ru═CHPh, c.f. Love, et al., Angew. Chem. Internat. Ed. 2002, 41, 4035.) The facially amphiphilic SMAMPs 5a-f were obtained by polymer analogous deprotection: the Boc protecting group was completely removed with trifluoroacetic acid according to NMR. Depending on the alkyl residue, the resulting polymers were water-soluble or dispersible after work up. For convenience, instead of referring to these polymers here by compound numbers, for example 5a-f, a polymer with R=propyl and a molecular weight of 3000 g/mol will be referred to as Propyl—3k. The organosoluble precursor polymers were analyzed by gel permeation chromatography (GPC) in DMF (Table 1a). The molecular weights obtained from GPC using polystyrene standards as a calibration are significantly larger than the ones expected from the reaction stoichiometry. It is well known from the literature that the G3 catalyst initiates quantitatively, so incomplete initiation is unlikely to be responsible for this disparity. (Slugovc, et al., Macromol. Rapid Common. 2004, 25, 1283.) Analysis of the Boc-protected Propyl—3k sample (Mn, GPC=9200 g/mol) by MALDI-TOF MS (Matrix Assisted Laser Ionization and Desorption Time Of Flight Mass Spectrometry) yields a distribution with a maximum intensity at m/z=3095, which is in excellent agreement with the targeted molecular weight. This means that GPC overestimates the molecular weight for these structures, which is further supported by additional oligomer data discussed.







TABLE 1





Homopolymers: a) Precursor polymer characterization by GPC (DMF,


0.01 mol L−1 LiCl, PS standards). b) Characterization of the


antimicrobial homopolymers by biological assays: Inhibitory activity


towards bacterial growth of E. coli and S. aureus bacteria.


(MIC90 = minimal inhibitory concentration preventing


90% bacterial growth) and hemolytic activity towards red blood


cells (HC50 = concentration lysing 50% of blood cells).







Table 1a)













Mn, Target
GPC Mn



Sample
Monomer
g mol−1
g mol−1
Mw/Mn





Methyl_3k
Methyl
3000
11200
1.08


Ethyl_3k
Ethyl
3000
9200
1.10


Propyl_3k
Propyl
3000
9200
1.10


Butyl_3k
Butyl
3000
11500
1.08


Isopentyl_3k
Isopentyl
3000
11400
1.11


Hexyl_3k
Hexyl
3000
11000
1.08


Methyl_10k
Methyl
10000
49000
1.05


Ethyl_10k
Ethyl
10000
11300
1.10


Propyl_10k
Propyl
10000
28200
1.07


Butyl_10k
Butyl
10000
26700
1.07


Isopentyl_8k
Isopentyl
8000
22600
1.06


Isopentyl_10k
Isopentyl
10000
51500
1.16


Hexyl_10k
Hexyl
10000
27600
1.04










Table 1b)











MIC90 μg mL−1
HC50
Selectivty












Sample
E. coli
S. aureus
μg mL−1
E. coli
S. aureus





Methyl_3k
>200
100
2000
<10
20


Ethyl_3k
50
50
1400
28
28


Propyl_3k
6.25
15
50
8.3
3.3


Butyl_3k
15
25
<50
<3.3
<2.0


Isopentyl_3k
12.5
50
<50
<4.0
<1.0


Hexyl_3k
>200
>200
<50
<0.3
<0.3


Methyl_10k
>200
>200
50
<10
<10


Ethyl_10k
>200
>200
1250
<6.3
<6.3


Propyl_10k
3.75
200
<50
13
<0.25


Butyl_10k
20
>200
<50
<2.5
<0.3


Isopentyl_8k
50
>200
<50
<0.3
<0.3


Isopentyl_10k
50
200
<50
<0.3
<0.3


Hexyl_10k
100
>200
n.d.
n.d.
n.d.









Biological Activity of Homopolymers. The biological properties of the homopolymers, i.e. their antibacterial activity, MIC90, towards growth of Escherichia coli and Staphylococcus aureus bacteria, and their hemolytic activity, HC50, towards red blood cells, were tested as described previously. (MIC90—minimal inhibitory concentration preventing 90% bacterial growth, HC50=hemolytic concentration lysing 50% of blood cells, c.f. Rennie, et al., J. Industrial Microbial. Biotechnol. 2005, 32, 296.) HC50/MIC90 quantifies the antimicrobial selectivity. The biological data obtained for these homopolymers are included in Table 2. The MIC90 and HC50 values are also plotted in FIGS. 2a and 2b. As can be seen, the polymer with the highest selectivity for bacterial over mammalian cells is Ethyl—3k, with a good selectivity of 28 for both E. coli and S. aureus. 
For comparison, MSI-78, a derivative of the natural host-defense peptide magainin, has a selectivity of 10. (Gabriel, et al., Materials Sci. Engineering Rev. 2007, 57, 28) For the 3k series shown in FIG. 2a, the following trend is observed: starting with the non-toxic (HC50=2000 μg/mL) and inactive (MIC90>200 μg/mL) Methyl—3k, the antibacterial activity peaks for the Propyl—3k (MIC90=6.25 μg/mL for E. coli), and there the polymers also start becoming more hemolytic (HC50≦50 μg/mL). From Butyl—3k to Hexyl—3k the activity decreases again until the polymers become inactive (MIC90>200 μg/mL), but this time strongly hemolytic. The MIC90s for S. aureus show the same trend, but the activities are generally lower. The data for the 10k series are compiled in FIG. 2b. The MIC90s for the 10k polymers are similar to the ones for the 3k series, with a maximum activity at Propyl—10k, but these samples are generally less active against E. coli than the 3k polymers, and inactive against S. aureus. 
These observations can be rationalized by taking the different compositions of mammalian and bacterial membranes into account. Human red blood cells (RBC) are predominantly composed of cholesterol and phosphtidylcholine (outer leaflet). In contrast, the membranes of Gram-negative bacteria like E. coli consist mostly of phosphatidylethanolamine and anionically charged phosphatidylglycerol (PG) while Gram-positive bacteria like S. aureus have membranes that consist mainly of anionically charged PG and cardiolipin. Thus, bacterial membranes are more negatively charged than RBC membranes. (Som, et al., Journal of Physical Chemistry B 2008 3495-3502.) Just as observed for natural AMPs, the anionic surface charge may attract the positively charged SMAMP. Due to their hydrophobic component they can subsequently penetrate the lipid bilayer if the SMAMP's facial amphiphilicity is rightly balanced. An overly hydrophilic SMAMP, like Methyl—3k, is not able to penetrate the hydrophobic core of the lipid bilayer and is therefore inactive.
Alternatively, such a hydrophilic molecule may prefer to remain in solution as opposed to being adsorbed to the membrane. Their high hydrophilicity also prevents Methyl—3k and Ethyl—3k from lysing RBCs—only the more hydrophobic homologues (Propyl—3k onwards) cause significant hemolysis (FIG. 2a). An overly hydrophobic SMAMP like Hexyl—3k has strong membrane activity and as a result is very hemolytic. The maximum activity against E. coli and S. aureus for the 3k series was from the Propyl—3k polymer, which seems to have the optimal facial amphiphilicity to penetrate the bacterial membrane.
While Propyl—3k was also found to be the most toxic polymer of the 3k series against S. aureus, the 10k polymers were all inactive against this pathogen (FIG. 2b). This may be rationalized as follows: Gram-positive bacteria have a 15-80 nm thick negatively charged murein layer around the cell membrane. As is well known from the polyelectrolyte literature, complexation of one polyion with an oppositely charged polyion (symplex formation) is essentially irreversible, while complexes of a polyion with a less charged species are reversible. (Dautzenberg, et al., Polyelectrolytes. Hanser/Gardner: Munich, 1994.) Thus, assuming that the negatively charged murein layer forms a polyion-polyion complex with the positively charged SMAMPs, the dissociation of such a complex becomes increasingly more difficult with increasing molecular weight (more charges on the SMAMP). The higher molecular weight SMAMPs get stuck in the murein layer of Gram-positive bacteria before reaching the plasma membrane and as a result do not kill S. aureus cells irrespective of their amphiphilicity. The appropriately hydrophobic polymers may be active against Gram-negative bacteria because they have a much thinner (2 nm) murein layer which is located between the outer and cytoplasmic membranes, consistant with the data shown in FIG. 2b. For example, Propyl—10k has an MIC90 of 3.75 μg/mL against E. coli while its MIC90 against S. aureus is 200 μg/mL, resulting in a polymer that is doubly selective, which is >50 times more active against E. coli than against S. aureus, and 13 times more active against E. coli than against RBCs.
Oligomer Synthesis and Molecular Weight Impact on Biological Activity.
All monomers were deprotected (with trifluoroacetic acid as described for the polymers), subjected to MIC90 testing, and found to be inactive (MIC90s>200 μg/mL). This shows that a minimum chain length is necessary to obtain any antibacterial activity. Consequently, choosing the propyl monomer as an example, small molecular weight oligomers were synthesized and analyzed (see Table 3a). The GPC traces for the oligomers (selected samples for clarity), along with the monomer, are shown in FIG. 3a. The peak maxima of all samples are in the expected order: the higher molecular weight oligomers elute before the lower molecular weight ones. MALDI-TOF MS was used to determine the actual oligomer molecular weight. The peaks of the MALDI-TOF mass spectra are shown in FIG. 3b as a distribution function (normalized intensity vs. m/z). From the relative peak intensities and m/z ratio of each peak, the number average molecular weight, Mn, and the polydispersity of the samples were calculated. Those distributions were monomodal, unlike some the GPC traces which appear to be multimodal due to the column resolution. These data are included in Table 3a. The results show once more that GPC curves calibrated with poly(styrene) standards heavily overestimates the molecular weight, while the average number of repeat units nMALDI is in much better agreement with the calculated number of repeat units. However, the smallest oligomer obtained had a degree of polymerization of 3.7 instead of the target of 2 due to the reaction kinetics of this particular solvent/catalyst system.







TABLE 3





Oligomers: a) Precursor oligomer characterization by GPC (DMF,


0.01 mol L−1 LiCl, PS standards). b) Characterization of


the antimicrobial oligomers by biological assays: Inhibitory


activity towards bacterial growth of E. coli and S. aureus bacteria.


(MIC90 = minimal inhibitory concentration preventing


90% bacterial growth) and hemolytic activity towards red blood


cells (HC50 = concentration lysing 50% of blood cells).







Table 3a)
















GPC

GPC
MALDI





Mn Target
Mn g

Mw/
Mn g
MALDI



Sample
g mol−1
mol−1
nGPC
Mn
mol−1
Mw/Mn
nMALDI





Monomer
370
1080
2.9
1.04
—
—
—


(3c,









hydrolyzed)









Oligo 1
740
3300
8.9
1.08
1529
1.11
3.7


Oligo 2
1110
4300
11.6
1.09
2151
1.09
5.3


Oligo 3
1480
5800
15.7
1.07
2642
1.07
6.7


Oligo 4
1850
5900
16.0
1.09
2873
1.11
7.0


Oligo 5
2220
6900
18.6
1.10
3260
1.08
8.2


Oligo 6
2590
7400
20
1.08
3935
1.09
9.7










Table 3b)











MIC90 μg mL−1
HC50 
Selectivty












Sample
E. coli
S. aureus
μg mL−1
E. coli
S. aureus





Monomer (3c,
>200
>200
n.d.
n.d.
n.d.


hydrolyzed)







Oligo 1
200
<3.75
1050
5.25
280


Oligo 2
100
6.25
800
8.0
128


Oligo 3
200
25
1250
6.3
50


Oligo 4
>200
50
>2000
>10
40


Oligo 5
100
50
1000
10
20


Oligo 6
50
25
150
3.0
6.0


Propyl_3k
6.25
15
50
8.3
3.3


Propyl_10k
3.75
200
<50
<13
<0.25









The biological data for the propyl series are summarized in FIG. 4 and Table 3b. Within the error limits of the method, all oligomers are equally non-hemolytic up to Oligo 5. Hemolytic activity increases by one order of magnitude with Oligo 6 (HC50=150 μg/mL) and increases further for Propyl—3k (HC50=50 μg/mL) and Propyl—10k (HC50<50 μg/mL). FIG. 4 shows that the small molecular weight oligomers are inactive against E. coli. Starting with Oligo 5, a marked increase in activity against E. coli (MIC90=100 μg/mL) is observed, which reaches a maximum for Propyl—10k with an MIC90 of 3.75 μg/mL. For S. aureus, the opposite trend is observed (FIG. 4). The best activities were found for the small oligomers (MIC90s=6.25 μg/mL and <3.75 μg/mL for Oligo 1 and Oligo 2, respectively). Activity is progressively lost as the molecular weight increases up to Propyl—10k (MIC90=200 μg/mL). The results of the molecular weight dependent propyl series show that molecular weight can be used as a parameter to selectively target either E. coli or S. aureus, and possibly Gram-negative or Gram-positive bacteria in general.
Previous research indicated that some SMAMPs have slight molecular weight dependent activity. Ilker et al. concluded for their system that the biological activity is independent of molecular weight (for Mn=1600 to 137000 g/mol). Poly2 and poly4 (MIC90s of 200 μg/mL against E. coli) did not show a molecular weight dependence. Poly3 was reported with Mn ranging from 1,650 g/mol to 57,200 g/mol and MIC90s of 25 μg/mL to 80 μg/mL, respectively. (Ilker, et al., J. Am. Chem. Soc. 2004, 126, 15870.) DeGrado reported poly(methacrylate) copolymers with Mn around 1600 g/mol having an MIC90 of 16 μg/mL while polymers above 7900 g/moL exhibited an MIC90 of 80 μg/mL against E. coli. (Kuroda, et al., J. Am. Chem. Soc. 2005, 127, 4128.) In contrast, Oligo 1 has an Mn equal to 740 g/mol and MIC90s<3.75 μg/mL against S. aureus and 200 μg/mL against E. coli while Propyl—10k has MIC90s of 200 μg/mL against S. aureus and 3.75 μg/mL against E. coli. Thus, antimicrobial activity is molecular weight dependent for these SMAMPs.
Tuning the Selectivities by Copolymer Synthesis: The incorporation of highly active but hemolytic repeat units into an otherwise non-active and non-hemolytic polymer can dramatically increase the selectivity. (Ilker, et al., J. Am. Chem. Soc. 2004, 126, 15870.) Here, the Methyl—3k homopolymer qualifies as inactive and non-toxic, whereas the Propyl—3k homopolymer is active and hemolytic. The Ethyl—3k homopolymer is neither most active nor toxic, but has the highest selectivity. Thus, methyl, ethyl and propyl monomers were chosen for copolymerization. Three series of copolymers with a target molecular weight around 3000 g/mol and a varying monomer feed ratio were synthesized and studied (Table 4a). The copolymer composition was checked by NMR spectroscopy, which revealed that the polymer composition matched with the monomer feed ratio.







TABLE 4





Copolymers: a) Precursor polymer characterization by GPC (DMF, 0.01 mol L−1


LiCl, PS standards). b) Characterization of the antimicrobial copolymers by


biological assays: Inhibitory activity towards bacterial growth of E. coli and S. aureus


bacteria. Sample labeling: e.g. E1:P9 is a copolymer with 10 mol % ethyl and 90 mol %


propyl monomer.







Table 43a)















Mn Target
GPC Mn




Sample
Monomers
g mol−1
g mol−1
Mw/Mn







E1:P9
Ethyl-Propyl
3500
11600
1.08



E1:P2
Ethyl-Propyl
3400
7000
1.34



E1:P1
Ethyl-Propyl
3000
11000
1.08



E2:P1
Ethyl-Propyl
3500
10200
1.15



E9:P1
Ethyl-Propyl
3400
7600
1.30



M9:E1
Methyl-Ethyl
3400
13600
1.10



M1:E1
Methyl-Ethyl
3500
14400
1.08



M1:E9
Methyl-Ethyl
3500
14500
1.08



M9:P1
Methyl-Propyl
3400
15300
1.07



M1:P1
Methyl-Propyl
3600
15900
1.07



M1:P9
Methyl-Propyl
3700
14700
1.07











Table 4b)












Mn. Target
MIC90 μg mL−1
HC50
Selectivty














Sample
Monomer
g mol−1
E. coli
S. aureus
μg mL−1
E. coli
S. aureus





Propyl_3k
Propyl
3000
6.25
15
50
8.0
3.3


E1:P9
Ethyl:Propyl
3500
15
15
<50
<3.3
<3.3


E1:P2
Ethyl:Propyl
3400
15
15
<50
<3.3
<3.3


E1:P1
Ethyl:Propyl
3000
100
200
1000
10
5.0


E2:P1
Ethyl:Propyl
3500
>200
200
>2000
10
>10


E9:P1
Ethyl:Propyl
3400
>200
50
500
<2.5
10


Ethyl_3k
Ethyl
3000
50
50
1400
28
28


Methyl_3k
Methyl
3000
>200
100
2000
<10
20


M9:E1
Methyl-Ethyl
3400
>200
6.25
700
<3.5
112


M1:E1
Methyl-Ethyl
3500
>200
12.5
1200
<6.0
96


M1:E9
Methyl-Ethyl
3500
>200
12.5
1500
<7
120


Ethyl_3k
Ethyl
3000
50
50
1400
28
28


Methyl_3k
Methyl
3000
>200
100
2000
10
20


M9:P1
Methyl-Propyl
3400
>200
<3.75
>2000
10
>533


M1:P1
Methyl-Propyl
3600
>200
<3.75
>2000
10
>533


M1:P9
Methyl-Propyl
3700
>200
<3.75
>2000
10
>533


Propyl_3k
Propyl
3000
6.25
15
50
8.3
3.3









The biological data are compiled in Table 4b, and in FIGS. 5a to 5c. For the ethyl-propyl series, none of the copolymers reached the high selectivity of the parent Ethyl—3k polymer: with increasing propyl content, the ethyl-propyl copolymers become more active, but also more toxic. Both series of methyl copolymers showed the expected trend: they become extremely active even with little amount (10 mol %) of the more active monomer but stay non-hemolytic. They are potently active against S. aureus (MIC90<3.75 μg/mL) and remain totally inactive against E. coli (MIC90>200 μg/mL). These copolymers are again doubly selective: >533 for bacterial over mammalian cells, and >53 for S. aureus over E. coli. This double selectivity is inverse to the one described earlier.
Experimental Details
General: Maleic anhydride, furane, 4-dimethyl aminopyridine (DMAP), 1,3-dicyclohexylcarbodiimide (DCC), methanol, ethanol, 1-propanol, 1-butanol, isopentanol, 1-hexanol, ethylvinyl ether and trifluoroacetic acid (TFA) were obtained as reagent grade from Aldrich, Fluka or Acros and used as received.
Third generation Grubbs catalyst (Dichloro-di(3-bromopyridino)-N,N′-Dimesitylenoimidazolino-Ru═CHPh; G3) was synthesized as described previously by Grubbs et al. (Love, et al., Angew. Chem. Int. Ed. 2002, 41, 4035.) The HPLC grade solvents N,N-dimethylformamide (DMF), toluene, ethyl acetate and hexane were purchased from Aldrich, Fisher Scientific or Acros and used as received. THF (HPLC grade, Fisher Scientific) was distilled from sodium/benzophenone under nitrogen. Dichloromethane (HPLC grade, Fisher Scientific) was distilled from CaH2 under nitrogen.
Gel permeation chromatography (DMF/0.01 M LiCl, calibrated with polystyrene standards, toluene as flow marker, 50° C.) was measured on a PL50 GPC setup (Polymer Laboratories, Amherst, Mass.) with a PL Gel 5 μm pre-column and two 10 μm analytical Mixed-D columns (Polymer Laboratories, Amherst, Mass.). NMR spectra were recorded on a Bruker DPX300 spectrometer (Bruker, Madison, Wis.). High resolution mass spectra were obtained from a JEOL JMS 700 instrument (JEOL, Peabody, Mass.); Matrix Assisted Laser Desorption and Ionization Time of Flight Mass Spectra (MALDI-TOF MS) were measured on a Bruker Daltonics Reflex III (Bruker, Madison, Wis.). The biological activity of the polymer samples (the minimal concentration inhibiting 90% of bacterial growth (MIC90) for Escherischia coli (D31) and Staphylococcus aureus (ATCC25923), and the concentration that lyses 50% of red blood cells (HC50) were determined as reported previously. (Rennie, et al., J. Industr. Microbio. Biotech. 2005, 32, 7, 296.)




(i) Maleic anhydride (100 g, 1.02 mol) was dissolved in 1 L toluene. 150 mL (140.7 g, 2.05 mmol) furan were added. The solution was stirred for 3 days. The product (1) was then filtered, washed with toluene and dried under vacuum. A colorless powder was obtained. Yields and spectroscopic data matched those reported previously. (Mantovani, et al., J. Am. Chem. Soc. 2005, 127, 2966.)
(ii) 1 (5 g, 30.0 mmol) was dissolved in THF. Two equivalents of the respective alcohol (e.g. 5.26 g (60 mmol) isopentanol for R=isopentyl) and 10 mol % DMAP were added. After stirring over night; the solvent was removed by vacuum evaporation at room temperature. The unreacted alcohol was removed by dynamic vacuum (5·10−2 mbar). Crystallization from dichloromethane/hexane yielded, e.g. for R=isopentyl, 4.7 g (18.5 mmol, 62%) of the product (2).




2a: R=Methyl: colorless solid, 1H-NMR (300 MHz, CDCl3): 2.86 (m, 2H, H3 & H3′), 3.71 (s, 3H, CH3), 5.27 & 5.32 (m, 2H, H2 & H2′), 6.47 (m, 2H, H1 & H1′).
2b: R=Ethyl: colorless solid, 1H-NMR (300 MHz, CDCl3): 1.25 (t, J=7.2 Hz, 3H, CH2—CH3), 2.83 (m, 2H, H3 & H3′), 4.16 (q, J=7.1 Hz, 2H, CH2—CH3), 5.25 & 5.31 (s, 2H, H2 & H2′), 6.46 (m, 2H, H1 & H1′), 7.53-8.21 (br s, 1H, OH).
2c: R=Propyl: colorless solid, 1H-NMR (300 MHz, CDCl3): 0.92 (t, J=7.3 Hz, 3H, CH2—CH3), 1.63 (m, 2H, β-CH2), 2.84 (m, 2H, H3 & H3′), 4.05 (m, 2H, α-CH2), 5.23 & 5.29 (m, 2H, H2 & H2′), 6.46 (m, 2H, H1& H1′), 9.12-9.68 (br s, 1H, OH).
2d: R=Butyl: colorless oil, 1H-NMR (300 MHz, CDCl3): 0.85 (t, J=7.1 Hz, 3H, CH2—CH3), 1.30 (m, 2H, γ-CH2), 1.54 (m, 2H, β-CH2), 2.78 (m, 2H, H3 & H3′), 4.04 (m, 2H, α-CH2) 5.21 & 5.27 (m, 2H, H2 & H2′), 6.39 (m, 2H, H1& H1′).
2e: R=iso-Pentyl: colorless oil, 1H-NMR (300 MHz, CDCl3): 0.90 (d, J=6.6 Hz, 6H, CH2—CH3), 1.53 (m, 1H, CH—CH3), 1.68 (m, 2H, β-CH2), 2.84 (m, 2H, H3 & H3′), 4.08 (m, 2H, α-CH2) 5.25 & 5.31 (m, 2H, H2 & H2′), 6.45 (m, 2H, H1 & H1′), 8.70-9.68 (br s, 1H, OH).
2f: R=Hexyl: colorless solid, 1H-NMR (300 MHz, CDCl3): 0.88 (t, J=6.4 Hz, 3H, CH2—CH3), 1.28 (m, 6H, γ- to ε-CH2), 1.61 (m, 2H, β-CH2), 2.85 (m, 2H, H3 & H3′), 4.13 (m, 2H, α-CH2) 5.25 & 5.29 (m, 2H, H2 & H2′), 6.47 (m, 2H, H1 & H1′).
(iii) 1 eq 2a-f (e.g. 2 g (7.88 mmol) for R=isopentyl) was dissolved in 25 mL dichloromethane. 1 eq (1.27 g, 7.88 mmol) boc-protected 2-amino ethanol and 10 mol % of DMAP were added. The solution was cooled to 0° C. 1 eq (206.3 g, 7.88 mmol) DCC was added, and the suspension was stirred over night. It was then filtered through a short alumina column (5 cm neutral Al2O3/dichlormethane). The solvent was removed by vacuum evaporation and the crude product was chromatographed (15 cm silica gel, ethyl acetate:hexane 9:1 to 1:1). Evaporation of the solvent yielded the pure monomer. The yield ranged from 70-80%.




3a: R=Methyl: colorless solid, 1H-NMR (300 MHz, CDCl3): 1.43 (s, 9H, H9), 2.82 (m, 2H, H3 & H3′), 3.40 (m, 2H, H6), 3.73 (s, 3H, CH3), 4.16 (m, 2H, H5), 5.03 (br s, 1H, NH), 5.25 & 5.29 (m, 2H, H2 & H2′), 6.46 (m, 2H, H1& H1′). 13C-NMR (75 MHz, CDCl3): 28.4 (C9), 39.5 (C6), 46.9 & 47.1 (C3, C3′), 52.4 (R, CH3), 64.8 (C5), 79.3 (C8), 80.4 & 80.6 (C2, C2′), 136.6 & 136.7 (C1, C1′), 171.6 (C4, C4′). HR-MS (FAB): calc. 341.36. found 342.15.
3b: R=Ethyl: colorless solid, 1H-NMR (300 MHz, CDCl3): 1.27 (t, J=7.2 Hz, 3H, CH2—CH3), 1.44 (s, 9H, H9), 2.81 (m, 2H, H3 & H3′), 3.39 (m, 2H, H6), 4.16 (m, 4H, CH2—CH3 and H5), 5.05 (br s, 1H, NH), 5.25 & 5.31 (m, 2H, H2 & H2′), 6.46 (m, 2H, H1 & H1′). 13C-NMR (75 MHz, CDCl3): 14.4 (R, CH3), 28.4 (C9), 39.4 (C6), 46.8 & 47.1 (C3, C3′), 61.3 (R, CH2), 64.8 (C5), 79.3 (C8), 80.4 & 80.7 (C2, C2′), 136.6 & 136.7 (C1, C1′), 155.9 (C7), 171.6 & 171.7 (C4, C4′). HR-MS (FAB): calc. 355.39. found 356.17.
3c: R=Propyl: colorless solid, 1H-NMR (300 MHz, CDCl3): 1.25 (m, 3H, CH2—CH3), 1.43 (s, 9H, H9), 1.67 (m, 2H, β-CH2), 2.81 (q, 2H, H3& H3′), 3.40 (m, 2H, H6), 4.12 (m, 4H, α-CH2 and H5), 5.07 (br s, 1H, NH), 5.24 & 5.30 (m, 2H, H2 & H2′), 6.46 (m, 2H, H1 & H1′). 13C-NMR (75 MHz, CDCl3): 10.4 (R, CH3), 21.9 (R, β-CH2), 28.4 (C9), 39.5 (C6), 46.8 & 47.2 (C3, C3′), 64.8 (C5), 66.9 (R, α-CH2), 79.3 (C8), 80.4 & 80.7 (C2, C2′), 136.6 & 136.7 (C1, C1′), 155.9 (C7), 171.6 & 171.8 (C4, C4′). HR-MS (FAB): calc. 369.42. found 370.19
3d: R=Butyl: colorless oil, 1H-NMR (300 MHz, CDCl3): 0.93 (m, 3H, CH2—CH3), 1.37 (m, 2H, γ-CH2), 1.44 (s, 9H, H9), 1.62 (m, 2H, β-CH2), 2.82 (q, 2H, H3 & H3′), 3.39 (m, 2H, H6), 4.12 (m, 4H, α-CH2 and H5), 5.06 (br s, 1H, NH), 5.23 & 5.29 (m, 2H, H2 & H2′), 6.46 (m, 2H, H1 & H1′). 13C-NMR (75 MHz, CDCl3): 13.7 (R, CH3), 19.0 (R, γ-CH2), 28.3 (C9), 30.4 (R, β-CH2), 39.4 (C6), 46.7 & 47.1 (C3, C3′), 64.7 (C5), 65.1 (R, α-CH2), 79.2 (C8), 80.4 & 80.6 (C2, C2′), 136.5 & 136.6 (C1, C1′), 155.9 (C7), 171.5 & 171.8 (C4, C4′). HR-MS (FAB): calc. 383.45. found 384.21.
3e: R=iso-Pentyl: colorless oil, 1H-NMR (300 MHz, CDCl3): 0.92 (d, J=6.4 Hz, 6H, CH—CH3), 1.44 (s, 9H, H9), 1.53 (m, 1H, CH—CH3), 1.69 (m, 2H, β-CH2), 2.82 (q, 2H, H3 & H3′), 3.41 (m, 2H, H6), 4.18 (m, 4H, α-CH2 and H5), 5.07 (br s, 1H, NH), 5.23 & 5.29 (m, 2H, H2 & H2′), 6.46 (m, 2H, H1 & H1′). 13C-NMR: n.d., HR-MS (FAB): n.d.
3f: R=Hexyl: colorless oil, 1H-NMR (300 MHz, CDCl3): 0.88 (t, J=6.6 Hz, 3H, CH2—CH3), 1.43 (s, 9H, t-butyl), 1.37 (m, 6H, γ-ε-CH2) 1.62 (m, 2H, β-CH2), 2.82 (m, 2H, H3 & H3′), 3.42 (m, 2H, H6), 4.14 (m, 4H, α-CH2 and H5), 5.07 (br s, 1H, NH), 5.23 & 5.29 (m, 2H, H2 & H2′), 6.46 (m, 2H, H1 & H1′). 13C-NMR (75 MHz, CDCl3): 10.9 (R, CH3), 22.5 (R, δ-CH2), 15.4 (R, γ-CH2), 28.3 (C9), 28.5 (R, β-CH2), 39.3 (C6), 46.6 & 46.9 (C3, C3′), 64.4 (C5), 65.2 (R, α-CH2), 78.9 (C8), 80.3 & 80.5 (C2, C2′), 136.4 & 136.6 (C1, C1′), 155.8 (C7), 171.5 & 171.7 (C4, C4′). HR-MS (FAB): calc. 411.50. found 412.24.




(i) The monomer 3a-f and G3-catalyst amounts (see Table 5 for details) were dissolved in 1 mL dichloromethane each and subject to three freeze-thaw cycles. The catalyst was added in one shot to the vigorously stirring monomer solution at room temperature under argon. After 30 min, the living polymer chain was end-capped with an excess of ethylvinyl ether (1 mL, 754 mg, 10.5 mmol). The solution was allowed to stir over night. After evaporation of the solvent and drying, an aliquot of each polymer was taken for GPC and NMR analysis. The product was a brown solid.







TABLE 5







Experimental parameters for homopolymer synthesis



















MnTarget
MMonomer
nMonomer
mMonomer
MCatalyst
nCatalyst
mCatalyst


Sample
Monomer
Nrepeat units
g mol−1
g mol−1
mmol
mg
g mol−1
mmol
mg



















Methyl_3k
Methyl
9.7
3000
341
0.290
100
885
0.030
29.0


Methyl_10k
Methyl
29
10000
341
0.290
100
885
0.010
8.9


Ethyl_3k
Ethyl
8.6
3000
355
0.563
200
885
0.065
57.6


Ethyl_10k
Ethyl
28
10000
355
0.563
200
885
0.020
17.7


Propyl_3k
Propyl
8.4
3000
369
0.282
104
885
0.034
29.7


Propyl_10k
Propyl
29
1000
369
0.278
103
885
0.010
8.50


Butyl_3k
Butyl
7.2
3000
383
0.525
202
885
0.073
64.5


Butyl_10k
Butyl
27
1000
383
0.565
217
885
0.021
18.6


Isopentyl_3k
Isopentyl
7.9
3000
397
0.515
205
885
0.065
57.5


Isopentyl_8k
Isopentyl
20.1
8000
397
1.926
766
885
0.096
85.0


Isopentyl_10k
Isopentyl
26
10000
397
0.508
202
885
0.020
17.5


Hexyl_3k
Hexyl
7.7
3000
412
0.516
212
885
0.067
59.0


Hexyl_10k
Hexyl
25
10000
412
0.493
203
885
0.020
17.7















4a: R=Methyl: 1H-NMR (300 MHz, CDCl3): 1.43 (s, 9H, H9), 3.12 (br m, 2H, H3 & H3′), 3.34 (br m, 2H, H6), 3.71 (s, 3H, CH3), 4.16 (br m, 2H, H5), 4.71 (m, 1H, H2 & H2′ trans), 5.10 (br s, 1H, H2 & H2′ cis), 5.40 (br s, 1H, NH), 5.58 (br m, 1H, H1 & H1′ cis) and 5.88 (br m, 1H, H1 & H1′ trans).
4b: R=Ethyl: 1H-NMR (300 MHz, CDCl3): 1.24 (s, 3H, CH2—CH3), 1.42 (s, 9H, H9), 3.09 (br m, 2H, H3 & H3′), 3.34 (br m, 2H, H6), 4.16 (br m, 4H, CH2—CH3 and H5), 4.72 (br m, 1H, H2 & H2′ trans), 5.10 (br m, 1H, H2 & H2′ cis), 5.30 (br s, 1H, NH), 5.58 (br m, 1H, H1 & H1′ cis), 5.88 (br m, 1H, H1 & H1′ trans).
4c: R=Propyl: 1H-NMR (300 MHz, CDCl3): 0.92 (m, 3H, CH2—CH3), 1.43 (s, 9H, H9), 1.62 (m, 2H, β-CH2), 3.12 (br m, 2H, H3 & H3′), 3.34 (br m, 2H, H6), 4.10 (m, 4H, α-CH2 and H5), 4.69 (br m, 1H, H2 & H2′ trans), 5.12 (br m, 1H, H2 cis & H2′), 5.31 (br m, 1H, H1 & H1′ cis), 5.59 (br s, 1H, NH), 5.88 (br m, 1H, H1 & H1′ trans).
4d: R=Butyl: 1H-NMR (300 MHz, CDCl3): 0.87 (m, 3H, CH2—CH3), 1.29 (m, 2H, γ-CH2), 1.43 (s, 9H, H9), 1.59 (m, 2H, β-CH2), 3.11 (br m, 2H, H3 & H3′), 3.37 (br m, 2H, H6), 4.10 (m, 4H, α-CH2 and H5), 4.73 (br m, 1H, H2 & H2′ trans), 5.11 (br m, 1H, H2 & H2′ cis), 5.35 (br s, 1H, NH), 5.59 (br m, 1H, H1 & H1′ cis), 5.88 (br m, 1H, H1 & H1′ trans).
4e: R=iso-Pentyl: 1H-NMR (300 MHz, CDCl3): 0.91 (m, 6H, CH—CH3), 1.43 (s, 9H, H9), 1.52 (m, 1H, CH—CH3), 1.62 (m, 2H, β-CH2), 3.10 (br m, 2H, H3 & H3′), 3.35 (br m, 2H, H6), 4.15 (m, 4H, α-CH2 and H5), 4.69 (br m, 1H, H2 & H2′ trans), 5.11 (br m, 1H, H2 & H2′ cis), 5.34 (br s, 1H, NH), 5.59 (br m, 1H, H1 & H1′ cis), 5.88 (br m, 1H, H1 trans).
4f: R=Hexyl: 1H-NMR (300 MHz, CDCl3): 0.88 (m, 3H, CH2—CH3), 1.29 (m, 2H, γ-ε-CH2), 1.43 (s, 9H, H9), 1.56 (m, 2H, β-CH2), 3.10 (br m, 2H, H3 & H3′), 3.36 (br m, 2H, H6), 4.10 (m, 4H, α-CH2 and H5), 4.70 (br m, 1H, H2 & H2′ trans), 5.12 (br m, 1H, H2 & H2′ cis), 5.38 (br s, 1H, NH), 5.58 (br m, 1H, H1 & H1′ cis), 5.89 (br m, 1H, H1 & H1′ trans).
(ii) The crude polymers were dissolved in 2 mL dichloromethane. An excess of TFA (2 mL, 2.97 g, 26.0 mmol) was added and the solution was stirred at room temperature over night. The excess acid was removed by azeotropic distillation with dichloromethane (2×15 mL) and methanol (1×15 mL) at the rotary evaporator. The samples were dried in vacuum over night and dissolved in 30 mL Milli-Q water or DMSO depending on solubility. The samples were dialyzed against Milli-Q water until the conductivity of the water was 0.1 μS after 12 h of dialysis (total dialysis time 4-7 days). The hydrolyzed polymers were then freeze dried.




5a: R=Methyl: 1H-NMR (300 MHz, DMSO): 3.04 (br m, 2H, H3 & H3′), 3.30 (br m, 2H, H6), 3.59 (s, 3H, CH3), 4.16 (br m, 2H, H5), 4.57 (m, 1H, H2 & H2′ trans), 4.91 (br m, 1H, H2 & H2′ cis), 5.61 (br m, 1H, H1 & H1′ cis), 5.82 (br m, 1H, H1 & H1′ trans).
5b: R=Ethyl: 1H-NMR (300 MHz, D2O): 1.28 (br s, 3H, CH2—CH3), 3.12 (br m, 2H, H3 & H3′), 3.32 (br m, 2H, H6), 3.49-3.81 (br m, 4H, CH2—CH3 and H5), 5.10-5.40 (br m, 2H, H2 & H2′), 5.30-5.90 (br m, 2H, H1& H1′).
5c: R=Propyl: 1H-NMR (300 MHz, D2O): 0.94 (br m, 3H, CH2—CH3), 1.67 (br m, 2H, β-CH2), 3.31 (br m, 2H, H3 & H3′), 3.50 (br m, 2H, H6), 4.12 (m, 4H, α-CH2 and H5), 4.39 (br m, 1H, H2 & H2′ trans), 5.12 (br m, 1H, H2 & H2′ cis), 5.31 (br m, 1H, H1 & H1′ cis), 5.88 (br m, 1H, H1 & H1′ trans).
5d: R=Butyl: 1H-NMR (300 MHz, D2O): 0.93 (m, 3H, CH2—CH3), 1.36 (m, 2H, γ-CH2), 1.61 (m, 2H, β-CH2), 3.10 (br m, 2H, H3 & H3′), 3.48 (br m, 2H, H6), 4.14 (m, 4H, α-CH2 and H5), 4.38 (br m, 1H, H2 & H2′ trans), 5.11 (br m, 1H, H2 & H2′ cis), 5.35-6.20 (br m, 2H, H1 & H1′).
5e: R=iso-Pentyl: 1H-NMR (300 MHz, DMSO): 0.91 (m, 6H, CH—CH3), 1.55 (m, 1H, CH—CH3), 1.69 (m, 2H, β-CH2), 3.31 (br m, 2H, H3 & H3′), 3.48 (br m, 2H, H6), 4.15 and 4.40 (m, 4H, α-CH2 and H5), 4.69 (br m, 1H, H2 & H2′ trans), 5.11 (br m, 1H, H2 & H2′ cis), 5.34 (br m, 1H, H1 & H1′ cis), 5.88 (br m, 1H, H1 & H1′ trans).
5f: R=Hexyl: 1H-NMR (300 MHz, D2O): 0.90 (m, 3H, CH2—CH3), 1.31 (m, 2H, γ-ε-CH2), 1.62 (m, 2H, β-CH2), 3.30 (br m, 2H, H3 & H3′), 3.47 (br m, 2H, H6), 4.13 and 4.40 (m, 4H, α-CH2 and H5), 4.70-5.12 (br m, 2H, H2 & H2′), 5.50-6.20 (br m, 2H, H1 & H1′).
Copolymer Synthesis, Deprotection and Characterization: Copolymers were synthesized and hydrolyzed using the same methods as for the homopolymers. Each comonomer was dissolved in 0.7 mL dichloromethane separately under argon. The monomer solutions were mixed, and the respective amount of G3 catalyst in 1 mL dichloromethane was added in one shot. Details are given in Table 6. The NMR spectroscopic data for each repeat unit proton matched the assignment of the analogous protons in the homopolymers. Where possible (no peak overlap), integration revealed that the copolymer composition matched the monomer feed ratio. Deprotection was performed as described for the homopolymers.







TABLE 6





Experimental parameters for copolymer synthesis: a) ethyl-propyl copolymers, b) methyl-


ethyl copolymers, c) methyl-propyl copolymers.







a)



















Mn Target
nEthyl
mEthyl
nPropyl
mPropyl
nCatalyst
mCatalyst


Sample
Monomers
Nrepeat units
g mol−1
mmol
mg
mmol
mg
mmol
mg





E1:P9
Ethyl:Propyl
10
3500
0.06
20.2
0.54
192
0.060
53.1


E1:P2
Ethyl:Propyl
10
3400
0.20
68.3
0.40
137
0.060
53.1


E1:P1
Ethyl:Propyl
 9
3000
0.14
48.0
0.14
51.4
0.037
32.2


E2:P1
Ethyl:Propyl
10
3500
0.40
130
0.20
70.0
0.060
53.1


E9:P1
Ethyl:Propyl
10
3400
0.54
183
0.06
20.1
0.060
53.1






















Mn Target
nMethyl
mMethyl
nEthyl
mEthyl
nCatalyst
mCatalyst


Sample
Monomers
Nrepeat units
g mol−1
mmol
mg
mmol
mg
mmol
mg










b)
















M1:E9
Methyl-Ethyl
10
3400
0.81
276.5
0.09
32.0
0.09
79.7


M1:E1
Methyl-Ethyl
10
3500
0.45
153.6
0.45
159.9
0.09
79.7


M9:E1
Methyl-Ethyl
10
3550
0.09
30.7
0.81
287.9
0.09
79.7







c)
















M1:P9
Methyl-Propyl
10
3400
0.81
276.5
0.09
33.2
0.09
79.7


M1:P1
Methyl-Propyl
10
3600
0.45
153.6
0.45
166.2
0.09
79.7


M9:P1
Methyl-Propyl
10
3700
0.09
30.7
0.81
299.2
0.09
79.7









Propyl Oligomer Synthesis

Propyl Oligomers were synthesized and hydrolyzed using the same methods as for the homopolymers. Experimental data are included in Table 7. GPC traces and distribution functions obtained from the MALDI-TOF MS spectra of all oligomers are included in FIG. 6.







TABLE 7







Experimental parameters for oligomer synthesis

















Mn Target
nPropyl
mPropyl
nCatalyst
mCatalyst


Sample
Monomers
Nrepeat units
g mol−1
mmol
mg
mmol
mg

















Oligo 1
Propyl
2
740
0.18
66.5
0.09
79.7


Oligo 2
Propyl
3
1100
0.27
99.7
0.09
79.7


Oligo 3
Propyl
4
1480
0.36
133.0
0.09
79.7


Oligo 4
Propyl
5
1850
0.45
166.2
0.09
79.7


Oligo 5
Propyl
6
2220
0.54
199.5
0.09
79.7


Oligo 6
Propyl
7
2600
0.18
66.5
0.09
79.7









Biological Data

Representative curves for the determination of the MIC90 as well as the HC50 values are given here. (Rennie, et al., J. Industr. Microbio. Biotech. 2005, 32, 7, 296) The MIC90 data was obtained from the plot of % growth vs. concentration. The concentration value below 10% growth was taken as the MIC90. Controls with inactive polymers and polymers with known activity were included on each plate to ensure data reproducibility. Additionally, the data was collected in quadruplicate. Representative examples are given below (FIGS. 7-8). An example of the dose-response behavior for the different bacteria is shown in FIG. 7 c) and d). The shape of the dose-response curve is largely the same for both bacteria. Hydrophobic polymers such as Propyl—3k sometimes appear to become less toxic at higher concentrations due to an artifact that comes from decreased solubility or aggregation at these concentrations. When the bacterial count is determined in these wells, there are zero remaining viable bacteria, or CFUs. Examples of typical HC50 curves are shown in FIG. 9. The HC50 value was taken as the average value of the data at 50% lysis.
To compare the differences between the reported MIC90 (Tables 1b, 32b and 4b), and the value of MIC100 which is sometime reported, Table 8 reports these MIC100 values. When they differ from the MIC90 values in the main text, they are highlighted in bold.







TABLE 8





Characterization of the antimicrobial homopolymers by biological


assays: Inhibitory activity towards bacterial growth of E. coli and


S. aureus bacteria. (MIC100 = minimal inhibitory concentration


preventing 100% bacterial growth) and hemolytic activity


towards red blood cells (HC50 = concentration lysing 50% of


blood cells). a) Homopolymers, b) oligomers, and c) copolymers.





















MIC100 μg mL−1
HC50

Selectivty













Sample
E. coli
S. aureus
μg mL−1
E. coli
S. aureus










a)












Methyl_3k
>200
>200
2000
<10
10


Ethyl_3k
50
200
1400
28
7


Propyl_3k
6.25
25
50
8.0
2.0


Butyl_3k
15
25
<50
<3.3
<2.0


Isopentyl_3k
12.5
50
<50
<4.0
<1.0


Hexyl_3k
>200
>200
<50
<0.3
<0.3


Methyl_10k
>200
>200
50
<10
<10


Ethyl_10k
>200
>200
1250
<6.3
<6.3


Propyl_10k
6.25
>200
<50
<8.0
<0.25


Butyl_10k
20
>200
<50
<2.5
<0.3


Isopentyl_8k
50
>200
<50
<0.3
<0.3


Isopentyl_10k
50
200
<50
<0.3
<0.3


Hexyl_10k
100
>200
n.d.
n.d.
n.d.







b)












Monomer (3c,
>200
>200
n.d.
n.d.
n.d.


hydrolyzed)


Oligo 1
>200
6.25
1050
5.25
168


Oligo 2
200
12.5
800
4.0
64


Oligo 3
200
25
1250
6.3
50


Oligo 4
>200
50
>2000
>10
40


Oligo 5
100
>200
1000
10
5.0


Oligo 6
50
50
150
3.0
3.0


Propyl_3k
6.25
25
50
8.3
2.0


Propyl_10k
6.25
>200
<50
<8.0
<0.25










c)











MIC90 μg mL−1
HC50
Selectivty













Sample
Monomer
E. coli
S. aureus
μg mL−1
E. coli
S. aureus





Propyl_3k
Propyl
6.25
25
50
8.0
2.0


E1:P9
Ethyl:Propyl
50
15
<50
<1.0
<3.3


E1:P2
Ethyl:Propyl
15
15
<50
<3.3
<3.3


E1:P1
Ethyl:Propyl
100
200
1000
10
5.0


E2:P1
Ethyl:Propyl
>200
200
>2000
10
>10


E9:P1
Ethyl:Propyl
>200
50
500
<2.5
10


Ethyl_3k
Ethyl
50
200
1400
28
7


Methyl_3k
Methyl
>200
>200
2000
<10
<10


M9:E1
Methyl-Ethyl
>200
12.5
700
<3.5
56


M1:E1
Methyl-Ethyl
>200
25
1200
<6.0
48


M1:E9
Methyl-Ethyl
>200
12.5
1500
<7
120


Ethyl_3k
Ethyl
50
200
1400
28
7


Methyl_3k
Methyl
>200
>200
2000
10
<10


M9:P1
Methyl-Propyl
>200
3.75
>2000
10
533


M1:P1
Methyl-Propyl
>200
3.75
>2000
10
533


M1:P9
Methyl-Propyl
>200
3.75
>2000
10
533


Propyl_3k
Propyl
6.25
25
50
8.3
2.0









b. Doubly Selective Antimicrobial Polymers

The desirable properties of SMAMPs are a high antibacterial activity (a low MIC90) value) and low red blood cell lysis (a high HC50 value), leading to a high selectivity for bacteria over the mammalian host cells. SMAMPs that are equally active against Gram-positive bacteria (e.g. S. aureus) and Gram-negative bacteria (e.g. E. coli) are well described in the literature. (Gabriel, et al. Mater. Sci. Eng., R: Rep. 2007, 57, 28; Kuroda, et al., J. Am. Chem. Soc. 2005, 127, 4128; Sambhy, et al., Angew. Chem. Inter. Ed. 2008, 47, 1250; Mowery, et al., J. Am. Chem. Soc. 2007, 129, 15474.)
The morphological differences between Gram-negative and Gram-positive cells are significant. The Gram-negative cell (FIG. 10a) has an outer and a plasma membrane, with a thin layer of loosely cross-linked peptidoglycan in the periplasmic space between the two membranes. On the other hand, the Gram-positive bacteria cell (FIG. 10b) has a 20 to 80 nm thick, highly cross-linked peptidoglycan layer surrounding a single bilayer lipid membrane. Additionally, the membranes consist of different phospholipids. Generally, Gram-negative bacteria (and specifically E. coli) membranes mainly consists of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), while the membrane of S. aureus, like most other Gram-positive bacteria, is made predominantly from cardiolipin (CL). (Alberts, et al. Molecular Biology of the Cell, 4th Edition ed., Garland Science, New York, 2002; Som, et al, J. Phys. Chem. B 2008, 112, 3495.)
Recently, Kallenbach et al. reported a synthetic peptide with a selectivity of 4 for E. coli over S. aureus (which compares to the natural AMP gentamicin with a selectivity of about 40). (Liu, et al., Chem Bio Chem 2007, 8, 2063.) Muehle and Tam reported a design concept for the synthesis of AMPs with Gram-negative selectivity through binding to the lipopolysaccharide layer of Gram-negative bacteria. (Muhle, et al., Biochemistry 2001, 40, 5777.)
The molecular origin for the observed double selectivity could be the general structural difference, specifically the different lipid composition, of the plasma membranes. The additional peptidoglycan layer of S. aureus might be responsible for a molecular weight dependence seen in many SMAMPs, namely that antimicrobial activity is lost with increasing molecular weight. Using dye-leakage experiments on bacteria-mimicking model vesicles, the observed doubly selectivity of a model SMAMP (FIG. 11) are related to the morphological differences between these two bacterial types. (Lienkamp, et al, Chem. Eur. J. submitted.)
The SMAMP shown in FIG. 11 proved to be a suitable probe to test the above hypotheses, as it is doubly selective and has a molecular weight-dependent antimicrobial activity against S. aureus. It is generally accepted that SMAMPs, like AMPs, interact with bacteria membranes such that the membranes are corrupted (although other targets can exist). (Ikeda, et al., Biochim. Biophys. Acta Biomembr. 1990, 1021, 56.) Therefore, dye-leakage experiments on unilamellar model vesicles, which probe membrane activity, were performed. (Som, et al. J. Phys. Chem. B 2008, 112, 3495.) (FIG. 14a.)
In a first series of experiments, the model vesicles for E. coli, containing PE/PG lipid, and S. aureus bacteria, made from CL lipid, were exposed to the same concentration of the SMAMP. The dye-leakage data obtained showed that the SMAMP, regardless of its molecular weight, was membrane active against both model membranes (FIGS. 14b and 14c). As dye-leakage data obtained at a single polymer concentration is not necessarily the most quantitative measure of membrane activity, a concentration dependent leakage curve was obtained (FIG. 12a). Fitting this data to the Hill equation gave the polymer concentration that leads to 50% polymer-induced leakage, EC50. (Som, et al, J. Phys. Chem. B 2008, 112, 3495.) For the PE/PG vesicles, EC50 was 1.57 μg mL−1 and for the CL vesicles 2.64 μg mL−1; additionally, both vesicle types have the same maximum leakage. Therefore it is clear that the SMAMP is equally membrane-active against S. aureus and E. coli mimics, i.e. the different membrane composition is not responsible for the observed differences in antimicrobial activity, specifically the inactivity against E. coli. 
Although vesicle studies are common in the (SM)AMP literature, these unilamellar vesicles are understood to be extremely simple models of the plasma membrane. They might be a reasonable model for S. aureus, with one membrane, but E. coli has a double membrane structure. This additional membrane effectively creates a gradient in SMAMP concentration (FIG. 10a). The outer membrane sees a concentration c, that causes membrane disintegration; however, the periplasmic space sees a significantly reduced concentration c2, which is insufficient to damage the plasma membrane. As the experiments proved that the different nature of the lipids are not crucial for the observed double selectivity, the double membrane structure might be responsible for the inactivity of the SMAMP against E. coli, consistent with previous observations demonstrating that the mere loss of the outer membrane integrity was not sufficient to kill E. coli bacteria. (Shai, Biochim. Biophys. Acta Biomembr. 1999, 1462, 55.)
A second series of experiments were designed to study the peptidoglycan layer in S. aureus in connection with the observed loss of antimicrobial activity with increasing molecular weight. According to the dye-leakage data in FIG. 14c, for the S. aureus-like membranes, all SMAMPs were membrane-active regardless of their molecular weight Mn. It is proposed that the higher Mn samples of the SMAMP cannot reach the plasma membrane of S. aureus due to the highly cross-linked peptidoglycan layer (FIG. 10b), which effectively reduces their concentration c2 at the plasma membrane and renders them inactive in the MIC90 experiments. The peptidoglycan layer consists of anionically charged, alternating copolymers of β-(1,4)-linked N-acetylmuramic acid and N-glucosamine, which are cross-linked by peptide chains. There are two possible Mn-dependent modes of interaction between this layer and SMAMPs. First, there may be SMAMP-peptidoglycan binding: Whatever the nature of this binding, whether it is charge-, polarity- or hydrophobicity-driven, the number of binding sites per chain will increase with Mn, making the binding gradually more irreversible. Second, the peptidoglycan may simply act as a sieve. Thus, the dimensions of the SMAMP can be crucial; the higher its Mn, the larger its hydrodynamic volume, which might prevent larger SMAMPs from passing through the peptidoglycan mesh.
As a mimic of this highly cross-linked peptidoglycan layer, peptidoglycan extract from S. aureus bacteria was used in the dye-leakage experiments. While this extract does not form a dense, uniform layer around the vesicle when added to a dye-leakage experiment and thus is not a perfect model for the sieving properties of the peptidoglycan layer of a real S. aureus cell, it is a very good model to investigate SMAMP-peptidoglycan binding.
The following experiments were designed to investigate the effect of peptidoglycan, and to differentiate between the two proposed modes of SMAMP-peptidoglycan interaction: (1) CL vesicles were incubated with the peptidoglycan extract for 10 min and 24 h, respectively, after which times the vesicles were exposed to the SMAMP. While the extract itself did not cause leakage, all molecular weights of the SMAMP were membrane-active in its presence for both incubation times. (2) CL vesicles were exposed to samples containing constant concentrations of the 3,000 g/mol polymer, which were previously incubated for 24 h with different amounts of peptidoglycan extract. Plotting the concentration of the peptidoglycan versus the induced leakage percentage, a peptidoglycan concentration-dependent curve was obtained (FIG. 12b). The data shows that the polymer-peptidoglycan ratio is critical for the amount of leakage reduction. At a sufficiently high peptidoglycan concentration, the leakage was near-quantitatively quenched, i.e. saturation was reached. (3) CL vesicles were mixed with SMAMP solutions that had been incubated with the previously determined saturation amount of peptidoglycan for 10 min and 24 h, respectively. In the case of short incubation time, all polymers were membrane active in the presence of the extract, meaning that no peptidoglycan-SMAMP complex formed. However after 24 h, as shown in FIG. 12c, the membrane activity of all samples was lost, indicating binding. The results of these experiments are illustrated in FIG. 13. SMAMP-peptidoglycan binding does occur, but it is a relatively slow process. On the timescale of an MIC90 experiment, the loss of SMAMP activity with increasing Mn due to binding is not responsible for the observed antimicrobial activity.
The results show that the CL model vesicles with non cross-linked peptidoglycan are not able to differentiate between different Mn polymers because the added peptidoglycan does not form a uniform layer on the vesicle, which is why both the high and the low Mn SMAMP are active when incubated for 10 min. In the parent bacteria, however, the peptidoglycan layer is thick, uniform and the pores have a finite size, meaning that only small molecules can diffuse through easily (FIG. 10b). Thus, the high molecular weight SMAMPs could only pass this layer by a reptation-like mode, meaning that the polymer coil would have to unravel. This process is so slow that, on the timescale of the MIC experiment, no antimicrobial activity is observed. This is consistent with the observation that only proteins with a diameter of up to 2 nm were able to pass cross-linked peptidoglycan layers that had been isolated from E. coli and B. subtilis. (Demchick, et al., J. Bacteriol. 1996, 178, 768.)
Experiments discussed herein give insight into the molecular origins of ‘double selectivity’ in SMAMPs: Lipid composition was shown not to be the decisive factor for losing antimicrobial activity against E. coli. It was further shown that the molecular weight dependence of the MIC90 data for S. aureus is not due to binding to the peptidoglycan layer, but most likely a sieving effect.
Examples
General: All chemicals were obtained as reagent grade from Aldrich, Fluka or Acros and used as received. 3rd generation Grubbs catalyst (Dichloro-di(3-bromopyridino)-N,N′-Dimesitylenoimidazolino-Ru═CHPh, G3) was synthesized as described previously by Grubbs et al. (Klein, et al., Emerg. Infect. Dis. 2007, 13, 1840.) HPLC grade solvents N,N-dimethylformamide (DMF) were purchased from Aldrich, Fisher Scientific or Acros and used as received. THF (HPLC grade, Fisher Scientific) was distilled from sodium/benzophenone under nitrogen. Dichloromethane (HPLC grade, Fisher Scientific) was distilled from CaH2 under nitrogen. Gel permeation chromatography (DMF/0.01 M LiCl, calibrated with polystyrene standards, toluene as flow marker, 50° C.) was measured on a PL50 GPC setup (Polymer Laboratories, Amherst, Mass.) with a PL Gel 5 μm pre-column and two 10 μm analytical Mixed-D columns (Polymer Laboratories, Amherst, Mass.). NMR spectra were recorded on a Bruker DPX300 spectrometer (Bruker, Madison, Wis.). High resolution mass spectra were obtained from a JEOL JMS 700 instrument (JEOL, Peabody, Mass.); Matrix Assisted Laser Desorption and Ionization Time of Flight Mass Spectra (MALDI-TOF MS) were measured on a Bruker Daltonics Reflex III (Bruker, Madison, Wis.). The biological activity of the polymer samples (the minimal concentration inhibiting 90% of bacterial growth (MIC90) for Escherischia coli (D31) and Staphylococcus aureus (ATCC25923), and the concentration that lyses 50% of red blood cells (HC50) were determined as reported previously. (Huang, Biochemistry 2000, 39, 8347.) Fluorescence measurements were performed on a Perkin-Elmer-LS55 luminescence spectrometer and Jobin Yvon Fluorolog-3. The phospholipids 1,1′-2,2′-tetraoleoyl cardiolipin sodium salt (CL), 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (PE) and 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (PG) were obtained as chloroform solutions from Avanti Polar Lipids, Inc. and used as received. Peptidoglycan extract from S. aureus was obtained from Fluka and used as received.
Polymer Synthesis: The synthesis of the model SMAMP is shown in Scheme 3. (Brodgen, Nature Rev. Microbiol. 2005, 3, 238.) The characterization results are given in Table 9.











TABLE 9







Analytical results for the polymers.














Mn. Target
GPC Mn

MIC90 μg mL−1
HC50
Selectivity















Sample
g mol−1
g mol−1
Mw/Mn
E. coli
S. aureus
μg mL−1
E. coli
S. aureus


















Diamine_3k
3000
8900
1.15
>200
15
1000
<5
67


Diamine_5k
5000
12900
1.08
>200
25
1000
<5
40


Diamine_10k
10000
22500
1.08
>200
50
1000
<5
20


Diamine_50k
50000
77000
1.07
>200
200
2000
<10
10









Dye-Leakage Experiments:

Vesicle Preparation: Two buffers were prepared. Buffer A consisted of 1 mmol (142 mg) Na2HPO4 in 100 mL H2O (c=10 mmol L−1), which was then adjusted to pH=7.0 with 0.1 mol L−1 NaOH. Buffer B was made from 10 mmol (1.42 g) Na2HPO4 in 1 L H2O (c=10 mmol L−1) and 90 mmol (5.26 g) NaCl (c=90 mmol L−1). 0.4 mmol (249 mg) of calcein dye was dissolved in 10 mL buffer A. 0.1 mol NaOH was added dropwise until all calcein dissolved and the pH was 7.0, yielding a solution of 40 mmol L−1.
The lipid solutions were added to thoroughly clean round bottom flask. For the CL vesicles, 600 μL (10 mmol) CL in chloroform was used, for the PE/PG 4:1 vesicles, 297 μL (8 mmol) PE and 159 μL (2 mmol) PG in chloroform were mixed. The solvent was evaporated to obtain a lipid film, which was hydrated with 1 mL of calcein solution in buffer. The suspension was stirred for 1 h and then subject to five freeze-thaw cycles (liquid nitrogen/water at room temperature). After that it was extruded ten times through a polycarbonate membrane with 400 nm pore diameter (Avanti Polar Lipids Mini-Extruder, Whatman membrane). The dye-filled vesicles were separated from the excess dye using an Sephadex G-50 and buffer B as an eluent. The vesicle fraction from the column was diluted by a factor of 20 for the dye-leakage experiments.
Fluorescence measurement: The polymer-induced leakage was monitored, as described previously, by recording the increase of calcein fluorescence intensity at a wavelength 515 nm (excitation wavelength=490 nm, slit width 3.0). (Som, et al., J. Phys. Chem. B 2008, 112, 3495.) To normalize the dye-leakage data, a base line of calcein fluorescence without polymer addition was observed for each sample. Also, after each measurement, 20 μL Trition X (20% in DMSO) was added to determine the fluorescence intensity corresponding to 100% leakage. With these data points, the measured fluorescence intensity was converted in % leakage (FIG. 14a). The polymer samples were dissolved in DMSO at various concentrations. Pure DMSO has been tested previously and was shown not to cause leakage. For the concentration-dependent EC50 curves, the leakage percentage after 800 sec was plotted versus the concentration in a semi-logarithmic plot. The EC50 concentration, which corresponds to 50% polymer-induced leakage, was determined using the Hill equation,




  
    
      y
      ⁡
      
        (
        c
        )
      
    
    =
    
      
        y
        max
      
      +
      
        
          
            y
            min
          
          -
          
            y
            max
          
        
        
          1
          +
          
            
              (
              
                c
                
                  EC
                  50
                
              
              )
            
            n
          
        
      
    
  
  ,




where y(c) is the leakage percentage at a given concentration c, ymin is the leakage percentage corresponding to no polymer addition, ymax is the leakage percentage at high polymer concentration (saturation), and n is a fit parameter, which may be interpreted as a measure for the number of molecules participating in the binding. The exact amounts of polymer and vesicles for each experiment is compiled in Tables 10 (experiments without peptidoglycan) and Tables 11 (experiments with peptidoglycan). All samples were filled up to a total volume of 2 mL with buffer B.













TABLE 10







Vesicle






type
Vesicle V
Polymer (in DMSO)
Polymer V






















CL
20 μL
Diamine_3k, 2 g/L
20
μL





Diamine_5k, 2 g/L





Diamine_10k, 2 g/L





Diamine_50k, 2 g/L



PE/PG
10 μL
Diamine_3k, 2 g/L
10
μL





Diamine_5k, 2 g/L





Diamine_10k, 2 g/L





Diamine_50k, 2 g/L



CL
20 μL
Diamine_3k, 4 g/L
40
μL






20
μL






10
μL






5
μL






2.5
μL






1
μL





Diamine_3k, 0.25 g/L
4
μL






1
μL



PE/PG
10 μL
Diamine_3k, 4 g/L
40
μL





Diamine_3k, 0.25 g/L
20
μL






10
μL






5
μL






2.5
μL






1
μL






4
μL






1
μL






0.5
μL




















TABLE 11







Vesicle
Polymer
Pepti-
Incubation












Type
V
c (in DMSO)
V
doglycan
time
















CL
10 μL
Diamine_3k, 4 g/L
10 μL
400
μg
24 h






200
μg






100
μg






40
μg






20
μg






10
μg






5
μg






2.5
μg






0.5
μg


CL
20 μL
Diamine_3k, 2 g/L
20 μL
100
μg
24 h




Diamine_5k, 2 g/L




Diamine_10k, 2 g/L




Diamine_50k, 2 g/L











Diamine_3k, 4 g/L
—
















—
 0 μL
100
μg



CL
20 μL
Diamine_3k, 2 g/L
 6 μL
25
μg
10 min




Diamine_50k, 2 g/L




Diamine_3k, 2 g/L



24 h




Diamine_50k, 2 g/L









c. Manipulating Antimicrobial Properties by Organic Counterion and Charge Density Variation

There have not been much literature on the effect of different counterions on the antimicrobial properties of a polymer has been essentially neglected in the SMAMP literature. (Ilker, et al., Journal of the American Chemical Society 2004, 126, 15870; Lienkamp, et al., J Am Chem Soc 2008, 130, 9836; Gabriel, et al., Chemistry—A European Journal 2009, 15, 433.) The few studies published so far investigate the effect of different inorganic counterions on polymer properties. Kanazawa et al. postulated an inverse correlation between the tightness of the ion pair of a polymer-counterion system and its antimicrobial properties (Cl−>BF4−>ClO4−>PF6−). (Kanazawa, et al, Journal of Polymer Science, Part A: Polymer Chemistry 1993, 31, 1441.) Chen et al. saw higher activities for bromide than for chloride counterions with their polycationic dendrimers, while Panarin et al. did not see any significant differences between chloride, bromide, and iodide. (Chen, et al., Biomacromolecules 2000, 1, 473; Panarin, et al., Makromol. Chem. Suppl. 1985, 9, 25.) The ranking of ‘activity’ in these studies was based on relative bacterial survival rates in time-kill studies. However, none of these studies quantified antimicrobial activity in terms of MIC data. This makes it difficult to assess how much the antimicrobial activity is actually affected by the counterion exchange, and makes comparison between these studies even more difficult. While the previous examples focused on inorganic counterions, the effect of different organic counterions such as tosylate or hexanoate, to our knowledge, has not been studied.
Counterions of varying hydrophobicity may provide an easy and versatile tool to tune the hydrophobicity and thus the antimicrobial properties of the polymer. This approach of tuning the hydrophobicity requires much less synthetic effort than the covalent attachment of hydrophobic groups, both on the monomer and the polymer synthesis level. For example, replacing the trifluoroacetate counterions of a hydrophilic model SMAMP by hydrophobic organic counterions can influence the polymer's antimicrobial properties and its membrane activity.
The effect of molecular charge density may also be employed on the antimicrobial properties such as hydrophobicity. A series of polymers were made with gradually varied cationic charge, but with minimal changes to the overall polymer structure and hydrophobicity. A copolymer with a selectivity as high as 650 for Staphylococcus aureus over mammalian cells was discovered.
Examples
1. Diamine Homopolymer Synthesis. A predominantly hydrophilic model polymer with two charges per repeat unit was synthesized (Scheme 4). Base-catalyzed ring-opening of the anhydride 1 with a Boc-protected amino ethanol gave the monoester 2. DCC coupling of 2 with a second equivalent of the alcohol yielded the diamine monomer 3, which was polymerized by ring-opening metathesis polymerization (ROMP) using Grubbs' third generation catalyst. (Jennifer, et al., Angewandte Chemie International Edition 2002, 41, 4035.) The obtained Boc-protected precursor polymer 4 was deprotected with trifluoroacetic acid (TFA) to yield the cationic SMAMP 5 (Scheme 4). To determine the optimum activity of polymer 5, a range of molecular weights was synthesized. The protected polymer 4 was characterized by gel permeation chromatography (GPC). All polymers had narrow polydispersities (1.07-1.15, Table 12); however polystyrene-calibrated GPC in DMF severely overestimated their molecular weights. This was confirmed by MALDI-TOF mass spectrometry, which showed that the molecular weights Mn of those polymers were close to the targeted ones. When plotting Mn.Target versus the molecular weights obtained from GPC, a straight line is obtained, indicating that the polymerization was controlled (FIG. 15).




The biological activities, MIC90 and HC50, of the deprotected SMAMPs (5) were tested using standard procedures. (Rennie, et al., Journal of Industrial Microbiology & Biotechnology 2005, 32, 296.) The results are summarized in Table 12, where Diamine—3k refers to a sample of polymer 5 with a molecular weight of 3000 g/mol. The data shows that, while all polymers were inactive against E. coli, the antimicrobial activity against S. aureus was highest for the 3000 g/mol polymer, with an MIC90 of 15 μg/mL. Upon increasing the molecular weight, the antimicrobial activity was lost, while the hemolytic activity HC50 was about the same for all molecular weights.







TABLE 12







Characterization of SMAMP homopolymers.















GPC







Mn. Targeta)
Mna) g

MIC90 μg mL−1 b)
HC50b)
Selectivityb)















Sample
g mol−1
mol−1
Mw/Mna)
E. coli
S. aureus
μg mL−1
E. coli
S. aureus


















Diamine_3k
3000
8900
1.15
>200
15
1000
<5
67


Diamine_5k
5000
12900
1.08
>200
25
1000
<5
40


Diamine_10k
10000
22500
1.08
>200
50
1000
<5
20


Diamine_50k
50000
77000
1.07
>200
200
2000
<10
10





a)GPC analysis was performed on the protected polymers 4 (in DMF, 0.01 M LiCl, calibrated with polystyrene standards):


b)Biological properties (minimum inhibitory concentration, hemolytic activity and selectivity) were determined for the corresponding deprotected polymers 5, with TFA counterions.






Effect of the Counterions. As it was found that Diamine—3k was the most active polymer of the series, the ion exchange studies were performed with this sample. As a result of the deprotection conditions, the initial counterion of the polymer was TFA. By passing the polymer solution through an ion exchange column, TFA was exchanged for hydroxide. Immediately after the ion exchange, excess of the acid form of the target counterion (HX) was added. This neutralized the hydroxide before side reactions such as transamidation or base-catalyzed ester hydrolysis of the side chains could occur, and left X− as a counterion. Excess acid was removed by dialysis. Using this method, the hydrophobic organic counterions X−=hexanoate, dodecanoate, benzoate and tosylate were introduced. 1H-NMR spectra of the freeze-dried polymers revealed that the counterion exchange was successful. These hydrophobic organic counterions were chosen because their distribution coefficient would be high. (In this context, the distribution coefficient is defined as the counterion concentration near the non-polar polymer backbone divided by the counterion concentration in the bulk polar buffer medium of the MIC experiment.) Thus, unlike more hydrophilic counterions such as TFA, they would not diffuse away from the polymer backbone and the effect of the counterion on the antimicrobial properties would be observed in the MIC experiment. The MIC90 results for the series of ion exchanged polymers are summarized in Table 13. To investigate the effect of a covalently attached ‘intramolecular counterion’, which certainly would not be able to diffuse away from the polymer backbone, the monoester 2 was also polymerized, yielding polymer 6. After deprotection with TFA, the zwitterionic polymer 7 was obtained (Scheme 5). That MIC data is also included in Table 13. Here, the 3000 g/mol sample of polymer 7 will be referred to by Zwitterion—3k for convenience.











TABLE 13







Minimum inhibitory concentration of the zwitterionic and ion-exchanged


polymers, determined for the deprotected polymers; ion exchanged


polymers are referred to by their counterion, e.g. Tosylate_3k


is the 3000 g/mol diamine polymer with a tosylate counterion.


Diamine_3k has the original TFA counterion.










MIC90 μg mL−1












Sample
S. aureus
E. coli















Diamine_3k
15
>200



Tosylate_3k
>200
>200



Benzoate_3k
>200
>200



Hexanoate_3k
>200
>200



Dodecanoate_3k
>200
>200



Zwitterion_3k
50
>200



Zwitterion_10k
>200
>200










Table 13 shows that the presence of the organic counterions influences the antimicrobial activity of the polymers: All polymers with hydrophobic counterions stayed inactive against E. coli and were significantly less active against S. aureus than Diamine—3k, although no discrimination between their relative activities within the range below 200 μg/mL could be made. Not only does this data confirm that the hydrophobic counterions stay close to the polymer backbone, but in fact they bind so strongly that they eliminate the antimicrobial activity.
There are two possible interpretations for this. First, the hydrophobic counterions could reduce the polymer solubility to such an extent that the SMAMP is unable to interact with the bacteria membrane and thus appears inactive in the MIC experiment. Second, the counterions could form such a tight ion pair with the ammonium groups that the overall positive charge of the polymer is effectively masked and the SMAMP is rendered inactive. One way to differentiate between these two possibilities is to perform dye-leakage experiments. (Abhigyan, et al., J Phys Chem B 2008, 112, 3495.) These experiments are designed to probe the membrane activity of a polymer. A polymer that is poorly soluble will still be active in dye-leakage experiments, because these studies are much more sensitive. An example of a polymer that is poorly soluble and therefore inactive in the MIC experiment, but retains significant membrane activity in the dye leakage experiment, is the previously reported Hexyl—3k. (Lienkamp, et al., J Am Chem Soc 2008, 130, 9836.) However, if the counterions of the ion exchanged polymers mask the positive charge of the polymer backbone, these polymers will be inactive both in the MIC and the dye-leakage experiments. Using standard procedures, dye-loaded unilamellar vesicles made from cardiolipin, the predominant component of the plasma membrane of S. aureus bacteria, were obtained. Leakage of the self-quenching dye from the vesicle led to fluorescence, which was monitored as a function of time. (Som, et al, J Phys Chem B 2008, 112, 3495.) The vesicles were exposed to a 20 μg/mL solution of the ion-exchanged polymers at t=100 sec. (The polymer concentration in the dye leakage experiments is 20 μg/mL. This makes the ratio of polymer to vesicles much higher than the ratio of polymer to bacteria in the MIC experiment. Although this polymer concentration is lower than in the MIC experiments, the much higher ratio of polymer to vesicle concentration would be expected to show leakage if the molecules are membrane disruptive. (Som, et al., Biopolymers (Peptide Science) 2008, 90, 83.) The resulting dye-leakage curves are plotted in FIG. 16 as % Leakage, normalized to the standard Triton X-100, versus time. The dye-leakage results from the S. aureus-mimicking vesicles closely follow the MIC trend against those bacteria: the polymers with hydrophobic counterions were membrane-inactive (<5% leakage), whereas the parent polymer Diamine—3k showed membrane activity (>50% leakage).
As illustrated in FIG. 17, the Hexanoate—3k and Hexyl—3k polymers are structurally similar, as each contains one hexyl chain per amine group, however Hexyl—3k (MIC90>200 μg/mL) causes 38% leakage at 20 μg/mL, whereas Hexanoate—3k causes <5% leakage. This supports the second interpretation, that the hydrophobic counterions form tight ion pairs and thereby dramatically reduce the positive charge of the polymer, resulting in a loss of membrane activity and, consequently, of the antimicrobial activity.
Interestingly, the zwitterionic sample (Zwitterion—3k), with an MIC of 50 μg/mL, was more membrane active than the ion exchanged samples (FIG. 16), but less active than the Diamine—3k polymer. Whereas the other anions could diffuse freely to the positive charges on the polymer backbone, this is not possible for the build-in counterions of the zwitterionic sample. However, because the zwitterionic polymers are structurally irregular, this leads to an overall irregular charge distribution along the backbone, leading to local cationic and anionic patches, as illustrated in FIG. 18. Due to these residual charges, neither the antimicrobial activity nor membrane activity of Zwitterion—3k vanishes completely.
2. Charge Variation by Copolymerization. The charge per repeat unit was diluted gradually from two to one by copolymerizing the diamine monomer 3 with structurally similar monoamine-alkyl monomers (FIG. 19). (Lienkamp, et al., J Am Chem Soc. 2008, 130, 9836.)
Four series of copolymers were synthesized with R varying from Methyl to Butyl on the monoamine-alkyl monomer. The polymer characterization data of the protected copolymers, as well as the biological data of the deprotected copolymers, is summarized in Table 14 and FIG. 20. The data for certain homopolymers is included for comparison. (Lienkamp, et al., J Am Chem Soc. 2008, 130, 9836.) M9:D1 refers to a copolymer with a molar ratio of methyl:diamine=9:1, while Methyl designates the monoamine-methyl homopolymer.







TABLE 14







Copolymer Characterization.















Co-
Mn, Targeta)
GPC Mna)

MIC90 μg mL−1 b)
HC50b)
Selectivityb)
















Sample
monomer
g mol−1
g mol−1
Mw/Mna)
E. coli
S. aureus
μg mL−1
E. coli
S. aureus



















Diamine_3k
—
3000
8900
1.15
>200
15
1000
5
66


M1:D9
Methyl
3500
17355
1.03
>200
12.5
1400
<7
112


M1:D1

4100
15400
1.07
>200
6.25
1400
<7
224


M9:D1

4600
17400
1.07
>200
4
2600
<13
650


Methyl
—
3000
11200
1.08
200
100
2000
<10
20


E1:D9
Ethyl
4600
13100
1.06
>200
50
3100
<15.5
62.0


E1:D1

4100
14700
1.08
>200
15
1400
<7.0
93.3


Ethyl
—
3000
9200
1.10
50
50
1400
28
28


P1:D9
Propyl
4600
13700
1.06
>200
15
4800
<24.0
320.0


P1:D1

4200
13900
1.05
>200
50
1400
<7.0
28.0


Propyl
—
3000
9200
1.10
6.25
25
50
8.3
3.3


B1:D9
Butyl
4600
14200
1.06
>200
15
1200
<6.0
80.0


B1:D1

4300
14700
1.07
<15
25
<50
>3.3
<2.0


Butyl
—
3000
11500
1.08
15
25
<50
<3.3
2.0





a)GPC analysis was performed on the protected polymers (DMF, 0.01 M LiCl, polystyrene standards);


b)Biological properties (minimum inhibitory concentration, hemolysis and selectivity) were determined for the corresponding deprotected polymers.






As this data indicates, all the methyl and ethyl copolymers are non-hemolytic (FIGS. 20a and 20b) and inactive against E. coli bacteria, whereas the propyl and butyl copolymers become very hemolytic but at the same time more active against E. coli with increasing alkyl comonomer content (FIGS. 20c and 20d). This suggests that the properties of these polymers are dominated by the hydrophobicity of those R groups. Meanwhile, the methyl copolymers are obviously the least hydrophobic as observed by their limited activity against E. coli even for the methyl homopolymer, and their high HC50 values. This lack of activity due to limited hydrophobicity is consistent with reverse phase thin layer chromatography results, and with the polymer solubilities in water and DMF, which showed that the polarity of the monoamine-methyl homopolymer closely resembled that of the diamine homopolymer, while the ethyl to butyl derivatives were significantly more hydrophobic.
These studies confirm that the methyl copolymers are the most appropriate model system to investigate the effect of charge density on biological activity.
Starting with Diamine—3k, which has an HC50 of 1000 μg/mL, the hemolytic activity decreases across the series to M9:D1 with an HC50 of 2600 μg/mL. This was paralleled by an increase in the antimicrobial activity from 15 to 4 μg/mL against S. aureus for the same polymers (see Table 14). When going further down in charge (M9:D1 to Methyl), there is a sudden jump in the MIC from 4 to 100 μg/mL, indicating that the optimum value for both the HC50 and the MIC90 against S. aureus is obtained for M9:D1.
An intriguing aside of this study is the finding that the methyl copolymers and Diamine—3k are not only selective for bacterial over mammalian cells, but also for Staphylococcus aureus over Escherichia coli (FIG. 20a). Further MIC experiments with M9:D1 and other bacteria revealed that this polymer has a Gram-selectivity: while inactive against Gram-negative E. coli, Pseudomonas aeruginosa and Klebsiella pneumoniae (MIC90=100 μg/mL for the latter two organisms), it was found to be active against Gram-positive. S. aureus, S. epidermis and even the multiresistant MRSA (MIC90=12.5 μg/mL).
Disclosed here are several important parameters that influence the antimicrobial and hemolytic activity of SMAMPs were investigated. For example, exchanging the hydrophilic counterions of a ROMP-derived diamine polymer by hydrophobic organic counterions drastically reduced the antimicrobial properties of that polymer. It was shown by dye-leakage studies on model vesicles that this loss in antimicrobial activity was due to the loss of the positive charge of the polymer backbone by ion pair formation with the counterions. It is expected that by a partial exchange of the counterions, which would add hydrophobicity but at the same time retain the required minimum molecular charge, polymers with tunable properties—between those of the active Diamine—3k polymer and the inactive Hexanoate—3k polymer—could be obtained: this is however difficult to realize and quantify experimentally.
Copolymerization of two structurally similar monomers with one and two charges, respectively, led to a series of polymers with gradually decreased charge density but more or less constant hydrophobicity. These results indicate that, for a system of a given amphiphilicity, a minimum threshold of charge is required for obtaining favorable HC50 values and activities. Above that threshold, the biological properties of the polymer are not affected by further charge increase. For the particular diamine-methyl copolymer system investigated here, this threshold is reached for M9:D1, with an average charge of 1.1 per repeat unit. In the process of optimizing the charge density of the copolymer system, a polymer with an impressive selectivity of 650 for S. aureus over human red blood cells was obtained. Additionally, this polymer was shown to be Gram-selective for all bacterial strains tested, including MRSA, which makes this SMAMP a promising candidate for materials applications.
Examples
General: All chemicals were obtained as reagent grade from Aldrich, Fluka or Acros and used as received. 3rd generation Grubbs catalyst (Dichloro-di(3-bromopyridino)-N,N′-Dimesitylenoimidazolino-Ru═CHPh; G3) was synthesized as described previously by Grubbs et al. (Love, et al, Angew. Chem. Internat. Ed. 2002, 41, 4035.) HPLC grade solvents were purchased from Aldrich, Fisher Scientific or Acros and used as received. THF (HPLC grade, Fisher Scientific) was distilled from sodium/benzophenone under nitrogen. Dichloromethane (HPLC grade, Fisher Scientific) was distilled from CaH2 under nitrogen. Gel permeation chromatography (DMF/0.01 M LiCl and THF, each calibrated with polystyrene standards, toluene as flow marker, 50° C.) was measured on a PL50 GPC setup (Polymer Laboratories, Amherst, Mass.) with a PL Gel 5 μm pre-column and two 10 μm analytical Mixed-D columns (Polymer Laboratories, Amherst, Mass.). NMR spectra were recorded on a Bruker DPX300 spectrometer (Bruker, Madison, Wis.). High resolution mass spectra were obtained from a JEOL JMS 700 instrument (JEOL, Peabody, Mass.); Matrix Assisted Laser Desorption and Ionization Time of Flight Mass Spectra (MALDI-TOF MS) were measured on a Bruker Daltonics Reflex III (Bruker, Madison, Wis.). The biological activity of the polymer samples (the minimal concentration inhibiting 90% of bacterial growth (MIC90) for Escherischia coli (D31) and Staphylococcus aureus (ATCC25923), and the concentration that lyses 50% of red blood cells (HC50) were determined as reported previously. (Rennie, et al., Journal of Industrial Microbiology & Biotechnology 2005, 32, 296.) Amberlite IRA67 anion exchange resin (Aldrich) was used for counterion exchange. Dialysis was performed using Cellulose Ester dialysis tubes (SpectraPor6, SpectrumLabs) with a molecular weight cut-off of 1000 Da. Fluorescence measurements were performed on a Jobin Yvon Fluorolog-3. The phospholipid 1,1′-2,2′-tetraoleoyl cardiolipin sodium salt (CL), was obtained as chloroform solutions from Avanti Polar Lipids, Inc. and used as received.




Monomers: The monomer 2 was obtained from exo-7-oxabicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid anhydride 1 (5 g, 30.0 mmol), which was dissolved in CH2Cl2. 1.1 eq of N-(tert-butoxycarbonyl)ethanolamine (5.32 g, 33 mmol) and 10 mol % 4-dimethylaminopyridine (DMAP) were added. After stirring over night, the solution was concentrated. Ether was added to precipitate DMAP salt, and the solution was filtered. This step was repeated until no more DMAP salts precipitated and the pure zwitterion was obtained. The isolated yield was 60-70%.
1H-NMR (300 MHz, CDCl3): δ=1.41 (s, 9H, H9), 2.83 (m, 2H, H3 & H3′), 3.37 (m, 2H, H6), 4.18 (m, 2H, H5), 5.24 & 5.32 (s, 2H, H2 & H2′), 6.46 (m, 2H, H1 & H1′), 7.5-8.2 (br s, 1H, OH). HR-MS (FAB): calc. 299.31 g/mol. found 272.1 g/mol (M-t-Butyl).
The diamine monomer 3 was obtained in a one pot-synthesis from exo-7-oxabicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid anhydride 1 without isolating the intermediate 2:1 (5 g, 30.0 mmol) was dissolved in CH2Cl2. 1.1 eq of N-(tert-butoxycarbonyl)ethanolamine (5.32 g, 33 mmol) and 10 mol % 4-dimethylaminopyridine (DMAP) were added. After stirring over night at r.t., the solution was cooled to 0° C. 1.1 eq N-(tert-butoxycarbonyl)ethanolamine (5.32 g, 33 mmol) and 1.0 eq (6.19 g, 30 mmol) DCC(N,N′-dicyclohexylcarbodiimide) were added, and the mixture was stirred over night. The precipitate was filtered through a short alumina column (5 cm neutral Al2O3/dichlormethane) and a clear solution was obtained. The solvent was removed by vacuum evaporation and the crude product was chromatographed (15 cm silica gel, hexane:ethyl acetate gradient, 9:1 to 1:1). Evaporation of the solvent yielded the pure monomer. The isolated yield was 70 to 80%.




1H-NMR (300 MHz, CDCl3): δ=1.41 (s, 18H, H9), 2.81 (s, 2H, H3), 3.36 (m, 4H, H6), 4.17 (m, 4H, H5), 5.25 (s, 2H, H2), 6.44 (s, 2H, H1). 13C-NMR (75 MHz, CDCl3): δ=28.39 (C9), 39.44 (C6), 47.03 (C3), 64.79 (C5), 80.55 (C2), 136.66 (C1). HR-MS (FAB): calc. 470.52 g/mol. found 471.23 g/mol.
Monoamine-Alkylmonomers: Monomers for the copolymers M, E, P and B (R=Methyl, Ethyl, Propyl and Butyl) were synthesized and characterized. (Lienkamp, et al., J. Am. Chem. Soc. 2008, 130, 9836.)
Homopolymerization: The monomers (2, 3 or 9) and the respective amount of G3-catalyst (see Table 15 for details) were dissolved in 1 mL dichloromethane each and subject to three freeze-thaw cycles. The catalyst was added in one shot to the vigorously stirring monomer solution at room temperature under N2. After 30 min, the living polymer chain was end-capped with an excess of ethylvinyl ether (1 mL, 754 mg, 10.5 mmol). The solution was allowed to stir for 2 hours. After evaporation of the solvent and drying, an aliquot of each polymer was taken for GPC and NMR analysis. The product was a brown solid. GPC was performed in DMF (0.01 M LiCl, polystyrene standards) for polymers 4 and 10, and in THF (polystyrene standards) for polymers 6. NMR and MALDI-TOF data is given below.




4: 1H-NMR (300 MHz, CDCl3): 1.42 (s, 9H, H9), 3.15 (br m, 2H, H3), 3.36 (br m, 2H, H6), 4.16 (br m, 2H, H5), 4.72 (m, 1H, H2 trans), 5.10 (br s, 1H, H2 cis), 5.42 (br s, 1H, NH), 5.60 (br m, 1H, H1 cis) and 5.89 (br m, 1H, H1 trans). MALDI-TOF MS (for Diamine—3k): Mn=3409 g/mol, Mw/Mn=1.07, Mrepeat unit=469.6 g/mol.




6: 1H-NMR (300 MHz, THF-d8): 1.40 (s, 9H, H9), 3.10 (br m, 2H, H3 & H3′), 3.28 (br m, 2H, H6), 4.07 (br m, 2H, H5), 4.71 (m, 1H, H2 trans), 5.09 (br s, 1H, H2 cis), 5.59 (br s, 1H, NH), 5.91 (br m, 1H, H1 cis) and 6.12 (br m, 1H, H1 trans). MALDI-TOF MS: n.a.







TABLE 15







Experimental parameters for the homopolymer synthesis.



















Mn Target g
MMonomer
nMonomer
mMonomer
MCatalyst
nCatalyst
mCatalyst


Sample
Monomer
Nrepeat units
mol−1
g mol−1
mmol
mg
g mol−1
mmol
mg



















Diamine_3k
3
6.4
3000
470.5
0.425
200
885
0.067
59.0


Diamine_5k

11
5000




0.040
35.4


Diamine_10k

22
10000




0.020
17.7


Diamine_50k

106
50000




0.004
3.5


Diamine_100k

213
100000




0.002
1.8


Zwitterion_3k
2
10.0
3000
299.3
0.668
200

0.067
59.0


Zwitterion_10k

33.4
10000




0.020
17.7









Homopolymer deprotetction: The crude polymers 4 and 6 respectively, were dissolved in 2 mL dichloromethane. An excess of TFA (2 mL, 2.97 g, 26.0 mmol) was added and the solution was stirred at room temperature over night. The excess acid was removed by azeotropic distillation with dichloromethane (2×15 mL) and methanol (1×15 mL) at the rotary evaporator. The samples were dried in vacuo over night and dissoved in 30 mL Milli-Q water or DMSO depending on solubility. They were dialyzed against Milli-Q water until the conductivity of the water was 0.1 μS after 12 h of dialysis (total dialysis time 4-7 days). The hydrolyzed polymers (5 and 7) were then freeze dried




5: 1H-NMR (300 MHz, DMSO-d6): 3.01 (br m, 2H, H3), 3.47 (br m, 4H, H6), 4.11 (br m, 4H, H5), 4.44 (br m, 1H, H2 trans), 4.99 (br m, 1H, H2 cis), 5.66 (br m, 1H, H1 cis), 5.84 (br m, 1H, H1 trans).




7: 1H-NMR (300 MHz, DMSO-d6+D2O): 3.02 (br m, 2H, H3 & H3′), 3.21 (br m, 2H, H6), 3.70 (br m, 2H, H5), 4.28 (m, 1H, H2 trans), 4.42 (br s, 1H, H2 cis), 5.60 (br m, 1H, H1 cis) and 5.83 (br m, 1H, H1 trans).
Copolymerization: Copolymers were synthesized using the same methods as for the homopolymers. Specific reagent amounts are given in Table 16. Each comonomer was dissolved in 0.7 mL dichloromethane separately under argon. The monomer solutions were mixed, and the respective amount of G3 catalyst in 1 mL dichloromethane was added in one shot. The 1H-NMR spectroscopic data for each repeat unit proton matched the assignment of the analogous protons in the homopolymers. (Lienkamp, et al, J. Am. Chem. Soc. 2008, 130, 9836.) Where possible (no peak overlap), integration revealed that the copolymer composition matched the monomer feed ratio. For simplicity, the 1H-NMR peak positions are given for each repeat unit separately.




R=Amine: see above (Polymer 5)
R=Methyl: 1H-NMR (300 MHz, CDCl3): 1.43 (s, 9H, H9), 3.12 (br m, 2H, H3 & H3′), 3.34 (br m, 2H, H6), 3.71 (s, 3H, CH3), 4.16 (br m, 2H, H5), 4.71 (m, 1H, H2 & H2′ trans), 5.10 (br s, 1H, H2 & H2′ cis), 5.40 (br s, 1H, NH), 5.58 (br m, 1H, H1 & H1′ cis) and 5.88 (br m, 1H, H1 & H1′ trans).
R=Ethyl: 1H-NMR (300 MHz, CDCl3): 1.24 (s, 3H, CH2—CH3), 1.42 (s, 9H, H9), 3.09 (br m, 2H, H3 & H3′), 3.34 (br m, 2H, H6), 4.16 (br m, 4H, CH2—CH3 and H5), 4.72 (br m, 1H, H2 & H2′ trans), 5.10 (br m, 1H, H2 & H2′ cis), 5.30 (br s, 1H, NH), 5.58 (br m, 1H, H1 & H1′ cis), 5.88 (br m, 1H, H1 & H1′ trans).
R=Propyl: 1H-NMR (300 MHz, CDCl3): 0.92 (m, 3H, CH2—CH3), 1.43 (s, 9H, H9), 1.62 (m, 2H, β-CH2), 3.12 (br m, 2H, H3 & H3′), 3.34 (br m, 2H, H6), 4.10 (m, 4H, α-CH2 and H5), 4.69 (br m, 1H, H2 & H2′ trans), 5.12 (br m, 1H, H2 cis & H2′), 5.31 (br m, 1H, H1 & H1′ cis), 5.59 (br s, 1H, NH), 5.88 (br m, 1H, H1 & H1′ trans).
R=Butyl: 1H-NMR (300 MHz, CDCl3): 0.87 (m, 3H, CH2—CH3), 1.29 (m, 2H, γ—CH2), 1.43 (s, 9H, H9), 1.59 (m, 2H, β-CH2), 3.11 (br m, 2H, H3 & H3′), 3.37 (br m, 2H, H6), 4.10 (m, 4H, α-CH2 and H5), 4.73 (br m, 1H, H2 & H2′ trans), 5.11 (br m, 1H, H2 & H2′ cis), 5.35 (br s, 1H, NH), 5.59 (br m, 1H, H1 & H1′ cis), 5.88 (br m, 1H, H1 & H1′ trans).







TABLE 16







Experimental parameters for copolymer synthesis



















Mn Target
nDiamine
mDiamine
nComomomer
mComonomer
nCatalyst
mCatalyst


Sample
Comonomer
Nrepeat units
g mol−1
mmol
mg
mmol
mg
mmol
mg



















M1:D9
Methyl
10
4600
0.81
381
0.09
30.7
0.09
79.7


M1:D1


4100
0.45
212
0.45
154


M9:D1


3500
0.09
42.3
0.81
277


E1:D9
Ethyl

4600
0.81
381
0.09
32.0


E1:D1


4100
0.45
212
0.45
160


P1:D9
Propyl

4600
0.81
381
0.09
33.2


P1:D1


4200
0.45
212
0.45
166


B1:D9
Butyl

4600
0.81
381
0.09
34.5


B1:D1


4300
0.45
212
0.45
173









Copolymer deprotection: The copolymers were deprotected using the same methods as for the homopolymers, and characterized by 1H-NMR. Again, the NMR peaks are given for each repeat unit separately.




R=Diamine: see above (Polymer 6).
R=Methyl: 1H-NMR (300 MHz, DMSO-d6): 3.04 (br m, 2H, H3 & H3′), 3.30 (br m, 2H, H6), 3.59 (s, 3H, CH3), 4.16 (br m, 2H, H5), 4.57 (m, 1H, H2 & H2′ trans), 4.91 (br m, 1H, 1H, H2 & H2′ cis), 5.61 (br m, 1H, H1 & H1′ cis), 5.82 (br m, 1H, H1 & H1′ trans).
R=Ethyl: 1H-NMR (300 MHz, D2O): 1.28 (br s, 3H, CH2—CH3), 3.12 (br m, 2H, H3 & H3′), 3.32 (br m, 2H, H6), 3.49-3.81 (br m, 4H, CH2—CH3 and H5), 5.10-5.40 (br m, 2H, H2 & H2′), 5.30-5.90 (br m, 2H, H1 & H1′).
R=Propyl: 1H-NMR (300 MHz, D2O): 0.94 (br m, 3H, CH2—CH3), 1.67 (br m, 2H, β-CH2), 3.31 (br m, 2H, H3 & H3′), 3.50 (br m, 2H, H6), 4.12 (m, 4H, α-CH2 and H5), 4.39 (br m, 1H, H2 & H2′ trans), 5.12 (br m, 1H, H2 & H2′ cis), 5.31 (br m, 1H, H1 & H1′ cis), 5.88 (br m, 1H, H1 & H1′ trans).
R=Butyl: 1H-NMR (300 MHz, D2O): 0.93 (m, 3H, CH2—CH3), 1.36 (m, 2H, γ-CH2), 1.61 (m, 2H, β-CH2), 3.10 (br m, 2H, H3 & H3′), 3.48 (br m, 2H, H6), 4.14 (m, 4H, α-CH2 and H5), 4.38 (br m, 1H, H2 & H2′ trans), 5.11 (br m, 1H, H2 & H2′ cis), 5.35-6.20 (br m, 2H, H1 & H1′).
Ion Exchange: A 20 cm glass column was filled with Amberlite IRA resin and washed with 500 mL distilled water before use. The Diamine—3k polymer was dissolved in water (c=1 g/L), slowly passed through the column (1 drop/min), and washed down with 200 mL distilled water. Immediately after recovery, the solution containing the polymer with OH− counter ions was split into 4 parts. A ten fold excess of a DMSO solution of the respective counterion in its acid form (toluenesulfonic acid, benzoic acid, hexanoic acid and dodecanoic acid) was added. The mixture was dialyzed against 10 L distilled water until the conductivity of dialysis water was equal to that of fresh distilled water (0.1 μS). The polymer solution was then freeze dried. GPC data of the parent polymers of the ion-exchanged samples, together with their antimicrobial activities, is shown in







TABLE 17







1H-NMR revealed that ion exchange was successful


(additional aromatic signals for tosylate and benzoate, and


aliphatic signals for hexanoate and dodecanoate, respectively.












Mn. Targeta)
GPC Mna)

MIC90 μg mL−1 b)












Sample
g mol−1
g mol−1
Mn/Mna)
S. aureus
E. coli















Diamine_3kc)
3000
8900
1.15
15
>200


Tosylate_3k



>200
>200


Benzoate_3k



>200
>200


Hexanoate_3k



>200
>200


Dodecanoate_3k



>200
>200


Zwitterion_3k
3000
3100
1.21
50
>200


Zwitterion_10k
10000
10700
1.16
>200
>200





Table 17.


a)GPC data for the protected parent polymers,


b)MIC data for the hydrolyzed, ion-exchanged polymers.






Vesicle Preparation: Two buffers were prepared. Buffer A consisted of 1 mmol (142 mg) Na2HPO4 in 100 mL H2O (c=10 mmol L−1), which was then adjusted to pH=7.0 with 0.1 mol L−1 NaOH. Buffer B was made from 10 mmol (1.42 g) Na2HPO4 in 1 L H2O (c=10 mmol L−1) and 90 mmol (5.26 g) NaCl (c=90 mmol L−1). Calcein solution was prepared from 0.4 mmol (249 mg) of calcein dye in 10 mL buffer A. 0.1 mol L−1 NaOH was added dropwise until all calcein dissolved and the pH was 7.0, yielding a solution of 40 mmol L−1.
The CL lipid solution was added to thoroughly clean round bottom flask: 600 μL (10 mmol) CL in chloroform was used. The solvent was evaporated to obtain a lipid film, which was hydrated with 1 mL of calcein solution. The suspension was stirred for 1 h and then subject to five freeze-thaw cycles (liquid nitrogen/water at room temperature). After that it was extruded ten times through a polycarbonate membrane with 400 nm pore diameter (Avanti Polar Lipids Mini-Extruder, Whatman membrane). The dye-filled vesicles were separated from the excess dye using an Sephadex G-50 and buffer B as an eluent. The vesicle fraction from the column was diluted with buffer B by a factor of 20 for the dye-leakage experiments.
Dye-Leakage Experiments: The polymer-induced leakage was monitored by recording the increase of calcein fluorescence intensity at a wavelength 515 nm (excitation wavelength=490 nm, slit width 3.0). (Eren, et al., Macromol. Chem. Phys. 2008, 209, 516.) To normalize the dye-leakage data, a base line of calcein fluorescence without polymer addition was observed for each sample. Also, after each measurement, 20 μL Triton X-100 (20% in DMSO) was added to determine the fluorescence intensity corresponding to 100% leakage. With these data points, the measured fluorescence intensity was converted into % leakage. The polymer samples were dissolved in DMSO at 2 g/L concentrations. Pure DMSO, as well as the acid form of the respective counterions, have been tested previously and was shown not to cause leakage. 20 μL of the 1/20 dilution of vesicles was added to 1980 μL buffer B. 20 μL polymer solution was added at t=100 sec, followed by the addition of Triton X at t=800 sec. This procedure is illustrated in FIG. 21.
d. Facially Amphiphilic Versus Segregated Monomers in Antibacterial Copolymers
Altering the amphiphilicity of antimicrobial polymers has been accomplished most commonly through two methods (FIG. 23). First is the copolymerization of “segregated” monomers, relatively non-polar monomers are polymerized with a masked cationic monomer (the cationic group is produced after polymerization) to produce cationic amphiphilic random copolymers. By using structurally different non-polar monomers having a range of hydrophobicities or by adjusting feed ratios, the amphiphilicities of the copolymers can be straightforwardly varied. This strategy is exemplified by DeGrado, Gellman, and Sen, independently, and by us in this report (structures A-D respectively, FIG. 23). (Kuroda, et al. Journal of the American Chemical Society 2005, 127, 4128-4129; Mowery, et al. Journal of the American Chemical Society 2007, 129, 15474-15476; Sambhy, et al. Angewandte Chemie, International Edition 2008, 47, 1250-1254.)
The second method using “facially amphiphilic” (FA) monomers is a strategy pioneered in our laboratories and involves the synthesis of monomers carrying both a masked cationic group and a non-polar group. (Ilker, et al. J. Am. Chem. Soc. 2004, 126, 15870-15875) Therefore every repeat unit of the resulting polymer has a cationic group and a tunable non-polar moiety. The spatial arrangement of polymer E (FIG. 23) or the linker flexibilities of polymer F allows the charged and non-polar moieties to be possibly positioned on opposite sides of the polymer backbone as it conforms to interact with the amphiphilic phospholipid bilayer of bacterial membranes. In effect the polymer itself can become FA. The study of a series of these types of polymers led to the discovery of several very selective polymers in which the compounds preferentially target specific bacteria and not mammalian red blood cells (FIG. 23).
One approach is that the membrane of the bacteria induces a globally amphiphilic conformation of the SMAMP without the benefit of a preorganized FA secondary structure in solution. (Arnt, et al. Journal of Polymer Science Part A: Polymer Chemistry 2004, 42, 3860-3864; Arm, et al. Langmuir 2003, 19, 2404-2408; Arnt, et al. Journal of the American Chemical Society 2002, 124, 7664-7665; Mowery, et al. Journal of the American Chemical Society 2007, 129, 15474-15476.) In these cases it was hypothesized that the orientation of charged and non-polar groups does not necessarily have to conform to a defined secondary structure (helix or β-sheet for example) to exhibit potency. In other words, while AMPs such as human defensin and magainin from amphibians display a well-defined FA solution structure it has been shown that it is not a requirement for the activity of most SMAMPs. Several systems have thus shown that flexible polymers are presumably able to attain a globally FA conformation. (Schmitt, et al. Journal of the American Chemical Society 2007, 129, 417-428; Kuroda, et al. Journal of the American Chemical Society 2005, 127, 4128-4129; Arnt, et al. Journal of Polymer Science Part A: Polymer Chemistry 2004, 42, 3860-3864; Arm, et al. Langmuir 2003, 19, 2404-2408; Arm, et al. Journal of the American Chemical Society 2002, 124, 7664-7665.)
Second is the approach that the specific spatial arrangement of the charge and non-polar groups greatly influences the biological activity thoroughly demonstrated by Sen and coworkers. (Sambhy, et al. Angewandte Chemie, International Edition 2008, 47, 1250-1254.) They probed several series of pyridinium polymers that differ only in the spatial positioning of the positive charges and their non-polar alkyl tails (polymer C, FIG. 23). Some copolymers contained the cationic charge and various alkyl groups on the same pyridinium ring and in other copolymers these groups were positioned on different units. Most notably, their system is in contrast to many others which probe the effect of overall charge (and thus global amphiphilicity) and not the arrangement of charged and non-charged side groups locally along the polymer. (Sambhy, et al. Angewandte Chemie, International Edition 2008, 47, 1250-1254.)
The spatial arrangement is significant in determining a polymer's activity. Within a polynorbornene framework, this provides a direct comparison between polymers composed of FA monomers and polymers in which the charged and non-polar moieties are segregated on different monomers (FIG. 24). Overall, polyamine oxanorbornene (PAON) exhibited little antibacterial and hemolytic activity. New copolymers in which the amine and various alkyl chains reside on different monomers were designed to survey a wide range of amphiphilicities with the hope of attaining potent and selective polymers. This strategy of copolymerizing segregated monomers converted PAON into an active polymer, with some polymers with encouraging activity. No polymer though was found with selectivities superior to those constructed of very comparable FA monomers. Polymer-induced leakage studies using dye-filled liposomes corroborate a membrane-disruptive mechanism for the active copolymers while microscopy of stained cells in the presence of copolymer support membrane compromise, along with cell agglutination, as a key killing event.
Examples
Design and Synthesis. Random copolymers with monomers splitting the charged and non-polar groups (FIG. 25) were synthesized. Instead of installing hydrophobicity early in the synthesis as was done in the previous system using FA monomers, a universal oxanorbornene imide precursor was used and derivatized with different alkyl chains (FIG. 30). One boc-protected amine oxanorbornene monomer and eleven monomers carrying various alkyl chains were synthesized. As a starting point 50/50 mol % random copolymers were targeted at two molecular weights using ring opening metathesis polymerization catalyzed by Grubbs' third generation catalyst. (Love, et al. Angewandte Chemie, International Edition 2002, 41, 4035-4037.) Gel permeation chromatography gave monomodal signals with narrow polydispersity indices (1.04-1.15). With this strategy, copolymers with a broad range of amphiphilicities, but with approximately the same number of amines and thus charges, can be easily obtained just by varying the feed ratio of comonomers.
Biological Assays. MIC assays were used to evaluate the antibacterial properties of these polymers against Gram-negative Ec and Gram-positive Sa (Table 18). PAON was found to be inactive (MIC=400 μg/mL) against both bacteria while the MIC values of the low molecular weight copolymers against Ec showed a clear trend with the polymers of intermediate hydrophobicity (A4, A4′, A5, and A5′) having the most potent activities (MIC=50 μg/mL) (Table 18 and FIG. 26). The most hydrophilic copolymer A1 and the most hydrophobic copolymer A12 exhibited activities identical to PAON. A similar trend was seen against Sa although these copolymers performed slightly better against Gram-positive bacteria in general. Higher MW copolymers were, for the most part, fairly inactive compared to their lower MW analogues. In comparison, the previous polynorbornenes constructed of FA monomers identified a polymer with an MIC as low as 3.8 μg/mL.







TABLE 18







Antibacterial and Hemolytic Activities.




















PAO
A1
A2
A3
A4
A4′
A5
A5′
A6
A6′
A9
A12













Low MW polymersa



















MIC (Ec)
400
400
200
75
50
50
50
50
100
100
200
400


MIC (Sa)
400
100
50
25
50
50
75
50
100
50
125
100


HC
2000
200
250
500
<50
<50
<50
<50
<50
<50
<50
<50


Selectivity
—
—
1.3
6.7
<
<
<
<
<05
<05
<
<


Selectivity
—
20.0
5.0
20.
<
<
<
<
<
<
<
<









High MW polymers



















MIC (Ec)
>400
>
200
50
100
200
400
400
>
>
>
>


MIC (Sa)
400
200
200
200
100
200
200
400
200
400
200
400


HC
2000
250
250
50
<50
<50
<50
<50
<50
<50
<50
<50


Selectivity
—
—
1.3
1.0
<
<
—
—
—
—
—
—


Selectivity
—
1.3
1.3
0.3
<
<
<
—
<
—
<
—





aMIC and HC values are reported in μg/mL.


Ec = Escherichia coli,


Sa = Staphylococcus aureus,


Selectivity = HC/MIC.


Selectivity values are not given for inactive polymers (MIC ≧ 400 μg/mL).






Incorporating more hydrophobic groups onto the polyamine increased the copolymer's ability to lyse human red blood cells. A high hemolytic concentration (HC) is one measure of non-toxicity against mammalian cells and is important for polymers that may be used in the body as oppose to those developed for self-sterilizing surfaces and fixture. For the low MW copolymers, undesirable hemolytic activities (HC≦50 μg/mL) were measured for A4 and copolymers that were more hydrophobic than A4. The higher MW copolymers displayed undesirable hemolytic activities starting even sooner at A3. Finally, selectivity values (HC/MIC) greater than 10 (magainin has a selectivity of 10 for both Ec and Sa) were only observed for low MW A1 and A3 (Selectivity for Sa=20 for both copolymers). This value is far below that seen for the most selective polymers in the previous studies which were >100 (for the center polymer in FIG. 24) and >533 (for several polymers of structure F in FIG. 23).
The ability to adjust biological activities using the monomers that split the charged and non-polar moieties was encouraging. Feed ratios deviating from 50/50 were also explored. Analogues of low MW A1, which was inactive, and A5′ which was active but not selective were tested (Table 19). The biological activities did not improve dramatically. In particular, incorporating more hydrophobicity to A1 hardly improved the activity much while adding more charge to A5′ did not eliminate the copolymer's high hemolytic activity.







TABLE 19







Data for Non-50/50 Copolymers (Low MW).










A1 copolymers
A5′ copolymers
















A.81.2
A.61.4
A.41.6
A.21.8
A.85′.2
A.65′.4
A.45′.6
A.25′.8



















MIC (Ec)
400
400
250
200
50
25
100
250


MIC (Sa)
200
100
200
250
50
50
100
250


HC
250
2000
2000
500
<50
<50
<50
<50


Selectivity (Ec)
—
—
8.0
2.5
<1.0
<2.0
<0.5
0.6


Selectivity (Sa)
1.3
8.0
10.0
2.0
<1.0
<1.0
<0.5
0.6









The inability to tune the biological properties was surprising. It was thought that adding hydrophobicity to A1 via increasing the ratio of the non-polar moiety should theoretically produce a copolymer nearing a similar amphiphilicity as the more hydrophobic copolymers A4 and A5 both of which are active (Table 18). In other words, if global amphiphilicity is a key determinant of activity then an A1 copolymer with a high enough ratio of non-polar groups (>50%) should approach a hydrophobicity (and therefore activity) of A4 or A5, both of which have a non-polar ratio of 50% and are modestly antibacterial. Similarly, making A5′ more hydrophilic through feed ratio adjustments unpredictably did not decrease its hemolytic activity even though it was expected that a higher amine to non-polar monomer ratio could push the HC value of A5′ closer to that of the relatively less hydrophobic A1 (HC=2000 μg/mL).
These unexpected results for the non-50/50 copolymers, when compared to the 50/50 copolymers and especially when compared to the polymers from FA monomers, support the finding that spatial arrangement of charge and non-polar groups is important and not just the overall amphiphilicity. In fact there may be profound advantageous effects in how a polymer made from FA monomers interacts with the phospholipid bilayer that copolymerization of segregated monomers cannot possibly attain.
It may be possible that due to the imperfect statistical nature of random copolymerization, there may exist runs of polar units and non-polar units, at least for some percentage of the polymer population (FIG. 27 B shows a run of three cationic groups as well as sections of adjacent non-polar units). This arrangement may not be optimum for disrupting the lipid membrane bilayer especially if the polymer loops in and out of the membrane at every repeat unit rather than having close contacts with the membrane as postulated here for the polymers of FA units (FIG. 275 A). With polymers from FA monomers, 100% of the polymer population has every monomer containing the identical ratio of cationic character to hydrophobic character. Because of these results it was not believed that antimicrobial activity and specificity in membrane-active polymers are a matter of overall global charge density. The series of copolymers presented here surveyed a wide range of overall global charge densities. The local charge density and the consistency of that density throughout the polymer may be extremely important.
Membrane studies. Two experiments were used to probe the membrane-disruption activity of these copolymers: vesicle dye leakage and fluorescence microscopy. Polymer-induced leakage studies on dye-filled liposomes composed of Ec lipid extracts were performed and showed that the most active copolymers caused the most dye release (FIG. 28). These Ec vesicles seem to model membranes quite well with the dye-leakage results tracking approximately with the MIC values. Still, they are not perfect mimics of Ec as can be seen when comparing the leakage abilities of PAON and A12. Both PAON and A12 have an MIC of 400 against Ec yet A12 caused much more dye release. Presumably, in the complex interactions between polyelectrolytes and vesicle membranes one has to consider that more hydrophobic polymers can interact better with the long alkyl lipid chains buried in the membrane bilayer. Still A12 does not cause as much leakage as the less hydrophobic but more antibacterial A4 and A5.
Additionally, live Ec bacteria were incubated with a two-component stain and treated with and without polymer (FIG. 29). The stain is made up of a green-emitting dye (SYTO9 from Invitrogen) which has the ability to stain all cells and a red-emitting propidium iodide dye that can only enter cells with compromised membranes. Fluorescence microscopy showed that PAON essentially has the same effect on Ec as the negative control. Antibacterial A4 not only displayed more intense red-emission but also agglutination of the cells possibly due to the aggregation of patches of torn membrane.
Here, using a common copolymerization strategy (copolymerizing a masked amine monomer with different non-polar monomers and at different feed ratios), several antibacterial polyamine oxanorbornenes were identified. Another scheme using designed “facially amphiphilic” monomers, however, gave polymers with better activities and superior selectivities. An optimum hydrophobicity was observed for the most active 50/50 copolymers. Surprisingly, investigation of non-50/50 copolymers did not lead to improved activities or selectivities. This result supports that the balance of hydrophobic/hydrophilic areas at the local monomer level is much more critical to attain highly active and selective polymers rather than just the global amphiphilicity or overall charge density. FA monomers can be used for better control of biological properties.
Synthesis and Characterization
The synthesis of the non-polar oxanorbornene monomers, Monomer 1-12, involved the N-alkylation of a previously reported exo-Oxanorbornene imide (FIG. 30). Typically Oxanorbornene imide (1 eq.) is dissolved in CH3CN (Fisher Scientific) and the appropriate alkyl bromide (1.2 eq., Aldrich) and K2CO3 (2 eq., Fisher Scientific) are added, stirred vigorously, and refluxed for 4-8 h. The heterogeneous mixture is then filtered through a course fritted funnel and the CH3CN is evaporated. The residue is typically an oil which is dissolved in Et2O (Fisher Scientific) and washed with brine four times. The Et2O was dried with Na2SO4 (Fisher Scientific) and evaporated. The solid was then subjected to silica gel chromatography using a CH2Cl2/MeOH gradient which gave pure product in 55-82% yield depending on the monomer. NMR characterization of Monomer 1-12 follows below.
This monomers were copolymerized by ring-opening metathesis polymerization (ROMP) using Grubbs' 3rd generation catalyst, [(H2Imes)(3-Br-py)2-(Cl)2Ru═CHPh]. The polymerization entailed adding to a test tube the appropriate monomers (100 mg total) plus catalyst. The test tube was capped with a septum and purged with N2 for 5 min, then 1 mL dry CH2Cl2 (Acros, packed under N2 and molecular sieves) was injected. The N2 line was removed and the clear, brown solution was stirred at 28° C. for 30 min after which 0.4 mL ethyl vinyl ether was injected to terminate the polymer. After stirring for 15 min the solution was added dropwise to 300 mL of stirring pentane to precipitate the polymer. The pentane solution was stirred an additional 30 min and left standing undisturbed for an hour. The precipitate was then collected by a fine sinter funnel. The polymer was then redissolved in 1 mL of CH2Cl2, reprecipitated, collected, and then dried by vacuum for 8 h. NMR characterization of the Boc-protected PAON and A1-A12 follows below.
The Boc-protected polymers were deprotected by stirring 100 mg in 8 mL of 1:1 TFA:CH2Cl2 for 2 h. The solution was dried to an oil by rotary evaporator set at 40° C. and residual TFA was removed by sonicating the oil in more CH2Cl2 and evaporating the solvent again by rotary evaporator. The resulting solid was place under vacuum for 2 h. Finally, the solid was fully dissolved in 4 mL H2O and filtered through a polyethersulphone (PES) syringe filter (Whatman, 25 mm diameter, 0.45 μm pore) and freeze-dried for 48 h to give an eggshell colored soft solid. Final deprotected polymers were stored at −20° C.
Instrumentation. Gel permeation chromatography (GPC) was performed on the Boc-protected polymers with a Polymer Lab LC1120 pump equipped with a Waters differential refractometer detector. The mobile phase was THF with a flow rate of 1.0 mL/min. Separations were performed with 105, 104, and 103 Å Polymer Lab columns and molecular weights were calibrated versus narrow molecular weight polystyrene standards. The Boc-protected polymers are THF soluble and GPC was used to approximate Mn and polydispersity index (PDI). 1H-NMR and 13C-NMR spectra were obtained on a Bruker DPX-300 or Bruker Avance-400 NMR spectrometer.
Monomer 1. 1H NMR (400 MHz, DMSO-d6): δ=6.55 (2H, s) 5.12 (2H, s), 2.92 (2H, s), 2.81 (3H, s). 13C-NMR (100 MHz, CDCl3): δ=176.3, 136.5, 80.8, 47.5, 24.9. HR-MS (FAB+): calc. 179.06. found 179.05.
Monomer 2. 1H NMR (400 MHz, DMSO-d6): δ 6.55 (2H, s), 5.13 (2H, s) 3.36 (2H, q, J=7.2 Hz), 2.91 (2H, s), 1.12 (3H, t, J=7.2 Hz). 13C-NMR (100 MHz, CDCl3): δ=176.1, 136.6, 80.9, 47.4, 33.9, 13.0. HR-MS (FAB+): calc. 193.07. found 193.07.
Monomer 3. 1H NMR (400 MHz, DMSO-d6): δ 6.55 (2H, s), 5.13 (2H, s), 3.31 (2H, t, J=7.5 Hz), 2.92 (2H, s), 1.46 (2H, m), 0.78 (3H, t, J=7.3 Hz). 13C-NMR (100 MHz, CDCl3): δ=176.4, 136.6, 81.0, 47.8, 40.5, 21.0, 11.1. HR-MS (FAB+): calc. 207.09. found 207.08.
Monomer 4. 1H NMR (400 MHz, DMSO-d6): δ=6.55 (2H, s), 5.12 (2H, s), 3.34 (2H, t, J=7.1 Hz), 2.92 (2H, s), 1.42 (2H, m), 1.21 (2H, m), 0.85 (3H, t, J=7.2 Hz). 13C-NMR (100 MHz, CDCl3): δ=176.5, 136.5, 81.0, 47.3, 38.9, 28.6, 19.7, 13.7. HR-MS (FAB+): calc. 221.11. found 221.01.
Monomer 4′. 1H NMR (400 MHz, DMSO-d6): δ=6.55 (2H, s), 5.13 (2H, s), 3.16 (2H, d, 7.5 Hz), 2.92 (2H, s), 1.88 (1H, m), 0.79 (6H, d, J=6.6 Hz). 13C-NMR (100 MHz, CDCl3): δ=176.5, 136.5, 81.0, 47.3, 46.2, 27.0, 19.9. HR-MS (FAB+): calc. 221.11. found 221.10.
Monomer 5. 1H NMR (400 MHz, DMSO-d6): δ=6.55 (2H, s), 5.12 (2H, s), 3.33 (2H, t, J=3.6 Hz), 2.91 (2H, s), 1.43 (2H, m), 1.23 (4H, m), 0.831 (3H, t, J=7.2 Hz). 13C-NMR (100 MHz, CDCl3): δ=176.3, 136.6, 80.9, 47.4, 39.0, 28.8, 27.3, 22.2, 13.9. HR-MS (FAB+): calc. 235.12. found 235.12.
Monomer 5′. 1H NMR (400 MHz, DMSO-d6): δ=6.55 (2H, s), 5.12 (2H, s), 3.34 (2H, t, J=7.4 Hz), 2.91 (2H, s), 1.48 (1H, m), 1.31 (2H, q, 7.2 Hz), 0.86 (6H, d, J=6.9 Hz). 13C-NMR (100 MHz, CDCl3): δ=176.3, 136.6, 80.9, 47.4, 37.5, 36.3, 25.9, 22.3. HR-MS (FAB+): calc. 235.12. found 235.10.
Monomer 6. 1H NMR (400 MHz, DMSO-d6) δ=6.55 (2H, s), 5.12 (2H, s), 3.33 (2H, t, J=7.1 Hz), 2.91 (2H, s), 1.43 (2H, s), 1.22 (6H, br), 0.84 (3H, t, J=6.6 Hz). 13C-NMR (100 MHz, CDCl3): δ=176.3, 136.6, 80.9, 47.4, 39.0, 31.3, 27.6, 26.3, 22.5, 14.0. HR-MS (FAB+): calc. 249.14. found 249.12.
Monomer 6′. 1H NMR (400 MHz, DMSO-d6): δ=6.55 (2H, s), 5.12 (2H, s), 3.24 (2H, d, J=7.2 Hz), 2.92 (2H, s), 1.56 (1H, m), 1.20 (4H, m), 0.81 (6H, t, J=7.4 Hz). 13C-NMR (100 MHz, CDCl3): δ=176.6, 136.5, 81.0, 47.3, 42.5, 38.6, 23.0, 10.3. HR-MS (FAB+): calc. 249.14. found 249.11.
Monomer 9. 1H NMR (400 MHz, DMSO-d6): δ=6.55 (2H, s), 5.11 (2H, s), 3.32 (2H, t, J=7.1 Hz), 2.91 (2H, s), 1.43 (1H, m), 1.22 (12H, br), 0.85 (3H, t, J=6.8 Hz). 13C-NMR (100 MHz, CDCl3): δ=176.3, 136.6, 80.9, 47.4, 39.1, 31.8, 29.4, 29.2, 27.6, 26.7, 24.9, 22.7, 14.1. HR-MS (FAB+): calc. 291.18. found 291.18.
Monomer 12. 1H NMR (400 MHz, DMSO-d6): δ=6.55 (2H, s), 5.11 (2H, s), 3.32 (2H, br), 2.91 (2H, s), 1.42 (2H, s), 1.23 (18H, br), 0.85 (3H, t, J=6.6 Hz). 13C-NMR (100 MHz, CDCl3): δ=176.3, 136.6, 80.9, 47.4, 32.1, 31.9, 29.6, 29.5, 29.4, 29.3, 29.1, 27.6, 26.7, 22.7, 21.4, (FAB+): calc. 333.23. found 333.23.
Boc-protected PAON, high MW. 1H NMR (300 MHz, DMSO-d6): δ=7.61 and 6.96 (1H total, br), 5.95 (trans) and 5.75 (cis) (2H total, br), 4.87 (cis) and 4.42 (trans) (2H total, br), 3.08 (4H, br), 1.35 (9H, s), cis:trans ratio=49:51 (For the lower MW homopolymer, cis:trans ratio=47:53).
PAON, high MW. 1H NMR (300 MHz, DMSO-d6): δ=8.04 (3H, br), 5.96 (1H, br), 5.76 (1H, br), 4.91 (1H, br), 4.48 (1H, br), 3.64 (2H, br), 3.00 (2H, br).
NMR data for copolymers. For the determination of copolymer composition by NMR, the integrations of the t-butyl group(s) of the Boc-protected charged monomers were compared with the methyl signal(s) assigned to the non-polar monomers, to give a charged:non-polar monomer ratio. Proton signals in the 3.3 to 3.5 ppm range were obscured by a water signal in the samples. The following NMR data are for the higher MW but the cis:trans ratio and the charged:non-polar monomer ratio for the low MW analogues are given as well. NMR data for the deprotected copolymers (TFA-salt analogues), A1-A12, are given also.
Boc-protected A1. 1H NMR (300 MHz, DMSO-d6): δ=6.91 (1H, br), 5.94 (2H, br), 5.74 (2H, br), 4.86 (2H, br), 4.42 (2H, br), 3.08 (2H, br), 2.82 (3H, br), 1.35 (9H, br), cis:trans ratio=54:46, A:1 mol ratio=51:49 (For the lower MW copolymer, cis:trans ratio=49:51, A:1 mol ratio=50:50).
A1. 1H NMR (300 MHz, DMSO-d6): δ=7.96 (3H, br), 5.96 (2H, br), 5.75 (2H, br), 4.89 (2H, br), 4.48 (2H, br), 3.64 (4H, br), 3.00 (2H, br), 2.85 (3H, br).
Boc-protected A2. 1H NMR (300 MHz, DMSO-d6): δ=6.91 (1H, br), 5.95 (2H, br), 5.74 (2H, br), 4.86 (2H, br), 4.42 (2H, br), 3.08 (2H, br), 1.35 (9H, br), 1.06 (3H, br), cis:trans ratio=52:48, A:2 mol ratio=46:54 (For the lower MW copolymer, cis:trans ratio=47:53, A:2 mol ratio=44:56).
A2. 1H NMR (300 MHz, DMSO-d6): δ=7.94 (3H, br), 5.96 (2H, br), 5.75 (2H, br), 4.88 (2H, br), 4.45 (2H, br), 3.62 (br), 2.99 (2H, br), 1.06 (3H, br).
Boc-protected A3. 1H NMR (300 MHz, DMSO-d6): δ=6.91 (1H, br), 5.95 (2H, br), 5.74 (2H, br), 4.86 (2H, br), 4.42 (2H, br), 3.08 (2H, br), 1.49 (2H, br), 1.35 (9H, br), 0.82 (3H, br), cis:trans ratio=50:50, A:3 mol ratio=47:53 (For the lower MW copolymer, cis:trans ratio=44:56, A:3 mol ratio=43:57).
A3. 1H NMR (300 MHz, DMSO-d6): δ=7.90 (3H, br), 5.96 (2H, br), 5.76 (2H, br), 4.88 (2H, br), 4.45 (2H, br), 3.62 (br), 2.99 (2H, br), 1.50 (2H, br), 0.82 (3H, br).
Boc-protected A4. 1H NMR (300 MHz, DMSO-d6): δ=6.91 (1H, br), 5.95 (2H, br), 5.74 (2H, br), 4.86 (2H, br), 4.42 (2H, br), 3.08 (2H, br), 1.45 (2H, br), 1.35 (9H, br), 1.25 (2H, br), 0.87 (3H, br), cis:trans ratio=51:49, A:4 mol ratio=47:53 (For the lower MW copolymer, cis:trans ratio=48:52, A:4 mol ratio=44:56).
A4. 1H NMR (300 MHz, DMSO-d6): δ=7.92 (3H, br), 5.96 (2H, br), 5.76 (2H, br), 4.88 (2H, br), 4.42 (2H, br), 3.63 (br), 2.99 (2H, br), 1.46 (2H, br), 1.23 (2H, br), 0.87 (3H, br).
Boc-protected A4′. 1H NMR (300 MHz, DMSO-d6): δ=6.90 (1H, br), 5.96 (2H, br), 5.75 (2H, br), 4.86 (2H, br), 4.42 (2H, br), 3.17 (2H, br), 3.08 (2H, br), 1.90 (1H, br), 1.35 (9H, br), 0.82 (6H, br), cis:trans ratio=51:49, A:4′ mol ratio=50:50 (For the lower MW copolymer, cis:trans ratio=46:54, A:4′ mol ratio=48:52).
A4′. 1H NMR (300 MHz, DMSO-d6): δ=7.93 (3H, br), 5.97 (2H, br), 5.76 (2H, br), 4.88 (2H, br), 4.42 (2H, br), 3.62 (br), 3.18 (2H, br), 2.99 (2H, br), 1.91 (1H, br), 0.83 (6H, br).
Boc-protected A5. 1H NMR (300 MHz, DMSO-d6): δ=6.90 (1H, br), 5.95 (2H, br), 5.74 (2H, br), 4.86 (2H, br), 4.41 (2H, br), 3.08 (2H, br), 1.47 (2H, br), 1.35 (9H, br), 1.23 (4H, br), 0.84 (3H, br), cis:trans ratio=52:48, A:5 mol ratio=46:54 (For the lower MW copolymer, cis:trans ratio=47:53, A:5 mol ratio=43:57).
A5. 1H NMR (300 MHz, DMSO-d6): δ=7.93 (3H, br), 5.96 (2H, br), 5.76 (2H, br), 4.88 (2H, br), 4.42 (2H, br), 3.63 (br), 2.99 (2H, br), 1.48 (2H, br), 1.24 (4H, br), 0.85 (3H, br).
Boc-protected A5′. 1H NMR (300 MHz, DMSO-d6): δ=6.90 (1H, br), 5.95 br), 5.74 (2H, br), 4.86 (2H, br), 4.41 (2H, br), 3.08 (2H, br), 1.49 (3H, br), 1.35 (9H, br), 0.88 (6H, br), cis:trans ratio=53:47, A:5′ mol ratio=49:51 (For the lower MW copolymer, cis:trans ratio=46:54, A:5′ mol ratio=49:51).
A5′. 1H NMR (300 MHz, DMSO-d6): δ=7.93 (3H, br), 5.96 (2H, br), 5.76 (2H, br), 4.88 (2H, br), 4.42 (2H, br), 3.62 (br), 2.99 (2H, br), 1.51 (1H, br), 1.37 (2H, br), 0.88 (6H, br).
Boc-protected A6. 1H NMR (300 MHz, DMSO-d6): δ=6.90 (1H, br), 5.95 (2H, br), 5.74 (2H, br), 4.85 (2H, br), 4.41 (2H, br), 3.08 (2H, br), 1.46 (2H, br), 1.35 (9H, br), 1.23 (6H, br), 0.84 (3H, br), cis:trans ratio=49:51, A:6 mol ratio=47:53 (For the lower MW copolymer, cis:trans ratio=45:55, A:6 mol ratio=41:59).
A6. 1H NMR (300 MHz, DMSO-d6): δ=7.96 (3H, br), 5.96 (2H, br), 5.76 (2H, br), 4.88 (21H, br), 4.42 (2H, br), 3.63 (br), 2.99 (2H, br), 1.47 (2H, br), 1.24 (6H, br), 0.85 (3H, br).
Boc-protected A6′. 1H NMR (300 MHz, DMSO-d6): δ=6.90 (1H, br), 5.96 (2H, br), 5.74 (2H, br), 4.85 (2H, br), 4.41 (2H, br), 3.08 (2H, br), 1.59 (1H, br), 1.35 (9H, br), 0.83 (6H, br), cis:trans ratio=52:48, A:6′ mol ratio=49:51 (For the lower MW copolymer, cis:trans ratio=47:53, A:6′ mol ratio=47:53).
A6′. 1H NMR (300 MHz, DMSO-d6): δ=7.92 (3H, br), 5.97 (2H, br), 5.76 (2H, br), 4.86 (2H, br), 4.42 (2H, br), 3.62 (br), 2.99 (2H, br), 1.59 (1H, br), 1.22 (4H, br), 0.83 (6H, br).
Boc-protected A9. 1H NMR (300 MHz, DMSO-d6): δ=6.90 (1H, br), 5.95 (2H, br), 5.74 (2H, br), 4.86 (2H, br), 4.41 (2H, br), 3.08 (2H, br), 1.46 (2H, br), 1.35 (9H, br), 1.23 (12H, br), 0.84 (3H, br), cis:trans ratio=51:49, A9 mol ratio=41:59 (For the lower MW copolymer, cis:trans ratio=45:55, A:9 mol ratio=46:54).
A9. 1H NMR (300 MHz, DMSO-d6): δ=7.94 (3H, br), 5.96 (2H, br), 5.76 (2H, br), 4.89 (2H, br), 4.42 (2H, br), 3.63 (br), 2.99 (2H, br), 1.47 (2H, br), 1.20 (12H, br), 0.85 (3H, br).
Boc-protected A12. 1H NMR (300 MHz, DMSO-d6): δ=6.90 (1H, br), 5.95 (2H, br), 5.74 (2H, br), 4.86 (2H, br), 4.41 (2H, br), 3.08 (2H, br), 1.45 (2H, br), 1.34 (9H, br), 1.22 (18H, br), 0.84 (3H, br), cis:trans ratio=51:49, A12 mol ratio=44:56 (For the lower MW copolymer, cis:trans ratio=45:55, A:12 mol ratio=44:56).
A12. 1H NMR (300 MHz, DMSO-d6): δ=7.94 (3H, br), 5.96 (2H, br), 5.76 (2H, br), 4.87 (2H, br), 4.42 (2H, br), 3.63 (br), 2.99 (2H, br), 1.46 (2H, br), 1.19 (18H, br), 0.85 (3H, br).
Microbial and Hemolysis Assays
Bacteria suspensions of Ec, and Sa (Escherichia coli D31 and Staphylococcus aureus ATCC 25923) were grown in Mueller-Hinton Broth (MHB) overnight at 37° C., diluted with fresh MHB to an optical density of 0.001 at 600 nm (OD600). This OD600 gave an initial bacterial concentration of  ̃105 cells/mL. This suspension was mixed with different concentrations of freshly prepared polymer solutions dissolved in DMSO (40 mg/mL stock solution) by serial dilutions in a 96-well plate, and incubated for 6 h at 37° C. The OD600 was measured for bacteria suspensions that were incubated in the presence of polymer solution or only MHB. Antibacterial activity was expressed as minimal inhibitory concentration (MIC), the concentration at which more than 90% inhibition of growth was observed after 6 h. All experiments were run in quadruplicate.
Hemolytic activity measurements were performed on all polymers. Freshly drawn human red blood cells (RBC, 30 μL), were suspended in 10 mL of Tris saline (10 mM Tris, 150 mM NaCl, pH 7.2, filtered through PES membrane with 0.22 μm pore size) and rinsed three times by centrifugation (5 min at 1500 rpm) and resuspended in Tris saline. Polymer solutions were prepared in DMSO at 40 mg/mL and further diluted as necessary. Freshly prepared polymer solutions with different concentrations were added to 100 μL of the above-prepared RBC suspension to reach a final volume of 200 μL on a 96-well plate. The resulting mixture was kept at 37° C. for 30 min on a stirring plate. Then the plate was centrifuged (IEC Centra-4B, 10 min at 1500 rpm), and the supernatant in each well was transferred to a new plate. Hemolysis was monitored by measuring the absorbance of the released hemoglobin at 414 nm. 100% Hemolysis was obtained by adding 10 μL of Triton-X (polyoxyethylene isooctylphenyl ether from Sigma-Aldrich) solution (20 vol % in DMSO), a strong surfactant, to the above-prepared HRBC suspension. The upper limit of polymer concentration that was required to cause 50% hemolysis is reported as HC50, and the absorbance from Tris saline containing no polymer was used as 0% hemolysis. All experiments were run in quadruplicate.
Dye-Leakage from Large Unilamellar Vesicles
The following is a general procedure to make large unilamellar vesicles (LUV). Chloroform solutions of Ec lipids (25 mg, Avanti Polar Lipids, Inc.) were mixed in a 10 mL round bottom flask and the chloroform was removed at room temperature by rotary evaporator to form a uniform film. The flask was placed under vacuum for an additional 6 h. The dried film was hydrated with 1 mL of a 40 mM aqueous calcein (Sigma-Aldrich) solution in Tris buffer without added NaCl. The calcein dye solution was adjusted to pH=7 prior to adding it to the film. The solution was subjected through five freeze/thaw cycles using liquid nitrogen and warm water. The entire volume of 1 mL of solution was subjected to extrusion (a total of 15 passes) through two stacked 400 nm pore PC membranes (Avanti Polar Lipids, Inc.) at room temperature. Finally, the solution was pressurized through a small column packed with Sephadex-25 (Sigma-Aldrich) eluting with Tris-saline buffer to remove non-trapped calcein dye. Fractions ( ̃5 drops each) were collected. The vesicle solution can be stored in a vial at 4° C. and diluted as needed for up to 4 days.
Diluted calcein-loaded vesicle fractions that afforded fluorescence intensities <80 a.u. without surfactant and >800 a.u. after addition of surfactant, were used (Ex.=490 nm used and Em.=515 nm monitored). 50 μL of 0.2% Triton-X was used as the strong surfactant which causes complete vesicle disruption and leakage. Typically, chosen vesicle fractions were diluted six-fold and 25 μL of this stock solution is added to 2 mL of Tris-saline buffer. A fluorescence intensity of <80 a.u. is verified to be stable over a minute before 25 μL of a polymer stock solution is added (2.5 μg/mL final concentration in cuvette), agitated briefly with the pipettor tip, then monitored at 515 nm. After 5 min Triton-X is added and the corresponding fluorescence was taken as 100% leakage.
Fluorescence Microscopy of Stained Bacteria Cells
An Olympus BX51 Reflected Fluorescence Microscope (Optical Analysis Corp. Nashua, N.H.) with a 100 W Mercury Lamp (Chin Technical Corp.) was used for fluorescence studies. A BacLightTM Kit L-7012 (Invitrogen Corp.) was used as the fluorescence dye in a mixture of 1:1 propidium iodide:SYTO9 to examine Sa in the presence of polymer. It is important to mention that an initial bacterial concentration of  ̃108 cells/mL was used for microscopy studies (three orders of magnitude higher than the MIC and bactericidal kinetics studies) for ease of visualization. The dye mixture was incubated with the bacteria at room temperature for 15 minutes before adding polymer solution (75 μg/mL final concentration). Solution of cells, dye, and polymer were allowed to sit for 30 min before 50 μL was placed on a slide, fitted with a coverslip, and visualized. Bacteria were viewed under a green filter (excitation/emission, 420-480 nm/520-800 nm) or a red filter (480-550 nm/590-800 nm).
e. A ROMP-based Platform for SMAMPs
From AMPs Via Foldamers to Romp-Based SMAMPs—an Evolution
AMPs are host-defense peptides found in many organisms from invertebrates to humans. Their important feature is that they are facially amphiphilic molecules, as illustrated in FIG. 31. They contain a face with hydrophilic, positively charged groups (blue) and a hydrophobic face (green). (Boman, Immunol. Rev. 2000, 173, 5.) This segregation of the hydrophilic and hydrophobic side chains onto two opposite sides of the molecule results from the amino acid sequence of the peptide, which also dictates the conformation of the backbone and thus the secondary structure of the molecule. While most traditional antibiotics have specific cellular targets, AMPs use non-receptor interactions, including direct action against the bacteria's membranes, although intracellular targets have been identified in some cases. (Brodgen, Nature Rev. Microbiol. 2005, 3, 238.) Their facial amphiphilicity enables them to insert into cell membranes and locally change the membrane lipid organization, leading to pore formation, membrane rupture, or other interactions that impact the membrane elasticity so that cell viability is compromised. Several mechanisms have been proposed for trans-membrane pore formation by AMPs (carpet, barrel-stave and toroidal pore model). (Brodgen, Nature Rev. Microbiol. 2005, 3, 238; Yang, et al., J. Am. Chem. Soc. 2007, 127, 12141; Zasloff, Nature 2002, 415, 389; Boman, Immunol. Rev. 2000, 173, 5; Hancock, et al., Trends Biotech. 1998, 16, 82; Yeaman, et al., Pharmacol. Rev. 2003, 55, 27.) The careful balance between cationic hydrophilic and hydrophobic groups allows AMPs to differentiate between the neutral, phosphatylcholine and cholesterol rich surface of mammalian cells and the negatively charged cell surface of bacteria.
As AMP extraction from natural organisms or their production in multi-step syntheses is tedious and expensive, SMAMP research strived for a platform that yielded a library of molecules which retained the antibacterial activities of AMPs, but would be obtained in a few synthetic steps and in large quantities. As such, many substance classes have been used, including α- and β-amino acids, peptoids, aromatic oligomers, steroids, and synthetic polymers. (Mowery, et al., J. Am. Chem. Soc. 2007, 129, 15474; Kuroda, et al., J. Am. Chem. Soc. 2005, 127, 4128; Arnt, et al., J. Polymer Sci. A—Polymer Chem. 2004, 42, 3860; Tew, et al., Proceed. Nat. Acad. Sci. USA 2002, 99, 5110; Zasloff, Proceed. Nat. Acad. Sci. USA 1987, 84, 5449; Castro, et al., Current Protein Peptide Sci. 2006, 7, 473; Chen, et al., J. Biol. Chem. 2005, 280, 12316; Won, et al., J. Biol. Chem. 2004, 279, 14784; Hamuro, et al., J. Am. Chem. Soc. 1999, 121, 12200; Porter, et al., Nature 2000, 404, 565; Liu, et al., J. Am. Chem. Soc. 2001, 123, 7553; Epand, et al., Biochemistry 2004, 43, 9527; Patch, et al., J. Am. Chem. Soc. 2003, 125, 12092; Brouwer, et al., Peptides 2006, 27, 2585; Haynie, et al., Antimicrob. Agents Chemother. 1995, 39, 301; Liu, et al., Angew. Chem. Internat. Ed. 2004, 43, 1158; Tang, et al., Chem. Comm. 2005, 1537; Savage et al., FEMS Microbio. Lett. 2002, 217, 1; Mason et al., Biophysical Journal 2007, 93, 4289; Van et al. Trends Pharmacol. Sci. 2008, 29, 124; Ilker, et al., J. Am. Chem. Soc. 2004, 126, 15870; Arnt, et al., J. Am. Chem. Soc. 2002, 124, 7664; Arnt, et al., Langmuir 2003, 19, 2404.) Initially, it was believed that the helical rigidity of the AMP backbone (FIG. 31) was a prerequisite for biological activity. As a result, early work focused on emulating the amphiphilic α-helical arrangement of side chains observed in the natural structures, leading to a large number of potent and selective antimicrobial peptides based on the natural amino acids. The availability of β-peptides provided another avenue to test and further elucidate the features required for the construction of SMAMPs. β3-peptides adopt “14-helices (14 residues within the repeating hydrogen-bonded rings), which have an approximate 3-residue geometric repeat. Thus, if polar and apolar sidechains are arranged with precise three-residue periodicity in the sequence of an appropriately designed β-peptide, they will segregate to opposite sides of the helix. Indeed, repeating tripeptides composed of hAla, hLeu and/or hVal (the β-amino acids were β3-substituted) were found to be have antimicrobial activity. Meanwhile, Gellman and coworkers described a potent and highly selective antimicrobial peptide based on cyclic β-amino acids. (Hamuro, et al., J. Am. Chem. Soc. 1999, 121, 12200; Porter, et al., Nature 2000, 404, 565; Liu, et al., J. Am. Chem. Soc. 2001, 123, 7553.) These studies, which were subsequently extended to a variety of different helical types formed by β-peptides, showed that charge, facial amphiphilicity, and an appropriate hydrophilic/hydrophobic balance were important to obtaining selective, non-toxic compounds.
All of these synthetic derivatives focused on helical secondary structures; however, the critical role of this secondary structural element was questioned early on as other folded forms of natural peptides were discovered. Within the synthetic analog community, early work on diastereomeric peptides containing D-amino acid substitutions believed to have little α-helix forming abilities but potent antibacterial activity supported this conclusion. (Shai, et al., Peptides 2001, 22, 1629; Hong, et al., Biochemistry 1999, 38, 16963; Oren, et al. Biochemistry 1997, 36, 1826.) Further support for the overall amphiphilicity being more important than a specific folded structure comes from recent work on scrambled sequence α/β-peptides which are selective antimicrobial agents without the ability to adopt globally amphiphilic helices. (Schmitt, et al., J. Am. Chem. Soc. 2007, 417.) Thus, it appears that there is no unique requirement for a rigid conformation so long the composition of that sequence is conducive for binding to the target membrane.
It is questionable whether this general approach could be extended to design much simpler oligomers and polymers that capture the essential biological and physiochemical properties of AMPs. In particular, one can consider two design strategies. On the one hand, the amphiphilic secondary and tertiary structures of natural AMPs can be mimicked by placing hydrophilic and hydrophobic groups on an appropriate framework. In this case, a preorganized backbone would help minimize the unfavorable conformational entropy of binding leading to good potency. On the other hand, if a rigid conformation is not an absolute requirement for activity, far more flexible (co)polymers might be expected to be active as they would be able to adopt the necessary conformations. In either case, it is important to optimize the structures for maximal activity by careful consideration of the molecular weight, charge and hydrophilic/lipophilic balance.
Probably the most (r)evolutionary step in SMAMP design simplification was to completely dispense with the helical motif in favor of a less complicated but rigid aromatic molecular scaffold. DeGrado and Tew developed aryl amide-based oligomeric SMAMPs with facially amphiphilic repeat units. (Tew, et al., Proceed. Nat. Acad. Sci. USA 2002, 99, 5110; Liu, et al., Angew. Chem. Internat. Ed. 2004, 43, 1158; Breitenkamp, et al., Polymers Adv. Technol. 2005, 16, 189.) These molecules were still potently antibacterial, and while they did not have the confinement of the helical secondary structure, the rigidity of the aryl amide backbone and hydrogen bonding between their functional groups ensured that one side of the molecule contained the hydrophobic groups and the opposite side the charged hydrophilic groups. This work clearly demonstrated that a helical backbone is not necessary. By synthesizing molecules with a phenylene ethynylene backbone, it was tested whether the configuration constraints of the backbone could be further relaxed. These molecules still possessed the rigidity of an aromatic backbone, but had no intramolecular hydrogen bonds. (Tew, et al., Biochim. Biophys. Acta-Biomembranes 2006, 1758, 1387; Arnt, et al., J. Polymer Sci. A—Polymer Chem. 2004, 42, 3860; Arnt, et al. J. Phys. Chem. B 2006, 110, 3527; Arnt, et al., Langmuir 2003, 19, 2404; Breitenkamp, et al., Polymers Adv. Technol 2005, 16, 189; Arnt, et al., Macromolecules 2004, 37, 1283.) While the aromatic backbone ensured an overall linear conformation, the repeat units were free to rotate around their single bonds. This allowed their functional groups to orient themselves to a facially amphiphilic conformation upon contact with a cell membrane or a similar hydrophilic-hydrophobic interface. The phenylene ethynylene polymers had the desired antibacterial activities; by small-angle X-ray scattering, it was shown that even phenylene ethynylene trimers were able to form pores when exposed to model membranes. (Yang, et al., J. Am. Chem. Soc. 2007, 127, 12141.)
The final move in SMAMP design was to get rid of the rigid aromatic backbone altogether and to equip synthetic polymers with hydrophobic and hydrophilic repeat units, hoping they would self-orient their functional groups to be facially amphiphilic and membrane active upon contact with cells. Several polymers were synthesised based on this idea, and it was demonstrated that, if the functional groups were adequately balanced and positioned, even this last constraint could be given up while the polymers still had superb antibacterial properties. (Sambhy, et al., Angew. Chem. Int. Ed. 2008, 47, 1250; Mowery, et al., J. Am. Chem. Soc. 2007, 129, 15474; Ilker, et al., J. Am. Chem. Soc. 2004, 126, 15870; Gabriel, et al., Chem. Eur. J. 2009, 15, 433; Lienkamp, et al., J. Am. Chem. Soc. 2008, 130, 9836.)
ROMP-Based Synthetic Mimics of Antibacterial Peptides
Over the last five years, a large number of ROMP-based SMAMPs were synthesized, and both their biological properties and mechanism of interaction with membranes were studied. The structures of these compounds are summarized in FIG. 34. This figure illustrates how, by gradual structural variation, a library of polymers with tunable antibacterial and hemolytic properties was obtained, and how the investigation of structure-property relationships of those polymers helped to elucidate key factors of SMAMP design.
ROMP was chosen as a synthetic platform as it is a living polymerization technique, yields molecules with low polydispersity over a wide range of molecular weights and is highly functional group tolerant. (Kiessling, et al., Vol. 3, Wiley-VCH, Weinheim, 2003; Trnka, et al., Acc. Chem. Res. 2001, 34, 18; Buchmeiser, Chemical Rev. 2000, 1565.) The variety of molecules, especially bioactive ones, that were accessible through ROMP has been reviewed elsewhere. (Smith, et al., Polymer Rev. 2007, 47, 419.)
The field of ROMP-based SMAMPs was pioneered by Ilker et al. (Ilker, et al., J. Am. Chem. Soc. 2004, 126, 15870.) They reported a series of poly(norbornene) derivatives with facially amphiphilic repeat units (poly1 to poly4, series 1 in FIG. 34). In this polymer series, the ratio of hydrophobic and hydrophilic moieties per repeat unit was gradually varied, and the effect of this variation on the antibacterial and hemolytic activities of these polymers was studied where a backbone-modification strategy was used to obtain the desired gradient in hydrophobicity across the polymer series, in which the different hydrophobic groups are attached in the first step of monomer synthesis (FIG. 33a).
These polymers are large molecular weight SMAMP-derivatives with good selectivity (>100). Also, this parent series with its norbornene-imide structure inspired many further modifications that allowed property fine tuning or more detailed investigations of certain design parameters, as illustrated in FIG. 34.
Unlike the imide-based series, whose polymers are more difficult to synthesize because the backbone modification comes in an early step (FIG. 33a), and thus each monomer requires different precursors to be prepared, ester-based monomers (2 in FIG. 34) can be obtained from the same precursor and the critical modification leading to installation of the different hydrophilic or hydrophobic groups is introduced independently and in either order in the last synthetic steps (FIG. 33b). Thus, a large variety of monomers can be obtained from the same precursors, which is why this ester-based platform is termed a ‘molecular construction kit’. (See Section a above; Lienkamp, et al., J. Am. Chem. Soc. 2008, 130, 9836.)
Structural Modifications and their Effects on SMAMP Activity
Increasing Hydrophobicity in a Series of Amphiphilic Homopolymers
Three series of polymers with facially amphiphilic repeat units and gradually increasing hydrophobicity have been synthesized. (Ilker, et al., J. Am. Chem. Soc. 2004, 126, 15870; Lienkamp, et al., J. Am. Chem. Soc. 2008, 130, 9836; Eren, et al., Macromolecular Chem. Phys. 2008, 209, 516.) The already mentioned poly(norbornene) imide series is shown as 1 in FIG. 34. In this series of molecules, the hydrophilic, cationic ammonium group was kept constant, and the hydrophobic group attached to the backbone was modified. The result of this structural modification on the biological properties is summarized in FIG. 35a. (Ilker, et al., J. Am. Chem. Soc. 2004, 126, 15870.) Eren et al. synthesized a series of poly(oxanorbornene) derivatives similar to poly1, the most hydrophilic polymer of series 1. They obtained different hydrophobicities by alkylation of the imide with side chains of different length containing quaternary pyridinium groups. (Eren, et al., Macromolecular Chem. Phys. 2008, 209, 516.) This yielded series 3, with R=ethyl to dodecyl, and R=phenylethyl. The biological data for these molecules are shown in FIG. 35b. Using the ester-based platform (Series 2 in FIG. 34), a systematic change of the hydrophobic group from R=methyl to hexyl led to a third series of hydrophobically modified polymers. The MIC90 and HC50 values for this series are shown in FIG. 35c. When comparing FIG. 35a to c, overall the same trends are observed in the antibacterial and hemolytic activities of these polymers. The HC50 values are highest for the most hydrophilic polymers and then decrease significantly as the polymers become more hydrophobic, which means that adding hydrophobicity makes the SMAMPs increasingly more hemolytic. The MIC90 values start off high for more hydrophilic polymers, meaning that those are inactive, and then go through a minimum for all three series, with poly3 (FIG. 35a), the octyl imide (FIG. 35b), and the propyl ester (FIG. 35c) being the most active structures in each series, respectively. At one point, however, the solubility of the SMAMP decreases so much that it severely aggregates and consequently becomes unavailable to interact with the bacteria membrane; therefore the MIC90 value goes up again. The overlapping trend of increasing activity and decreasing solubility then lead to the observed minimum in the MIC90 data for all three series of polymers. Thus, with increasing hydrophobicity the polymers become toxic to both bacteria and mammalian cells.
Copolymers—Facially Amphiphilic Versus Segregated Monomers
Since poly3 in Series 1 had the lowest MIC90, and poly2 the highest HC50. Ilker et al. attempted to increase the therapeutic window of these polymers by copolymerizing the corresponding two monomers at different ratios, and tested their activities. (Ilker, et al., J. Am. Chem. Soc. 2004, 126, 15870.) The biological data thus obtained is summarized in FIG. 36a. As can be seen from this data, for monomer feed ratios from 2:1 to 9:1, the resulting copolymers stayed non-hemolytic but became active even when the molar ratio the ‘antimicrobial’ component was 10%. This led to selectivities as high as 100 against both E. coli and S. aureus bacteria. Similar results were obtained when monomers of the ester series (2) were copolymerized. While the ethyl homopolymers of that series were the ones with the highest selectivity, the methyl polymers were the least hemolytic and the propyl polymers the most active. Thus, a systematic property variation was expected by copolymerization of these monomers. Three copolymer series (Series 4 in FIG. 34, with R1/R2=methyl/ethyl, methyl/propyl and ethyl/propyl) were thus obtained. The biological data from these series is summarized in FIG. 36b-d. Copolymerization of the ethyl with the methyl copolymer gave little improvement in the antimicrobial properties, and no significant difference in the HC50 data. Likewise, while incorporation of ethyl into the propyl copolymers made those less toxic, at the same time they lost their activity. The methyl-propyl copolymers, on the other hand, show the same trend as the poly2-poly3 copolymers, and can therefore be considered as the direct analogue to that series. From a feed ratio of 1:9 to 9:1, these polymers became more active against S. aureus bacteria, and at the same time less hemolytic. The selectivities of those polymers were >533. However, unexpectedly, these polymers lost their activity against E. coli and were therefore termed ‘doubly selective’ SMAMPs because they were active against bacteria, but did not lyse mammalian cells, and they preferentially killed one kind of bacteria over another.
The common structural feature of both the poly2-poly3 copolymers and the ester-based copolymers (4) is that both comonomers were facially amphiphilic. While successive structural simplification in going from AMPs via foldamers to SMAMPs showed that many design features, such as the rigid nature of the backbone and the presence of aromatic groups, were not essential for obtaining high selectivities for bacteria over mammalian cells, it was found by Gabriel et al. that the facial amphiphilicity on the monomer level was critical. (Gabriel, et al., Chem. Eur. J. 2009, 15, 433.) To obtain a polymer series with tunable antimicrobial properties without the need to go through the tedious synthetic procedures of the poly1-4 series (FIG. 34), SMAMP copolymers from one hydrophilic unit (the poly1 monomer) and comonomers that carried the hydrophobic group were made, with a feed ratio of 1:1 (Series 5 in FIG. 34). Due to the high structural similarity between these polymers and the poly1-4 series, one could expect that this approach would lead to polymers with similarly tunable properties. However, while these new polymers (series 5) followed the general trends that had been found before (a minimum value for the MIC90 and HC50 values that decreased with increasing hydrophobicity), the overall selectivities of these polymers remained much lower, with a maximum selectivity of 20 (FIG. 37a). Deviation from the 1:1 monomer feed ratio did not improve the selectivities. This was thought to be a result of the segregation of the functional groups onto two different monomers, which leads to runs of hydrophobic and hydrophilic groups in the statistical copolymer (FIG. 37b, top). Thus, the local hydrophobicity of these polymers was not uniform, whereas the polymers from facially amphiphilic monomers have a well-defined local hydrophobicity (FIG. 37b, bottom). This led to the reduced activity of the segregated SMAMPs when interacting with the bacterial membrane (FIG. 37b).
Adding Hydrophilicity
The observation that the least hydrophobic polymers of series 1, 2 and 3 were usually also the least hemolytic (FIG. 35) led to the idea that the hemolytic activity of an active but toxic polymer could be reduced by copolymerizing the respective monomer with a hydrophilic comonomer. poly3, the most active and hemolytic polymer in series 1, was modified by incorporating non-ionic and zwitterionic hydrophilic repeat units. (Colak, et al., 2009, Biomacromolecules, 10, 353.) This strategy is different from the above approach, where hydrophobicity is modified by varying R groups in a series of facially amphiphilic monomers: the hydrophilic moieties used are not facially amphiphilic, each carries a lot of functionalities that impart hydrophilicity, and as a result they are structurally very dissimilar to the previously used inactive comonomers. Also, these comonomers all had the same charge, while the hydrophilic repeat units used by Colak et al. were overall neutral. The hydrophilic moieties chosen were a sugar residue, a zwitterionic side chain, and a short poly(ethylene glycol) chain (Series 6 in FIG. 34). The activities of these polymers are shown in FIG. 38a-c, respectively. The data indeed shows that gradually making poly3 more hydrophilic systematically reduced its hemolytic activity; however the dilution of the active component, the ammonium group, also rendered these molecules increasingly inactive, and therefore the selectivities of these polymers remained low.
The Effect of Charge
Two series with systematic charge variation were investigated, one of them imide-based (Series 7 in FIG. 34), and the other ester-based (Series 8 in FIG. 34). (Al-Badri, et al., Biomacromolecules 2008, 9, 2805; Lienkamp, et al., J. Am. Chem. Soc. 2008, 130, 9836) For the imide-based series, poly1 and poly3, respectively, were taken as a starting point. Structurally alike polymers carrying two and three charges per repeat unit were made (Series 7 in FIG. 34), and their biological properties were compared to their parent compounds. (Al-Badri, et al., Biomacromolecules 2008, 9, 2805.) The data for these polymers is reported in FIG. 39. The hydrophobic poly3, which is active and toxic, became drastically less hemolytic and more active against E. coli as the charge doubled. However, further addition of charge did not improve the biological properties. On the other hand, the hemolytic activity of the already hydrophilic poly1 did not improve upon addition of more charge, yet the polymer became more active against S. aureus bacteria.
Charge and hydrophilicity are two parameters that are difficult to separate. When increasing the charge across a polymer series, the hydrophilicity is automatically altered also. The effect this has on the biological properties depends on the overall hydrophobicity of the polymer series. In an already hydrophilic polymer like poly1, adding charge does not alter the hydrophilicity dramatically, thus the overall properties of the polymer change minimally. However, as seen by the drastic jump in hemolytic activity the poly3, adding charge here drastically influences the overall hydrophilicity of the molecule. Thus the poly1 series is probably the better model to isolate the effect of charge on the biological properties.
Taking these considerations into account, the ester-platform was used to obtain four series of copolymers from a doubly charged and a singly charged repeat unit carrying a variable R group (R=methyl to butyl). The thus obtained polymer series had different overall hydrophobicities between each series. Additionally, different monomer feed ratios allowed the charge density to be continuously varied across the series in contrast to the ‘step-function’ (one, two, or three charges per repeat unit). The biological properties of those polymers are summarized in FIG. 40.
As this data indicates, all the methyl and ethyl copolymers are non-hemolytic, whereas the propyl and butyl copolymers become more hemolytic with high propyl and butyl comonomer content, respectively. Thus, the properties of those two series are dominated by the hydrophobicity of those R groups. It was found with complementary methods that the hydrophobicity of the monoamine-methyl homopolymer closely resembled that of the diamine homopolymer, while the ethyl to butyl homopolymers were significantly more hydrophobic. Thus, the methyl copolymers were found to be a suitable model system of polymers to study the effect of increasing charge density. Indeed, the properties of this polymer series (FIG. 40a) are very similar to those of the poly 1 derivatives (FIG. 39). With increasing charge, the hemolytic activity is slightly affected; however the activity against S. aureus dramatically improves. When going down in charge in the methyl-diamine series (from M9:D1 to Methyl in FIG. 40a), there is a sudden jump in the MIC from 4 to 100 μg/mL. The same is found in the poly1 derivatives when going from one to two charges per repeat unit. These findings, together with AMP literature data led to the postulation that there is a specific charge threshold that needs to be exceeded to obtain decent activities against S. aureus. (Pasupuleti, et al., Biochemistry 2008, 47, 9057.) Rather than a certain number of charges per repeat unit, this charge threshold is to be understood as a minimum charge density, or charge per unit volume, and the exact threshold number of charges per repeat unit will be slightly different for each SMAMP series depending on the molecular volume of the repeat units. On the molecular level, this postulated charge threshold translates into a minimum charge density that is necessary to trigger successful attachment of the SMAMP to the bacterial membrane. Once enough charge is present to enable this attachment, the hydrophobicity of the molecule will then determine to what extent the SMAMP is active. For S. aureus, the methyl-propyl copolymers 8 are active, for E. coli, apparently they are not hydrophobic enough, as no activity is seen against these bacteria. Preliminary evidence indicates these molecules bind to E. coli, meaning that the interpretation that these molecules have not reached the charge threshold for E. coli interactions, is unlikely.
Counterion Effects
To further probe the interaction between charge and hydrophobicity, the effect of counterion exchange on the biological properties of the series 2 polymers was studied. The hydrophilic counterions of the most hydrophilic ester-based polymer (polymer 9 in FIG. 34) were exchanged by hydrophobic organic counterions (e.g. hexanoate and tosylate). While the original idea was that exchanging these counterions would impart hydrophobicity onto the polymer and make it more active, it was found ion-exchanging these polymers completely eliminated the antibacterial activities (FIG. 41). Using dye-leakage studies, it was found that, while the parent polymer 9 was membrane active, the ion-exchanged polymers were not, meaning that the ammonium group of these polymers and the organic counterions formed such a tight ion pair that the overall positive charge of the polymer was masked.
The Effect of Molecular Weight
Ilker et al. found a weak molecular weight dependence for their poly3 compound, which at that time did not seem significant (FIG. 42a). (Ilker, et al., J. Am. Chem. Soc. 2004, 126, 15870.) In the case of the poly2 and poly4 series, no trend was observed as those polymers were in the inactive regime. Eren's low and high molecular weight polymers (Mn ̃3000 g/mol and 10000 g/mol, respectively) all had similar antibacterial and hemolytic activities, whether they were in the active or inactive regime. (Eren, et al., Macromolecular Chem. Phys. 2008, 209, 516.) For the ‘segregated’ copolymers, Gabriel found that the high molecular weight polymers (Mn ̃10,000 g/mol) were less active than the low molecular weight polymers (Mn ̃3000 g/mol) by a factor of 2-8. (Gabriel, et al., Chem. Eur. J. 2009, 15, 433.) Some of them were also slightly more hemolytic. The same general trend was found by Lienkamp et al. for the higher molecular weight ester-based polymers (Series 2 in FIG. 34, Mn ̃10,000 g/mol, biological data in FIG. 42b). Compared to their Mn ̃3000 g/mol analogues (FIG. 35c), these polymers were less active against E. coli, with the exception of the Propyl—10k polymer which was surprisingly active against that bacterial type. More notably, they were all inactive against S. aureus. (Lienkamp, et al., J. Am. Chem. Soc. 2008, 130, 9836.) Similarly, the diamine homopolymers (polymer 9 with TEA counterions in FIG. 34, biological data in FIG. 42c) showed a systematic decrease in activity against S. aureus with molecular weight, together with inactivity against E. coli at all molecular weights. This lead to the hypothesis that, at higher molecular weights, these particular polymers get stuck in the peptidoglycan layer of Gram-positive bacteria, as will be discussed in more detail below.
To investigate the molecular weight effect in more detail, especially in the low molecular weight region, a series of oligomers from the propyl polymer series 2 was prepared (FIG. 42d). As this data shows, the molecular weight dependence for both the hemolytic and antibacterial activities is highly nonlinear and different for each bacterial species involved. In the case presented here, Oligo 1 is selective for S. aureus over E. coli, while Propyl—3k shows the opposite tendency.
Mechanistic Studies of ROMP-Based SMAMPs
Vesicle Experiments and Fluorescence Microscopy
In the field of SMAMPs, the most popular method to probe the SMAMP-membrane interaction is the dye-leakage experiment. In this experiment, dye-filled lipid vesicles that are simplified models for bacteria and mammalian cell membranes are used. These vesicles capture the key features of cellular plasma membranes, and although they lack cell features such as the peptidoglycan cell wall of Gram-positive organisms, the double-membrane structure of Gram-negative bacteria, or the many proteins found in cell membranes, they are well-accepted methods to investigate the interaction of membranes with polymers or proteins, and can be used to correlate the membrane-disrupting properties of a compound to its biological activity. (Tew, et al., Proceed. Nat. Acad. Sci. USA 2002, 99, 5110; Liu, et al., J. Am. Chem. Soc. 2001, 123, 7553; Oren, Biochemistry 1997, 36, 1826.) The lipid composition of these model vesicles was chosen to closely match the cell type they are supposed to mimic; consequently the primary lipid(s) that make up the plasma membrane of that cell type was used to match properties like surface charge, fluidity, and lipid curvature as closely as possible within these simple models. For example, pure cardiolipin vesicles are most commonly used to mimic S. aureus bacteria, whereas a mixture of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) is used to mimic E coli, and phosphatidylcholine (PC) is used to mimic human red blood cells. (Som, et al., J. Phys. Chem. B 2008, 112, 3495.) Alternatively, a full lipid extract from the respective bacteria can also be used. Leakage of the self-quenching dye from the vesicle upon exposure to the SMAMPs leads to fluorescence, which is monitored as a function of time, or the leakage percentage upon SMAMP exposure can be plotted versus SMAMP concentration. (Som, et al., J. Phys. Chem. B 2008, 112, 3495.)
Ilker et al. investigated the lysis of neutral cholesterol:PC vesicles (as a mimic for erythrocytes) and of negatively charged phosphatidyserine:PC vesicles. They found that the lysing properties of poly2, poly3 and the poly2-3 copolymers were in good agreement with their antibacterial activities, poly2 being inactive and the other two polymers showing marked dye leakage. (Ilker, et al., Macromolecules 2004, 37, 694.) Gabriel et al. also studied the poly1 to poly3 series using E. coli mimicking PE:PG vesicles and found that their lysis behavior followed the MIC90 trend exactly. Eren et al. studied the effect of exposing their polymers to PC vesicles (erythrocyte mimics) and E. coli extract, and found a good correlation between the membrane disruptive properties of these polymers and their HC50 and MIC90 data, respectively. (Eren, et al., Macromolecular Chem. Phys. 2008, 209, 516.) However, in this special case the activity towards PE:PG vesicles did not correlate with the MIC90 data. Al-Badri et al. also found good agreement of the leakage from PC vesicles with the hemolysis data of his polymers. (Al-Badri, et al., Biomacromolecules 2008, 9, 2805.) Their samples also followed the general trend for both PE:PG and CL vesicles, i.e. that the poly3-derived samples were more membrane disruptive than the poly1-derived ones. For the segregated copolymers it was found that their activity against E. coli matched their membrane-disruptive potency towards vesicles made from E. coli lipid extracts.|57| (Gabriel, et al., Chem. Eur. J. 2009, 15, 433) Lienkamp et al. studied their polymers on S. aureus mimicking CL vesicles. They found a good correlation between the MIC90 for S. aureus and the vesicle leakage for the methyl to butyl homopolymers. (Lienkamp, et al., Chemistry Eur. J. 2009, submitted.) As this body of data shows, overall there is a good correlation between dye-leakage activity of a SMAMP and the corresponding biological activity. This led to the conclusion that the mechanism of antibacterial activity for all those polymers is by disruption of the plasma membrane of the bacteria, which then causes a breakdown of the membrane potential, leakage of the cell content, and, eventually, cell death.
When dye-leakage studies fail to model cell-SMAMP interactions, this can indicate that other components of the cell structure are important which are not adequately modeled by the simple bilayer membrane, such as the peptidoglycan cell wall of Gram-positive bacteria. Lienkamp et al. studied the behavior of their diamine homopolymers (polymer 9 with trifluoroacetate counterions in FIG. 34, Mn ̃3000 g/mol), towards both cardiolipin (S. aureus mimic) and PE:PG (E. coli mimic) vesicles. (Lienkamp, et al., Chemistry Eur. J. 2009, submitted.) These polymers had previously been found to be ‘doubly selective’ first for bacteria over mammalian cells, and for S. aureus (MIC90=15 μg/mL over E. coli (MIC90>200 μg/mL). (Lienkamp, et al., Chemistry Eur. J. 2009, submitted.) Surprisingly, although the MIC90s of the 3000 g/mol sample of polymer 9 were dramatically different for E. coli and S. aureus, this polymer had almost identical lysis percentages against both vesicle types (FIG. 43). This demonstrated that the differences in lipid composition of the two bacteria were not responsible for the observed differences in MIC90; it was therefore postulated that the cell selectivity was due to the double membrane structure of E. coli, while S. aureus only has one plasma membrane. Since it was observed that the 3000 g/mol polymer was active against S aureus, but that the 50,000 g/mol polymer was inactive, it was considered that the peptidoglycan cell wall found in Gram-positive organisms was an impenetrable barrier for large SMAMPs. This could be due to binding or due to steric hindrance.
In other cases, the cause for discrepancies between dye-leakage experiments and biological data might not have to do with the experiment at all, but with the SMAMP solubility. It was found that the two most hydrophobic polymers of series 5, with R=dodecyl, and series 2, with R=hexyl, did not follow the MIC90-dye leakage correlation,|57, 80| while the other molecules in both series were perfectly well-behaved. In both cases, a hydrophilic polymer of the same series with the same MIC90 as the ‘odd’ polymer caused much less dye leakage than the corresponding hydrophobic polymer. (Gabriel, et al., Chem. Eur. J. 2009, 15, 433; Lienkamp, et al., Chemistry Eur. J. 2009, submitted.) The reason for this is the low solubility of the hydrophobic polymer at the comparatively high concentrations of the MIC experiment. The SMAMP concentration in the dye leakage experiments is usually lower than in the MIC experiments, as these experiments are understood to be much more sensitive, and even at these lower polymer concentrations, the ratio of polymer to vesicles is much higher than the ratio of polymer to bacteria in the MIC experiment. (Som, et al., Biapolymers 2008, 90, 83.) If a polymer has poor solubility in aqueous media, it will seem less active in the MIC experiment, but it will still be active in the dye-leakage experiment, causing a discrepancy in the results from the two methods.
Another method to probe the membrane-polymer interaction is the so called ‘live-dead’ stain. This somewhat misleading name refers to an experiment in which the bacteria are incubated with a dye mixture of SYTO 9, giving green fluorescence, and propidium iodide, a red fluorescent dye. While SYTO 9 can diffuse through the membranes of both intact and membrane-compromised cells, propidium iodide can only enter the cells with damaged plasma membranes and competes with the green dye for binding sites. Thus, a ‘live’ cell will appear green when using the green filter of the fluorescence microscope, and not red under the red filter, whereas a membrane-compromised, ‘dead’ cell will appear red when the red filter is applied, and may or may not appear green under the green filter depending on the dye stoichiometry. Using fluorescence microscopy, it was shown that the antibacterially active butyl polymer of series 5 caused red fluorescence and severe aggregation in E. coli bacteria, whereas the inactive poly1 did neither (FIG. 44). (Gabriel, et al., Chem. Eur. J. 2009, 15, 433.) This demonstrates again that active SMAMPs compromise the bacteria cell membranes. Using the same method, when comparing different molecular weights of polymer 9 it was discovered that there is a correlation between SMAMP concentration and bacteria aggregation, as well as between SMAMP molecular weight and bacteria aggregation. At the same concentration, higher molecular weight SMAMPs cause more cell aggregation than lower molecular weight ones. Also, the higher the SMAMP concentration, the more cell aggregation was observed.
Other Techniques
In the most detailed physical study on ROMP-based SMAMPs to date, HPLC, dye-leakage studies, light-scattering, isothermal calorimetry and fluorescence microscopy were combined to investigate the mechanism of polymer-membrane interactions of the poly1 to poly4 series.|83| The combination of these methods allowed Gabriel et al. to elucidate the mechanism of SMAMP-bacteria interaction in unprecedented detail. As expected, they found a linear correlation between HPLC elution times and the alkyl side chain length of the polymers from poly1 to poly4, which proved the intuitive assumption that the hydrophobicity increases from poly1 to poly4. As mentioned above, they also demonstrated that the dye-leakage data of these polymers follows their MIC trend, i.e. the inactive poly1 does not lyse vesicles, whereas poly2 and poly3 are increasingly membrane active. (Gabriel, et al., Langmuir 2008, 24, 12489.) Dynamic light scattering was used to monitor the effect of SMAMP addition on the hydrodynamic radius of the vesicles a function of time. While the radii of vesicles exposed to poly1 remained unaltered, those exposed to poly2 and poly3 grew significantly over time (FIG. 45).
This is another indication that poly1 is not membrane-active, whereas poly2 and poly3 clearly are, although the light scattering studies do not capture the significant difference in the MIC90s of those polymers (200 vs. 25 μg/mL). It is also not clear whether the vesicle growth is due to aggregation or vesicle fusion. Consequently, this effect was further studies by fluorescence microscopy on dye-labeled vesicles and stained bacteria cells. (Gabriel, et al., Langmuir 2008, 24, 12489.) In the case of vesicles, giant fluorescent vesicles appeared upon exposure with poly3, while the E. coli bacteria aggregate, as has been observed with other SMAMPs. (Gabriel, et al., Chem. Eur. J. 2009, 15, 433.) These aggregation phenomena highlight that SMAMPs are not just a very complicated detergent. When added to vesicles, detergents would just dissolve the membranes, instead of causing vesicle fusion or aggregation. Also, detergents do not have the ability to differentiate between cells. The membrane-SMAMP interactions were further studies with isothermal calorimetry. These studies revealed that, while no binding interaction between the vesicles and poly1 or poly4, respectively, was observed, there was a strong binding event between the vesicles and both poly2 and poly3. (Gabriel, et al., Langmuir 2008, 24, 12489.) Fitting the data with modeling software revealed that the binding between the vesicle and the SMAMP is entropically favorable, and the overall free enthalpy of binding was about the same for both poly2 and poly3. However, marked differences were observed in the binding stoichiometry—the ratio of vesicle lipids to ammonium groups of the polymer was 0.4 for poly2, and 1.06 for the more active poly3.
Beyond SMAMPs—Cell Penetrating Peptides
Recently, an unusual SMAMP has been discovered (polymer 10 in FIG. 34). While all the previously discussed ROMP-based SMAMPS had amine groups as the positively charged moiety, this polymer contained guanidinium groups and had broad-spectrum antibacterial activity against both Gram-negative (E. coli and S. marcescens, MIC90=6 μg/mL, and 50 μg/mL, respectively) and Gram-positive (S. aureus and B. subtilis, MIC90=12 μg/mL for both) bacteria. Together with a remarkably low hemolytic activity (HC50=1500 μg/mL), this yields a selectivity for E. coli over red blood cells of 250, which is the highest selectivity so far observed for a broad-spectrum SMAMP, as the polymers of series 4 and 8 were only active against Gram-positive bacteria. Using bactericidal kinetics studies (also known as time-kill assays), it was shown that this polymer caused a 5 log reduction in less than 60 minutes at 4× the MIC90, meaning that the polymer is indeed bactericidal and not just bacteriostatic. (Gabriel, et al., Biomacromolecules 2008, 9, 2980.) Comparative dye-leakage studies with poly1, poly3 and polymer 10 showed that, in spite of its low MIC90 value, this polymer did not lyse model membranes (FIG. 46a). Similarly, while the active poly3 caused membrane damage and cell aggregation, as observed in fluorescence microscopy experiments (FIG. 46b, panel C and D), the red fluorescence caused by polymer 10 (FIG. 46b, panel E and F) was undistinguishable from that of the membrane-inactive poly1 (FIG. 46b, panel A and B). These indicate that the antimicrobial activity of this polymer is not due to membrane damage, leakage of cell content, and resulting cell death, as it is the case for the other SMAMPs. The guanidinium groups contained in this SMAMP are also found in poly(arginine) and other cell-penetrating peptides, and such cell-penetrating peptides are known to be able to cross membranes, transport cargo into cells and bind DNA. (Miyatake, et al., J. Am. Chem. Soc. 2006, 128, 12420; Henriques, et al., Biochemical J. 2006, 399, 1; Rothbard, et al., J. Med. Chem. 2002, 45, 3612; Futaki, et al., J. Biol. Chem. 2001, 276, 5836; Mitchell, et al., J. Peptide. Res. 2000, 56, 318; Sakai, et al., Soft Mater 2006, 2, 636; Pantos, et al., Biochim. Biophys. Acta 2008, 1778, 811; Schroeder, et al., J. Med. Chem. 2008, 51, 376; Deglane, et al., Chem Bio Chem 2006, 7, 684; Fillon, et al., J. Am. Chem. Soc. 2005, 127, 11798; Funhoff, et al., Bioconjugate Chem. 2004, 15, 1212.) In the light of this body of literature and the above results, it was postulated that polymer 10 may be antibacterially active by first penetrating the cell membrane without causing damage, and then interacting with an intracellular target, potentially the bacteria DNA, which leads to the cell's death. (Gabriel, et al., Biomacromolecules 2008, 9, 2980.)
Using ring-opening metathesis polymerization as a synthesis platform, a large variety of synthetic mimics of antimicrobial peptides (SMAMPs) was obtained. By carefully tuning the overall hydrophobicity and charge density of these molecules, polymers with tailor-made properties, from inactive/non-hemolytic via active/non-hemolytic to active/toxic, were obtained.
f. Hydrophilic Modifications and Hemolytic Activity
Here is disclosed an approach to fine-tune the properties of this interesting polymer, Poly3, by incorporating hydrophilic, biocompatible functionalities including sugar, zwitterionic, and polyethylene glycol (PEG) moieties (FIG. 47). Carbohydrate based sugars, in addition to being used as solubilizing agents, play an important role in application areas of bio-molecular chemistry. (Dwek, Chemical Reviews 1996, 96, 683-720; Goto, et al. Journal of Controlled Release 1994, 28, 223-33.) PEG and various zwitterions have been widely used for improving the water-solubility of compounds. (Travert-Branger, et al. Langmuir 2008, 24, 3016-3019; Guillemet, et al. Langmuir 2006, 22, 1875-1879.). Having similar properties, all these groups have been routinely employed in conjunction with each other as solubilizing agents in many biological systems, where they were found to be non-toxic, thus making them good candidates for this study. (Travert-Branger, et al. Langmuir 2008, 24, 3016-3019; Narain, et al. Journal of Polymer Science, Part A: Polymer Chemistry 2006, 44, 6558-6568; Yasugi, et al. Macromolecules 1999, 32, 8024-8032.) The importance of repeat unit level facial amphiphilicity, rather than globally (or the entire molecule), on the selectivity of SMAMPs was recently emphasized by Lienkamp et al. (Lienkamp, et al. Journal of the American Chemical Society 2008, 130, 9836-9843.) Easily synthesizable structures that contain both hydrophobic and hydrophilic units on the same monomer allowed successful tuning of the SMAMPs' selectivities. Gabriel et al. also emphasized the advantages of the facial amphiphilicity approach at the repeat unit level versus random copolymers of hydrophilic and hydrophobic monomers. (Gabriel, et al. Biomacromolecules 2008, 9, 2980.)
Here, facially amphiphilic monomers having the same hydrophobic backbone, but various hydrophilic pendant groups, were incorporated into Poly3 via ROMP (FIG. 47). The resulting effects on the overall biological activities of these polymers were studied via determination of MICs coupled with hemolytic activity (HC50) studies.
Examples
Materials and Instrumentation
Poly(ethylene glycol) methylether acrylate (PEG-acrylate) (99%), triphenylphosphine (Ph3P) (99%), diisopropylazodicarboxylate (DIAD) (94%), 2nd generation Grubbs' catalyst, and 3-bromopyridine (99%) were purchased from Sigma-Aldrich and used as received. Maleimide (98%) was obtained from Alfa Aesar and used as received. 1,2:3,4-Di-O-isopropylidene-D-galactopyranose (97%), N,N-dimethylethanolamine (99%), and ethylvinyl ether (EVE) (99%) were purchased from Acros Organics and used without further purification. 4-Bromobutanoic acid tert-butyl ester (95%) was purchased from Astatech, Inc. and used as is. Ethyl acetate, hexane, anhydrous diethyl ether, methanol, sodium bicarbonate (NaHCO3), citric acid, and trifluoroacetic acid (TFA) were purchased from Fisher Scientific and used as received. Tetrahydrofuran (THF) was obtained from Fisher Scientific and was distilled form sodium/benzophenone under nitrogen before use. Dichloromethane (DCM) (Fisher Scientific) was distilled from CaH2, under nitrogen. 3rd generation Grubbs' catalyst (G3) (dichloro-di(3-bromopyridino)-N,N′-dimethylenoimidazolino-Ru═CHPh) was synthesized according to a previously published procedure. (Love, et al. Angewandte Chemie, International Edition 2002, 41, 4035-4037.)
1H NMR and 13C NMR spectra were recorded on a Bruker DPX300 spectrometer (Bruker, Madison, Wis.). Gel permeation chromatography (GPC) was performed using a Polymer Laboratories PL-GPC50 (Amherst, Mass.) instrument equipped with a PL Gel 5 μm pre-column and two 5 μm Mixed D columns. The mobile phase was THF with a flow rate of 1 mL/min and toluene was used as a flow marker. The instrument was calibrated with narrow molecular weight polystyrene standards using a Knauer RI detector. High resolution mass spectra were obtained on a JEOL JMS 700 instrument (JEOL, Peabody, Mass.).
Synthesis of M1
Isopropylfulvene (7.0 g, 58.3 mmol) and maleimide (4.7 g, 48.6 mmol) were charged in a Schlenk flask and dissolved in toluene (100 mL). (Ilker, et al. Macromolecules 2004, 37, 694-700.) The reaction mixture was placed in an oil bath at 135° C., stirred for 12 h, then cooled to room temperature. Excess toluene was removed under reduced pressure to yield a mixture of exo/endo (9/1 by 1H NMR) product. The pure exo product 1 was isolated by crystallization from diethyl ether as a colorless solid. Yield: 60%. 1H NMR (300 MHz, CDCl3, ppm) δ: 0.87 (q, J=6.6 Hz, 6H), 2.3 (m, 1H), 2.77 (m, 2H), 3.37 (s, 1H), 3.74 (s, 1H), 4.76 (d, J=9.6 Hz, 1H), 6.4 (m, 2H), 8.24 (s, 1H). 13C NMR (75 MHz, CDCl3, ppm) δ: 22.97, 23.47, 28.10, 44.53, 48.61, 49.04, 120.90, 136.99, 137.54, 143.62, 178.10, 178.21.
Compound 1 (2.0 g, 9.2 mmol), Ph3P (2.4 g, 9.2 mmol), and 1,2:3,4-Di-O-isopropylidene-D-galactopyranose (2.4 g, 9.2 mmol) were dissolved in dry THF (50 mL) in a 250 mL round bottom flask, under N2 atmosphere. The solution was then cooled to 0° C. in an ice bath. DIAD (1.8 g, 1.8 mL, 9.2 mmol) was added drop wise to the cooled reaction mixture. After addition was complete, the ice bath was removed and the solution was stirred at room temperature for 24 h. Excess THF was removed under reduced pressure to yield a yellow, oily product, which was recrystallized from toluene. After discarding the white precipitate the excess toluene from the filtrate was removed under reduced pressure to yield an oily product. The pure M1 was isolated by crystallization from cold diethyl ether to afford a colorless solid. Yield: 50%. 1H NMR (300 MHz, CDCl3) δ: 0.77 (q, J=4.14 Hz, 3H), 0.85 (d, J=6.59 Hz, 3H), 1.29 (d, J=17.7 Hz, 6H), 1.45 (d, J=9.2 Hz, 6H), 2.23 (m, 1H), 2.76 (m, 2H), 3.27 (d, J=2.6 Hz, 1H), 3.34 (d, J=2.6 Hz, 1H), 3.69 (d, J=3.0 Hz, 1H), 3.95 (m, 1H), 4.17 (d, J=8.3 Hz, 2H), 4.26 (q, J=2.4 Hz, 1H), 4.58 (d, J=2.5 Hz, 1H), 4.65 (d, J=9.6 Hz, 1H), 5.43 (d, J=5.1 Hz, 1H), 6.38 (m, 2H). 13C NMR (75 MHz, CDCl3) δ: 23.82, 24.45, 25.33, 25.79, 26.54, 26.73, 28.89, 39.76, 45.24, 48.32, 48.64, 49.71, 64.87, 71.17, 71.64, 72.23, 97.05, 109.54, 110.56, 121.43, 137.80, 138.65, 144.81, 177.67, 178.69. HR-MS (FAB): calculated 459.53. found 460.20.
Synthesis of M2
Compound 1 (4.0 g, 18.4 mmol), Ph3P (4.8 g, 18.4 mmol), and N,N-dimethylethanolamine (1.6 g, 1.8 mL, 18.4 mmol) were dissolved in dry THF (150 mL) in a 250 mL round-bottom flask, under N2 atmosphere. The solution was then cooled to 0° C. in an ice bath. DIAD (3.7 g, 3.6 mL, 18.4 mmol) was added drop-wise to the cooled reaction mixture. After addition was complete, the ice bath was removed and the solution was stirred at room temperature for 24 h. Excess THF was removed under reduced pressure. The remaining product was extracted from ethyl acetate into citric acid solution (pH=4.0), pH of the solution was adjusted to 13.0 by the addition of saturated NaHCO3 solution. Compound 2 was then extracted with hexane/diethyl ether mixture. The excess solvent was removed under reduced pressure to yield a colorless powder. Yield=70%. 1H NMR (300 MHz, CD3OD) δ: 0.86 (q, J=6.8 Hz, 6H), 2.27 (m, 7H), 2.5 (t, J=6.7 Hz, 2H), 2.81 (dd, J=7.2 Hz, 2H), 3.26 & 3.66 (s, 2H), 3.53 (t, J=6.7 Hz, 2H), 4.63 (d, J=9.4 Hz, 1H), 6.46 (m, 2H). 13C NMR (75 MHz, CD3OD) δ: 23.49 & 24.01, 29.21, 36.93, 45.46 & 45.53, 49.07 & 49.13, 50.08, 57.06, 120.9, 138.22 & 138.95, 146.44, 179.39 & 179.52.
Compound 2 (2.0 g, 6.9 mmol) was dissolved in 50 mL of dry THF. To the solution 4-bromobutanoic acid ten-butyl ester (3.1 g, 13.8 mmol) was added and the reaction mixture was stirred at 50° C. for 36 h. The product was filtered, washed with excess dry THF, and dried under vacuum to yield M2 as a colorless powder. Yield=65%. 1H NMR (300 MHz, CD3OD) δ: =0.88 (q, J=6.7 Hz, 6H), 1.48 (s, 9H), 2.04 (m, 2), 2.29 (m, 1H), 2.43 (t, J=6.6 Hz, 2H), 2.9 (m, 2H), 3.19 (m, 6H), 3.45 (m, 4H), 3.72 (m, 2H), 3.91 (t, J=7.2 Hz, 2H), 4.67 (d, J=9.2 Hz, 1H), 6.48 (m, 2H). 13C NMR (75 MHz, CD3OD) δ: 19.8, 24.3, 25.2, 29.2, 30.1, 32.8, 33.7, 46.4, 52.8, 61.5, 65.3, 83.0, 121.9, 139.1, 139.8, 147.1, 173.8 (ester, —C═O), 179.7 (imide, —C═O). HR-MS (FAB): calculated 431.59. found 431.29.
Homo- and Copolymer Synthesis and Deprotection
Homopolymerization of M1 and M2: All the monomers were polymerized using 3rd generation Grubbs' catalyst as an initiator. The same procedures were followed for the homopolymerization of M1 and M2 and the deprotection of the corresponding polymers. In a typical experiment, M1 (0.4 g, 0.8 mmol) and G3 (0.013 g, 0.014 mmol) were weighed in separate reaction flasks and purged with N2 gas. The monomer was dissolved in 1 mL and the catalyst was dissolved in 0.5 mL of THF. In the homopolymerization of M2, the monomer was dissolved in 1 mL of methanol due to its insolubility in THF. Both solutions were degassed by three freeze-pump-thaw cycles. After warming the solutions to room temperature, the monomer solution was cannulated into the catalyst solution. The reaction mixture was stirred at 60° C. for 2 h. The reaction was cooled in an ice bath and terminated by the addition of EVE (0.3 g, 0.6 mL, 6.3 mmol). The reaction mixture was further stirred for 1 h. The resultant homopolymers, Poly1 and Poly2, were precipitated from excess diethyl ether to yield dark brown powders. Yield=95%.
Poly1: 1H NMR (300 MHz, CDCl3) δ: 0.92 (br m, 6H), 1.27 (br m, 3H), 1.33 (br m, 3H), 1.48 (br m, 3H), 1.59 (br m, 3H), 2.41 (br m, 1H), 3.04 (br m, 2H), 3.37 (br m, 2H), 3.77 (br m, 1H), 3.99 (br m, 1H), 4.18 (br m, 2H), 4.27 (br m, 1H), 4.59 (br m, 1H), 5.16 (br m, 1H), 5.42 (br m, 2H), 5.61 (br m, 1H). Poly2: 1H NMR (300 MHz, DMSO-d6) δ: 0.89 (br m, 6H), 1.42 (br m, 9H), 1.91 (br m, 2H), 2.32 (br m, 3H), 2.82 (br m, 2H), 3.15 (br m, 6H), 3.43 (br m, 4H), 3.64 (br m, 2H), 3.8 (br m, 2H), 4.6 (d, 1H), 5.18 (br m, 2H, cis), 5.33 (br m, 2H, trans).
Deprotection of Poly1 and Poly2: The deprotection reactions of all the polymers were done as follows: in a typical procedure, 0.25 g of Poly1 was dissolved in 3 mL of neat TFA and stirred at room temperature for 24 h. The deprotected polymer was precipitated from diethyl ether. The deprotected Poly1 was dried under vacuum for 24 h, then dissolved in 5 mL methanol and 25 mL RO water, and freeze-dried to remove all organic solvents. Yield=85%.
Deprotected Poly1: 1H NMR (300 MHz, DMSO-d6) δ: 0.87 (br m, 6H), 3.21 (br 2H), 3.49 (br m, 4H, —OH), 4.09 (br m, 1H), 4.84 (br m, 1H), 4.99 (br m, 1H), 5.14 (br m, 1H), 5.35 (br m, 1H), 5.58 (br m, 1H), 7.32 (br m, 2H, aromatic), 7.41 (br m, 3H, aromatic). Deprotected Poly2: 1H NMR (300 MHz, DMSO-d6) δ: 0.88 (br m, 6H), 1.92 (br m, 2H), 2.33 (br m, 3H), 2.81 (br m, 2H), 3.12 (br m, 6H), 3.4 (br m, 4H), 3.7 (br m, 4H), 5.2 (br m, 2H, cis), 5.3 (br m, 2H, trans).
Copolymerizations of M1 and M2 with M3: The same procedure described above for the homopolymerization of Poly1 and Poly2 was used for the synthesis of all Poly3-co-Poly1 and Poly3-co-Poly2 series. Corresponding monomers were mixed in the same reaction vessel prior to reacting with the catalyst. Copolymers with varying compositions were synthesized by adjusting the molar amounts of the monomers at the desired ratios.
The monomer composition of the copolymers was quantified by 1H NMR analysis. For the Poly3-co-Poly1 samples copolymer composition was determined by comparing the integration value of methyl protons (6H, at 0.92 ppm) at the backbone to the —NH3 (3H, at 7.88 ppm) protons of M3, after deprotection. In the case of Poly3-co-Poly2 copolymer composition was determined from the comparison of the integration value of methyl protons (6H, at 0.92 ppm) at the backbone to the methyl protons (9H, at 1.39 ppm) of the Teri-butyl group of the M2.
Poly3-co-Poly1 (50:50): 1H NMR (300 MHz, CDCl3) δ: 0.92 (br m, 6H), 1.27 (br m, 3H), 1.33 (br m, 3H), 1.41 (br m, 9H), 1.47 (br m, 6H), 2.41 (br m, 1H), 3.04 (br m, 2H), 3.29 (br 2H), 3.61 (br m, 2H), 3.78 (br m, 1H), 3.98 (br m, 1H), 4.17 (br m, 2H), 4.27 (br m, 1H), 4.60 (br m, 1H), 5.17 (br m, 2H), 5.42 (br m, 1H), 5.62 (br m, 1H). Deprotected Poly3-co-Poly1 (50:50): 1H NMR (300 MHz, DMSO-d6) δ: 0.88 (br m, 6H), 2.95 (br m, 2H), 3.21 (br m, 2H), 3.43 (br m, 4H, —OH), 3.61 (br m, 2H), 3.87 (br m, 1H), 4.07 (br m, 1H), 4.85 (br m, 1H), 5.02 (br m, 1H), 5.16 (br m, 1H), 5.29 (br m, 2H), 5.46 (br m, 1H), 5.59 (br m, 1H), 7.32 (br m, 2H, aromatic), 7.40 (br m, 3H, aromatic), 7.88 (br m, 3H, —NH3+).
Poly3-co-Poly2 (50:50): 1H NMR (300 MHz, CD3OD) δ: 0.92 (br m, 6H), 1.39 (br m, 9H), 1.44 (br m, 9H), 2.03 (br m, 1H), 2.39 (br m, 2H), 3.19 (br m, 2H), 3.29 (br m, 6H), 3.52 (br m, 2H), 3.95 (br m, 2H), 5.54 (br m, 1H), 5.71 (br m, 1H), 7.28 (br m, 2H), 7.38 br m, 2H). Deprotected Poly3-co-Poly2 (50:50): 1H NMR (300 MHz, DMSO-d6) δ: 0.87 (br m, 6H), 1.91 (br m, 1H), 2.33 (br m, 2H), 3.09 (br m, 2H), 3.39 (br m, 6H), 3.61 (br m, 2H), 3.91 (br m, 2H), 5.54 (br m, 1H), 5.62 (br m, 1H), 7.5 (br m, 5H), 8.04 (br m, 3H).
Synthesis of Poly3-co-PolyP: A post-functionalization approach was utilized for the synthesis of Poly3-co-PolyP copolymer. Poly3 was synthesized and deprotected according to a previous literature procedure. The repeat unit determination was done by GPC analysis. In a typical reaction, deprotected Poly3 (0.15 g, 0.4 mmol) was dissolved in 10 mL DMSO. Triethyl amine (0.02 g, 0.20 mmol) was added to the reaction mixture and stirred for 30 min followed by the addition of PEG-acrylate (0.15 g, 0.3 mmol). The reaction mixture was stirred at room temperature for 24 hours. The pure polymer was isolated by crystallization three times from cold diethyl ether affording a light brown solid. Yield: 70%.
Copolymer composition was determined by the ratio of the integration values of methyl protons of the backbone (6H) appearing at 0.75 ppm to the methylene peaks next to the ester linkage of the PEG-acrylate (2H) appearing at 4.07 ppm. 1H NMR (300 MHz, D2O) δ: 0.85 (br m, 6H), 2.42 (br m, 1H), 2.84 (br m, 2H), 3.05 (br m, 2H), 3.15 (br m, 3H, —CH3 of PEG), 3.46 (br m, 32H), 3.55 (br m, 2H), 4.07 (br m, 2H, —COOCH2—), 5.18 (br m, 1H), 5.48 (br m, 1H), 7.10 (br m, 5H, aromatic).
Polymer Synthesis and Characterization
Facial amphiphilicity at the repeat unit level has an important effect on the overall selectivity of the SMAMPs compared to random copolymers of hydrophobic and hydrophilic monomers, yielding an amphiphilic structure. (Ilker, et al. J. Am. Chem. Soc. 2004, 126, 15870-15875; Lienkamp, et al. J. Am. Chem. Soc. 2008, 130, 9836-9843; Gabriel, et al. Biomacromolecules 2008, 9, 29808).; Sambhy, et al. Angew. Chemie, Int'l Ed. 2008, 47, 1250-1254.) Selectivity values can be improved by fine tuning of these systems by copolymerization. Monomers composed of hydrophobic backbone and varying biocompatible hydrophilic pendant moieties were designed and synthesized (FIG. 47).
ROMP has been successfully used to polymerize norbornene based monomers bearing various functionalities to obtain well-defined systems. (Eren, et al. Macromolecular Chemistry and Physics 2008, 209, 516-524; Ilker, et al. Macromolecules 2004, 37, 694-700; Alfred, et al. Journal of Polymer Science, Part A: Polymer Chemistry 2008, 46, 2640-2648.) However, it has been also reported that the presence of particular additives such as pyridine, secondary amines, thiols, or benzoic acid, can significantly slow or shut down the kinetics of ROMP. (Slugovc, Macromolecular Rapid Communications 2004, 25, 1283-1297; Colak, et al. Macromolecules 2008, 41, 8436) Therefore, to prevent these retardation effects and to be able to successfully perform ROMP, a protecting group approach was utilized to synthesize the highly functionalized monomers (M1-M3). In addition to facilitating the polymerization process, the protective group approach also provided ease in the later polymer characterization by gel permeation chromatography (GPC) and nuclear magnetic resonance (NMR). Upon complete polymerization, the protective groups were removed to yield the corresponding antimicrobially active polymers.
The first step of the monomer synthesis is the Diels-Alder reaction of maleimide with isopropylfulvene to yield product 1. M1, the sugar functional norbornene based monomer, was synthesized via Misunobu coupling of 1 with the corresponding alcohol (Scheme 6). (Mitsunobu, Synthesis 1981, 1, 10-28.) The same reaction conditions were employed to obtain 2, which was further alkylated with 4-bromobutanoic acid tert-butyl ester to yield the positively charged M2 as the quaternary ammonium salt, leading to the zwitterionic functionality upon deprotection (Scheme 6). (Colak, et al. Macromolecules 2008, 41, 8436.) M3 was synthesized according to a previously published procedure. (Ilker, et al. Macromolecules 2004, 37, 694-700.) All monomers were characterized using NMR techniques. FIGS. 48 and 49 show the resulting 1H NMR spectra of M1 and M2, respectively.




All homopolymers (Poly1, Poly2, and Poly3) and copolymers (Poly3n-co-Poly1m, Poly3n-co-Poly2m, and Poly3n-co-PolyPm, where n indicates the mol fraction of M3 and m indicates the mol fraction of M1, M2, or (PEG functionality) in the copolymer composition) were synthesized using 3rd generation Grubbs' catalyst as an initiator. Upon reaction completion, the polymerizations were terminated via addition of excess ethyl vinyl ether. ROMP yielded polymers with approximate molecular weights of 3 kDa and narrow polydispersity indices ranging from 1.08 to 1.15, as determined by GPC (See Table 20). All polymers were deprotected using trifluoroacetic acid to obtain the antimicrobially active polymers.
Syntheses of Poly3n-co-Poly1m (sugar functional) and Poly3n-co-Poly2m (zwitterionic functional) copolymers were done via random copolymerization of the corresponding facially amphiphilic monomers. To vary the amphiphilicity of the resulting polymers, different molar ratios of the monomers were used in the copolymerization reactions (Table 20). Depending on the solubility of the monomers and the catalyst, different solvent systems were used to provide homogeneous reaction media for all polymerizations. Poly3n-co-Poly1m was synthesized using only THF, whereas Poly3n-co-Poly2m was synthesized in a THF-methanol solvent mixture (Scheme 7). The copolymerization kinetics were studied via 1H NMR analysis by recording spectra every 30 minutes, until the reaction was completed within 2 h. The constant decrease in the intensity of the broad peak at 6.5 ppm, corresponding to the alkene functionalities of both norbornene monomers, indicated successful copolymer formation. The complete disappearance of the same peak and the simultaneous appearance of two new peaks at approximately 5.4 and 5.6 ppm, corresponding to the cis and trans conformations of double bonds of the polymer backbone, indicated complete conversion.




Molecular weight characterization of Poly3n-co-Poly1m was done by gel permeation chromatography (GPC) with THF as the eluent, and calibration was done against polystyrene standards. The experimental molecular weights were in accordance with the theoretical (calculated) values (Table 20). Representative GPC traces of Poly1 (homopolymer of M1) and the copolymer, Poly30.5-co-Poly10.5, are shown in FIG. 50.
It is well known that GPC of charged polymers is challenging due to formation of ionic aggregation or interactions with the stationary phase of the chromatography column. (Volet, et al. Journal of Liquid Chromatography 1994, 17, 559-577.) Therefore, instead of performing GPC analysis on the charged polymers, 1H NMR end group analysis was used for the molecular weight determination of Poly3n-co-Poly2m copolymers. The ratio of the phenyl end group integral value to the methyl protons of the isopropylfulvene backbone yielded the degree of polymerizations, which was used to calculate the total molecular weight of the polymers.
Prior to any functionalization, M3 was polymerized to yield Boc-protected Poly3 which was then deprotected with TFA to yield the antimicrobially active polymer according to the previously published procedure. (Ilker, et al. Macromolecules 2004, 37, 694-700.) Positively charged ammonium groups were neutralized to yield the amine functionalities, which were then used to react with the acrylate end functionalized PEG via Michael-addition (Scheme 8). (Colak, et al. Journal of Applied Polymer Science 2007, 104, 2244-2253.) In the 1H NMR spectra of the purified polymers no residual acrylate peaks were observed and the appearance of a peak at 4.07 ppm corresponding to the methylene peaks next to the ester linkage of the PEG-acrylate indicated the successful addition.




To assess the biological activities of the polymers, the minimum inhibitory concentrations (MIC) against Eschericia coil (E. coli) and Staphylococcus aureus (S. aureus) and hemolytic activity (HC50) were determined according to standard procedures. (Rennie, et al. Journal of Industrial Microbiology & Biotechnology 2005, 32, 296-300.) All of the analyses were performed using the deprotected polymer samples. FIG. 51 shows the results of the Poly3n-co-Poly1m series. As the sugar content of the copolymer was increased from 0% to 100% (referring to the Poly1 homopolymer), a linear decrease in the biological activity was observed; MIC values increased from 50 to 200 μg/mL. This loss of activity might be attributed to the fact that, as the number of repeat units containing sugar residues is increased, the overall positive charge of the polymer is being reduced. It should also be noted that the sugar functional homopolymer, Poly1, is inactive towards bacteria. However, due to its biocompatible nature and overall neutral charge, its toxicity toward mammalian cells, HC50=2000 μg/mL, is much lower than that of Poly3, HC50<1 μg/mL. Thus, while increasing the sugar content of the Poly3n-co-Poly1m copolymers improved their hemolytic properties, it caused a reduction of the antibacterial activities.
The same behavior was observed for the Poly3n-co-Poly2m copolymer series until equal co-monomer composition was reached (Poly30.5-co-Poly20.5) (FIG. 52). As the number of zwitterionic residues was further increased, loss of antimicrobial activity along with higher hemolytic activity was observed. A possible explanation for the loss of activity with higher zwitterionic content could be the increased possibility of forming intra- or interchain associations, causing significant conformational changes in the polymer backbone, thus affecting overall facial amphiphilicity.
The highest biological activity was observed for Poly3n-co-PolyPm samples (FIG. 53). Upon incorporation of PEG groups up to 30%, lower MIC values were obtained compared to Poly3, accompanied with a slight improvement in the hemolytic activities. Compared to the sugar or zwitterionic functionalized copolymers, the presence of an amine (primary or secondary) on every repeat unit in the Poly3n-co-PolyPm structures, indicate that the overall charge remained constant even as PEG content increased. However, after 50% functionalization the same loss of activity was observed as for the other polymers.
Table 20 summarizes the resulting MIC and HC50 values of all the studied SMAMPs, as well as the selectivity values, calculated taking the ratio of HC50 and MIC. When the results are compared to that of Poly3, generally the same behavior is observed for all the samples; with increasing hydrophilicity, biological activity is reduced. However, due to the biocompatible nature of these hydrophilic groups, improvements of the hemolytic activities were observed (from <0.02 up to 7.5 μg/mL for Poly30.5-co-Poly20.5), thus higher selectivities were obtained.







TABLE 20







Biological activity data of Poly3n-co-Poly1m copolymers.












Copolymer




Selectivity


Ratio
Mn

MIC [μg/mL]
HC50
(HC50/MIC)














(n/m)
(kDa)
PDI
E. coli
S. aureus
[μg/mL]
E. coli
S. aureus

















Poly3
2.8a
1.15
<50
<50
<1
<0.02
<0.02


Poly30.7-co-
2.9a
1.10
100
100
50
0.2
0.2


Poly10.3


Poly30.5-co-
2.9a
1.10
200
200
100
0.5
0.5


Poly10.5


Poly30.3-co-
3.0a
1.09
>200
>200
400
<2.0
2.0


Poly10.7


Poly1
3.0a
1.08
>200
>200
2000
<10.0
<10.0


Poly30.7-co-
3.6b
n.d.
100
100
250
2.5
2.5


Poly20.3


Poly30.5-co-
4.1b
n.d.
150
200
1500
10.0
7.5


Poly20.5


Poly30.3-co-
4.3b
n.d.
>200
>200
750
<3.8
<3.8


Poly20.7


Poly2
4.0b
n.d.
>400
>400
750
<1.9
<1.9


Poly30.8-co-
3.3d
1.15
15
25
<50
<3.3
<2


PolyP0.2


Poly30.7-co-
3.6d
1.15
20
100
<50
<2.5
<0.2


PolyP0.3


Poly30.5-co-
4.1d
1.15
>200
>200
1000
<5
<5


PolyP0.5





aMn determined by GPC of the protected polymers.


bMn determined by 1H NMR of the deprotected polymers.


cMn reported after subtracting the corresponding weights of the protecting groups.


dMn includes the molecular weight of the PEG chains.













TABLE 21







Summary of M/I ratios of all the Poly3-co-Poly1 and Poly3-co-Poly2 copolymer series


and their corresponding homopolymers.













Monomer







Concentrations
Initiator
Degree of





(mmol/mL)
Concentrations
Polymerization
Mn theoretical
Mn experimental













Sample
M3
MXa
(mmol/mL)
(DP) = [M]/[I]
(kDa)
(kDa)
















Poly3
0.37
—
0.044
8.22
2.9
2.8b


Poly30.7-co-
0.14
0.06
0.027
7.40
2.9
2.9b


Poly10.3


Poly30.5-co-
0.11
0.11
0.030
7.33
3.0
2.9b


Poly10.5


Poly30.3-co-
0.08
0.12
0.027
7.40
3.1
3.0b


Poly10.7


Poly1
—
0.22
0.033
6.53
3.0
3.0b


Poly30.7-co-
0.17
 0.072
0.026
9.3
3.5
3.6c


Poly20.3


Poly30.5-co-
0.11
0.12
0.021
10.9
4.2
4.1c


Poly20.5


Poly30.3-co-
 0.062
0.14
0.019
10.6
4.2
4.3c


Poly20.7


Poly2
—
0.23
0.033
6.95
3.0
4.0c





aX-1 for Poly1, 2 for Poly2.


bMn determined by GPC of the protected polymers.


cMn determined by 1H NMR of the deprotected polymers.






g. Charge Density and Hemolytic Activity

A series of norbornene monomers were designed that carry one, two, and three amine groups (FIG. 55). ROMP of the Boc protected monomers, followed by deprotection, resulted in SMAMPs with multiple amine groups on each repeat unit (FIG. 56).
The syntheses of A1 and B1 were previously reported in the literature (FIG. 55). (Ilker, et al. J. Am. Chem. Soc. 2004, 126, 15870-15875.) The other monomers were prepared by Mitsunobu coupling of norbornene derivates with the corresponding alcohols. The Boc-protected polyA1-3 and polyB1-3 series (FIG. 56) were prepared via ROMP using Grubbs' 3rd generation catalyst. The number average molecular weights (Mn) of all polymers were approximately 3 kDa and the polydispersity indices (PDI) were 1.1 as measured by GPC. The amine protecting groups were then removed by treating the polymers with trifluoroacetic acid to afford the cationic polyA1-3 and polyB1-3 series. The synthesis and characterization of these monomers and polymers are reported in the supporting information.
Hemolytic and Antibacterial Activities
The biological activity of polyA1 indicates that it is non-selectively active, meaning it disrupts both mammalian and bacterial cell membranes. (Ilker, M. F.; Nusslein, K.; Tew, G. N.; Coughlin, E. B. J. Am. Chem. Soc. 2004, 126, 15870-15875.) The toxicity of polyA1 was attributed to its hydrophobic nature; the polymer backbone contains the isobutenyl functionality which increased the overall hydrophobicity of the polymer. The effect of doubling and tripling the number of amine groups per monomer on the biological activity of polyA1-3 was investigated.
The results show that increasing the number of amine groups significantly reduces the hemolytic behavior of polyA, (FIG. 57A). The hemolytic activities were tested against human red blood cells (RBCs) and reported as HC50 values. (HC50: Is the concentration required to lyse 50% of human red blood cells.) PolyA1 is very hemolytic (<1 μg/mL). Upon doubling and tripling number of amine groups the polymers become non-hemolytic with HC50 values of 700 and 500 μg/mL for polyA2 and polyA3, respectively. Meanwhile, the antimicrobial activities of polyA1-3, reported as the minimum inhibitory concentration required to restrain 90% of bacterial growth (MIC90), were tested against Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria (FIGS. 57B and 573C). The MIC90s show no significant change in the antimicrobial activity upon doubling or tripling number of amine groups.
The above results show that increasing the number of amine groups, while keeping the hydrophobic moieties the same has a favorable impact on the hemolysis but little influence on the antimicrobial activities of polyA1-3. This observation highlights the significance of optimizing the balance between the hydrophobic and hydrophilic components of the polymer. Because polyA2 and polyA3 were much less hemolytic while retaining their antimicrobial activity, the influence of increasing the number of amine groups on a non-active polymer, polyB1, was investigated. The results show no significant change in the hemolytic behavior of the polyB1-3 series and they remain inactive against RBCs, as expected, due to the overall hydrophilic nature of these polymers (FIG. 57A). The antimicrobial activities of these new polyB derivatives remain inactive against E. coli (FIG. 57B); however, polyB2 and polyB3 become active against S. aureus as the number of amine groups is doubled or tripled. This was manifested by the decrease in the MIC90 values from >200 μg/mL for polyB1 to 25 and 15 μg/mL for polyB2 and polyB3, respectively.
Model Membrane Studies
Understanding the interactions between SMAMPs and different cell membranes is of important scientific concern, although a complicated, multi-component problem. (Som, et al. Biopolymers 2008, 90, 83-93.) Apart from the differences in intracellular components and membrane proteins, bacterial cell walls and mammalian cell membranes are also very distinct with respect to their phospholipid compositions. (Som, et al. J. Phys. Chem. B 2008, 112, 3495-3502.) The outer surface of RBC membranes is devoid of anionic lipids and composed predominantly of zwitterionic phosphatidylcholine (PC). On the other hand, Gram-positive bacterial (e.g., S. aureus) membranes are mainly composed of anionic phospholipids (phosphatidylglycerol (PG) and cardiolipin (CL)), while the membranes of most Gram-negative bacteria are rich in zwitterionic lipid phosphatidylethanolamine (PE). For example the plasma membrane of E. coli is composed of 80% PE and  ̃20% PG. (Gabriel, et al. Mater. Sci. Eng., R2007, 57, 28-64.) The specific role of PE in SMAMP induced pore formation was recently reported. (Yang, et al. J. Am. Chem. Soc. 2007, 129, 12141-12147.)
To investigate the membrane activity of these new SMAMPs, polymer induced leakage from three different calcein dye loaded vesicles were examined: (i) 100% PC vesicles V1 to mimic RBC outer leaflets, (ii) 20/80 PG/PE vesicles V2 to mimic E. coli lipid membranes, and (iii) 100% CL vesicles V3 to mimic S. aureus lipid membranes. Membrane activity of these polymers against PC vesicles V1 (RBC mimic) was monitored and is shown in FIG. 58. The hemolytic polyA1 is highly membrane active against PC vesicles; however, the leakage activity is significantly reduced upon doubling (polyA2) or tripling (polyA3) the number of amine groups. The entire non-hemolytic polyB1-3 series remains inactive against PC vesicles. All of these experiments correlate with the hemolytic activities of the polyA1-3 and polyB1-3 series.
To understand the interactions of these SMAMPs with bacterial membrane mimics, calcein dye leakage from PG/PE vesicles (V2) and CL vesicles (V3) was studied. PolyA1 showed 80% leakage activity against both V2 and V3; doubling the number the of amine groups (polyA2) slightly reduces the leakage activity by 15% and 5% for V2 and V3, respectively (FIGS. 59A and 59B). Upon tripling the number of amine groups (polyA3) the leakage activity against V2 is similar to polyA2; however, the leakage activity against V3 is much less (FIGS. 59A and 59B). In contrast, polyB1 remains inactive against both V2 and V3, whereas polyB2 and polyB3 show increasing leakage activity from these vesicles with polyB2 being the most active (FIGS. 59C and 59D).
If membrane activity was predominantly based on hydrophobicity, polyA, would be active and polyB1 would be inactive against RBCs. By doubling or tripling the number of amine groups in the polyA1-3 series, the activity against PC vesicles is reduced, consistent with the observed HC50 values. In contrast, due to the hydrophilic nature of polyB1, the polymer shows no affinity towards RBCs or PC membranes and remains non-hemolytic upon increasing the number of amine groups.
While antimicrobial activity of the polyA1-3 series remains unchanged upon increasing charge density, the polyB1-3 series shows no activity against E. coli but enhanced activity against S. aureus (FIGS. 57B and 57C). This phenomenon could be due to the difference in the structural makeup of their cell membranes. Most Gram-positive bacteria are rich in negatively charged lipids. It is also possible that these highly hydrophilic cationic polymers (polyA3 and polyB2-3) interact with the outer murein layer which is composed of negatively charged peptidoglycans. (Navarre, et al. J. Biol. Chem. 1999, 274, 15847-15856.) The model membrane activities generally correlate with the biological activity of these SMAMPs. The unusual decrease in membrane activity for polyA3 and polyB2-3 against anionic CL vesicles (FIGS. 59B and 59D) indicate that polyA3 and polyB2-3 may not be membrane-lipid dependent, but rather interact with other extra-cellular and/or intra-cellular targets.
In order to increase the selectivity of SMAMPs there has to be a balance between the hydrophobic and hydrophilic moieties. In the case of polyA1-3, non-toxic polymers are obtained by doubling or tripling the number of amine group. The hemolysis of polyA1 was improved from <1 μg/mL to 700 μg/mL for polyA2. The polyB1-3 series remains non-hemolytic as expected; however, polyB2 and polyB3 show enhanced activity against Gram-positive bacteria. This enhanced antimicrobial activity of polyB2 and polyB3 coupled to their lack of hemolysis and membrane activity in the vesicle studies suggest that these SMAMPs may have other cell targets besides the membrane.
Examples
Instrumentation. NMR spectra were recorded on Bruker DPX-300 MHz (1H NMR; 13C NMR, 75 MHz) spectrometer. Chemical shifts are reported in δ (ppm), referenced to the 1H (of residual protons) and 13C signals of deuterated solvents. Molecular weights and polydispersity indices (PDIs) were measured by GPC in THF relative to polystyrene standards on systems equipped with two-column sets (Polymer Laboratories) and refractive-index detectors (HP1047A) at 40° C. with a flow rate of 1 mL/min. Fluorescence measurements were performed on a Jobin Yvon Fluorolog-3.
Materials. All reagents were purchased either from Acros Organics, Aldrich, or Strem and used without further purification. Phospholipids, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG); 1,1′2,2′-tetraoleoyl cardiolipin (TOCL) were purchased from Avanti Polar Lipids Inc. Calcein was purchased from Sigma, all salts and buffers of the best grade were available from Sigma-Aldrich, and used as received. Dichloromethane and THF were dried over CaH2 and sodium benzophenone ketyl, respectively. Compounds 3, 5, 7, and 3rd generation Grubbs' catalyst were synthesized according to the literature. Monomers A1-2 and B2-3 were synthesized according to Scheme 9. (Davis, et al. Bioorg. Med. Chem. 1999, 7, 2303; Stone, et al. J. Org. Chem. 1984, 49, 1849; Kwart, et al. J. Am. Chem. Soc. 1952, 74, 3094; Louie, et al. Angew. Chem. Int. Ed. 2001, 40, 247.)
Boc-protected bisamino alcohol 2. A solution of bis-amine 1 (3.00 g, 33.3 mmol) was dissolved in a minimal amount of water (1 mL) and added to a solution of di-tert-butyl dicarbonate (16.0 g, 73.3 mmol) in dioxane (400 mL). The reaction mixture was stirred for 24 hours at room temperature under nitrogen. The solvent was removed under reduced pressure. The resulting solid was dissolved in dichloromethane (300 mL), washed with water (300 mL), dried over MgSO4, and filtered. The solvent was removed under reduced pressure to afford a white solid. The white solid was washed with ether and filtered (9.18 g, 98%). 1H NMR (CDCl3): δ 5.34-5.30 (br, 2NH, 2H), 4.03 (s, 1H, OH), 3.73-3.68 (m, 1H, CH), 3.18-3.14 (m, 4H, 2CH2), 1.39 (s, 18H, 6CH3). 13C NMR (CDCl3): δ 157.2, 79.71, 70.67, 43.46, 28.39.
Boc-protected trisamino alcohol 4. A solution of tris-amine salt 3 (3 g, 12.37 mmol) dissolved in a minimal amount of water (1 mL) was added to a solution of triethylamine (5.7 mL, 40.81 mmol), and di-tert-butyl dicarbonate (8.9 g, 40.81 mmol) in dioxane (200 mL). The reaction mixture was stirred for 24 hours at room temperature under nitrogen. The solvent was removed under reduced pressure. The resulting solid was dissolved in dichloromethane (250 mL), washed with water (250 mL), dried over MgSO4, and filtered. The solvent was removed under reduced pressure to afford a white solid. The white solid was washed with ether and filtered (5.07 g, 95%). 1H NMR (CDCl3): δ 5.83 (t, J=6.6 Hz, 3H, 3NH), 4.34 (t, J=7.5 Hz, 1H, OH), 3.10 (d, J=7.5 Hz, 2H, CH2), 2.78 (d, J=6.6 Hz, 6H, 3CH2), 1.41 (s, 27H, 9CH3). 13C NMR (CDCl3): δ 157.8, 79.59, 60.24, 46.10, 38.98, 28.33.
Functionalized norbornene 6. A solution of 5 (6.81 g, 56.66 mmol) and maleimide (5 g, 51.5 mmol) in toluene (200 mL) was heated to 130° C. in a sealed tube for 24 hours. After removal of toluene under reduced pressure, the resulting solid was dissolved in a minimal amount of ether. Successive recrystallizations afforded the pure exo isomer 6 (3.6 g, 32%). NMR (CDCl3): δ 8.97 (s, NH, 1H), 6.36 (s, 2H, CH2), 4.71 (d, J=9.6 Hz, 1H, CH), 3.69 (s, 1H, CH), 3.32 (s, 1H, CH), 2.73 (s, 2H, CH2), 2.29-2.22 (m, 1H CH), 0.85 (d, J=6.6 Hz, 3H, CH3), 0.80 (d, J=6.6 Hz, 3H, CH3). 13C NMR (CDCl3): δ 178.2, 143.6, 137.5, 137.0, 120.9, 49.04, 48.61, 44.53, 28.10, 23.47, 22.97.
General Synthesis Procedure for Monomers A2-3 and B2-3. To a round bottom flask charged with compound 7 or 8, alcohol derivatives 2 or 4, and 1.1 equivalent of triphenylphosphine, THF was added. The solution mixture was then immersed in an ice bath and 1.1 equivalent of diisopropyl azodicarboxylate (DIAD) was added drop wise. After the addition of DIAD, the ice bath was removed and the reaction was allowed to stir at room temperature for 24 h. The solvent was removed under reduced pressure and the crude product was dissolved in toluene and kept at −20° C. for 24 h. The byproducts (white crystals) were removed by filtration and the mother liquor was concentrated. The product was crystallized from cold anhydrous diethyl ether (−20° C.). The mother liquor was concentrated and the remaining product was isolated by column chromatography.
Monomer A2. Norbornene 8 (2.37 g, 10.9 mmol), alcohol 2 (5.44 g, 12.0 mmol), triphenylphosphine (3.15 g, 12.0 mmol) DIAD (2.33 mL, 12.0 mmol). The product was purified by flash chromatography (Silica, 120 g ISCO column, 50:50 ethyl acetate/hexane). Yield; 2.88 g, 54%. 1H NMR (CDCl3): δ 6.43-6.35 (m, 2H, CH2), 5.07 (br, 2H, 2NH), 4.70 (d, J=9.6 Hz, 1H, CH), 4.15 (br, 1H, CH), 3.70-3.32 (m, 6H, 2CH, 2CH2), 2.74-2.64 (m, 2H, CH2), 2.33-2.21 (m, 1H, CH), 1.38 (s, 18H, 6CH3), 0.87-0.82 (m, 6H, 2CH3). 13C NMR (CDCl3): δ 177.9, 156.0, 145.0, 138.4, 137.1, 119.9, 79.41, 52.53, 48.84, 47.47, 44.30, 39.56, 28.24, 28.01, 23.21.
Monomer A3. Norbornene 8 (1.00 g, 4.60 mmol), alcohol 4 (2.19 g, 5.06 mmol), triphenylphosphine (1.33 g, 5.06 mmol), DIAD (0.98 mL, 5.06 mmol). The product was purified by flash chromatography (Silica, 120 g ISCO column, 10:90 acetonitrile/dichloromethane). Yield; 1.75 g, 60%. 1H NMR (CDCl3): δ 6.39 (s, 2H, CH2), 5.78 (br, 3H, 3NH), 4.64 (d, J=9.6 Hz, 1H, CH), 3.67 (s, 1H, CH), 3.33 (s, 1H, CH), 3.24 (s, 2H, CH2), 2.90-2.80 (m, 8H, 4CH2), 1.41 (s, 27H, 9CH3), 0.84 (d, J=6.6, 3H, CH3), 0.76 (d, J=6.6, 3H, CH3). 13C NMR (CDCl3): δ 178.7, 156.8, 144.0, 137.8, 137.2, 120.6, 79.27, 48.84, 47.73, 44.42, 41.42, 39.53, 28.39, 27.98, 23.56, 23.03.
Monomer B2. Oxanorbornene 7 (4.00 g, 24.2 mmol), alcohol 2 (7.73 g, 26.6 mmol), triphenylphosphine (6.99 g, 26.6 mmol), DIAD (5.17 mL, 26.6 mmol). The product was purified by flash chromatography (Silica, 120 g ISCO column, 10:90 acetone/dichloromethane). Yield; 6.32 g, 60%. 1H NMR (CDCl3): δ 6.48 (s, 2H, CH2), 5.20 (s, 2H, CH2), 4.97 (br, 2H, 2NH), 4.26 (br, 1H, CH), 3.51-3.38 (m, 2H, 2CH2), 2.79 (s, 2H, CH2), 1.36 (s, 18H, 6CH3). 13C NMR (CDCl3): δ 176.7, 155.9, 136.4, 81.16, 79.33, 52.88, 47.06, 39.09, 28.24.
Monomer B3. Oxanorbornene 7 (2.73 g, 16.5 mmol), alcohol 4 (7.87 g, 18.2 mmol), triphenylphosphine (4.78 g, 18.2 mmol) in dry THF (300 mL), DIAD (3.54 mL, 18.2 mmol). The product was purified by flash chromatography (Silica, 120 g ISCO column, 20:80 acetone/dichloromethane) Yield; 6.89 g, 70%. 1H NMR (CDCl3): δ 6.48 (s, 2H, CH2), 5.82 (br, 3H, 3NH), 5.23 (s, 2H, CH2), 3.36 (s, 2H, CH2), 2.88-2.87 (m, 8H, 4CH2), 2.84 (s, 2H, CH2), 1.40 (s, 27H, 6CH3). 13C NMR (CDCl3): δ 177.2, 156.9, 136.4, 80.93, 79.24, 47.30, 45.09, 41.22, 40.08, 28.33.
General Polymerization Procedure. The polymerization for monomers A2-3 and B2-3 were conducted in anhydrous THF and CH2Cl2, respectively (Scheme 10). A known amount of monomer was weighed into a Schlenk flask, placed under an atmosphere of nitrogen, and dissolved in anhydrous CH2Cl2 or THF (1 mL per 100 mg of monomer). Into a separate Schlenk flask, a desired amount of 3rd generation Grubbs' catalyst was added, flushed with nitrogen, and dissolved in a minimum amount of anhydrous CH2Cl2 or THF. Both flasks were degassed three times by freeze-pump-thaw cycles. The monomer was transferred to the flask containing the catalyst via a cannula. The reaction was allowed to stir until the polymerization is complete (as monitored by NMR and GPC). The polymerization for A2-3 series was conducted at 60° C. for 30 min, whereas for B2-3 series, the polymerization was conducted at room temperature for 10 min. The polymerization reaction was quenched with ethyl vinyl ether (0.2 mL). An aliquot was taken for GPC analysis. For polyA2-3, the solvent, THF, was first removed under reduced pressure. The polymers were then redissolved in CH2Cl2 and passed through a short silica plug. For polyB2-3, the polymer solutions were passed through a short silica plug to remove the catalyst. The solvent, CH2Cl2, was then removed under reduced pressure.
PolyA2. Monomer:catalyst ratio=6:1; monomer A2 (0.10 g, 0.20 mmol), Grubbs' 3rd generation catalyst (0.030 g, 0.033 mmol). Yield; 75%. 1H NMR (CDCl3): δ broad signals 5.61, 5.45, 5.12, 4.25, 3.76, 3.55, 3.38, 3.00, 2.53, 1.38, 0.92. 13C NMR (CDCl3): δ 178.8, 155.6, 138.8, 136.0, 131.5, 128.1, 125.9, 79.79, 78.92, 52.30, 51.02, 49.91, 49.36, 46.42, 38.86, 27.86, 22.77. Mn=3 kDa, PDI=1.10.
PolyA3. Monomer:catalyst ratio=5:1; monomer A3 (0.10 g, 0.16 mmol), Grubbs' 3rd generation catalyst (0.028 g, 0.032 mmol). Yield; 80%. 1H NMR (CDCl3): δ broad signals 5.84, 5.59, 5.42, 3.79, 3.33, 2.87, 2.43, 1.40, 0.88. 13C NMR (CDCl3): δ 179.2, 156.6, 136.7, 134.9, 129.7, 128.4, 126.1, 79.09, 51.46, 50.03, 46.69, 44.82, 41.19, 39.70, 28.18, 22.77. Mn=3 kDa, PDI=1.10.
PolyB2. Monomer:catalyst ratio=7:1; monomer B2 (0.10 g, 0.22 mmol), Grubbs' 3rd generation catalyst (0.028 g, 0.031 mmole). Yield; 90%. 1H NMR (CDCl3): δ 6.01, 5.74, br 5.50-4.55, 4.49, 4.20, br 3.55-3.48, 3.27, 1.39. 13C NMR (CDCl3): δ 176.2, 156.07, 130.9, 80.69, 79.50, 52.04, 39.12, 28.27. Mn=3 kDa, PDI=1.09.
PolyB3. Monomer:catalyst ratio=5:1; monomer B3 (0.10 g, 0.17 mmol), Grubbs' 3rd generation catalyst (0.030 g, 0.034 mmol). Yield; 85%. 1H NMR (CDCl3): δ 6.06, 5.83, br 5.750, br 5.00, 4.43, 3.34, 2.87, 1.39. 13C NMR (CDCl3): δ 176.6, 156.9, 131.9, 130.7, 80.58, 79.36, 52.24, 45.14, 41.22, 28.33. Mn=3 kDa, PDI=1.10.
General Deprotection Procedure. All polymers were dissolved in a minimal amount of CH2Cl2 and excess amount of trifluoroacetic acide (TFA) was added. The solution was allowed to stir at room temperature for 6 h. TFA was removed under reduced pressure and the polymers were dissolved in distilled water, passed through a short silica plug, and freeze dried for 48 h.
Liposome preparation. The following buffers were used: Buffer A, 40 mM calcein, 10 mM Na2HPO4 pH 7.0; buffer B, 10 mM Na2HPO4, 90 mM NaCl, pH 7.0. Homogeneous large unilamellar vesicles (LUVs) were prepared by the extrusion method. (Som, et al. J. Phys. Chem. B. 2008, 112, 3495-3502.) Vesicle abbreviations with the corresponding lipid compositions, inside and outside buffer are listed in Table 22.
General procedure for vesicle preparation: Appropriate volume of lipid stock solution (25 mg/mL CHCl3) were taken in 2 mL CHCl3 and evaporated slowly at reduced pressure and then placed under vacuum >2 h to prepare a thin film. The thin film was then hydrated for 1 h with 1 mL buffer A. The resulting suspension was subjected to five freeze-thaw cycles (using liquid nitrogen to freeze and warm water bath to thaw) and extruded >10 times using a mini-extruder through a polycarbonate membrane (Whatman, pore size 400 nm), stacked between two pairs of membrane supports. The external calcein was removed by gel filtration (Sephadex G-50) using buffer B and the resulting 1.0 mL vesicle solution was diluted with buffer B to 20 mL to give calcein loaded LUVs stock solution, having final lipid concentration of 0.5 mM. Following the general vesicle preparation procedure vesicles V1-V3 were prepared using appropriate lipid ratios.
Dye leakage assays. Calcein loaded LUVs (20 μL) were added to 1.98 mL buffer B in a fluorescence cuvette (final lipid concentration in the cuvette 5.0 μM). Fluorescence emission intensity It (λem=510 nm, λex=450 nm) was monitored as a function of time (t) during addition of 20 μL DMSO solution of polymer and 50 μL of 20% Triton X-100. Control experiments were performed adding only 20 μL DMSO. Flux curves were normalized to percentage leakage activity Y, where Y=[(It−I0)/(I∞−I0)]*100. I0, is It before addition of samples (polyA1-3 or polyB1-3), and I∞ is It after addition of 20% Triton-X.
Antimicrobial Studies. The antimicrobial activities of polyA1-3 and polyB1-3 were evaluated using standard methods. (Hamuro, et al. J. Am. Chem. Soc. 1999, 121, 12200-12201.) Stock solutions of polymers were made in DMSO and then diluted into 96-well plates with Mueller Hinton (MH) medium to a constant volume. All bacteria (used cell lines: Escherischia coli D31 and Staphylococcus aureus ATCC25923) were taken from stock glycerol solutions or from a frozen stock, diluted into MH medium, and grown overnight at 37° C. Sub-samples of these cultures were grown for 3 h, the OD600 was measured and then the cells were diluted to 0.001 OD600. The diluted cell solutions (approximately 105 cells/mL) were then added to the 96-well plate and incubated at 37° C. for 6 h. The MIC90 values reported are the minimum concentration necessary to inhibit 90% of the cell growth. Experiments were run in duplicate. The results are shown in Table 23.
Hemolysis Studies. Fresh human erythrocytes were obtained by centrifuging whole blood (3000 rpm, 10 min) to remove plasma and white blood cells. The RBCs (1 mL) were diluted with 9 mL of TBS buffer (10 mM Tris buffer, pH=7.0, 150 mM NaCl), and the suspension was further diluted by a factor of 40 to give a RBC stock suspension (0.25% blood cells). This RBC stock (120 μL), TBS buffer (15 μL), and the polymer stock solutions (15 μL) (or control solutions) were added to a 200 μL centrifugation tube and incubated at 37° C. for 1 h. The tube was centrifuged at 4000 rpm for 5 min. Supernatant (30 μL) was diluted with TBS buffer (100 μL), and OD414 of the solution was measured as hemoglobin concentration. A control solution containing only DMSO was used as a reference for 0% hemolysis. Complete hemolysis was measured by adding 1% Triton X-100 to the RBCs and measuring OD414. Nonlinear exponential curve-fitting plots of OD414 vs the concentration of polyA1-3 and polyB1-3 series determined the HC50, the hemolytic dose required to lyse 50% of the RBCs. The results are shown in Table 23.


















TABLE 22







Vesicles composition










Vesicles
Lipid Composition
Buffer inside
Buffer outside





V1
DOPC 100%
A
B


V2
DOPG/DOPE 20/80
A
B


V3
TOCL 100%
A
B
















TABLE 23







Antimicrobial and hemolytic activity.










MIC90 (μg/mL)













Polymers
E. coli
S. aureus
HC50 (μg/mL)
















polyA1
25
50
<1



polyA2
6
50
700



polyA3
25
25
500



polyB1
>400
>200
>2150



polyB2
200
25
1400



polyB3
200
15
1200










INCORPORATION BY REFERENCE
References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made in this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.
EQUIVALENTS
The representative examples are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples herein and the references to the scientific and patent literature cited herein. The examples contain important additional information, exemplification and guidance which can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

## Claims

1. A monomer unit having the structure of:



wherein one of R1 and R2 comprises a hydrophobic group and the other one of R1 and R2 comprises a hydrophilic group; and X is C═R3, O or S, wherein R3 is a C1-C12 alkyl or alkoxy group.

2. The monomer of claim 1, wherein the hydrophobic group comprises a substituted or unsubstituted alkyl group.

3. The monomer of claim 1, wherein the hydrophilic group comprises a group selected from ammonium ions, sulfonium ions, guanidinium ions, phosphonium ions, and mono- and di-alkyl substituted amino groups.

4. The monomer of claim 1, wherein X is O; R1 is a linear or branched C1-C12 alkyl group; and R2 is




or a guanidinium ion wherein p is an integer from 1-10.

5. The monomer of claim 1, wherein both —C(═O)O— groups in (I) occupy the exo-stereo position.

6. The monomer of claim 4, wherein R1 is a linear or branched C1-C6 alkyl group; and
p is an integer from 1-5.

7. The monomer of claim 1, wherein X is C═R3; R1 is a linear or branched C1-C12 alkyl group; and R2 is




or a guanidinium ion, wherein p is an integer from 1-10.

8. The monomer of claim 7, wherein R3 is a C1-C12 alkyl.

9. A polymer comprising a monomer unit having the structure of:



wherein one of R1 and R2 comprises a hydrophobic group and the other one of R1 and R2 comprises a hydrophilic group; and X is C═R3, O or S, wherein R3 is a C1-C12 alkyl or alkoxy group.

10. The polymer of claim 9, wherein the hydrophobic group comprises a substituted or unsubstituted alkyl group.

11. The polymer of claim 9, wherein the hydrophilic group comprises a group selected from ammonium ions, sulfonium ions, guanidinium ions, phosphonium ions, and mono- and di-alkyl substituted amino groups.

12. The polymer of claim 9, wherein X is O; R1 is a linear or branched C1-C12 alkyl group; and R2 is




or a guanidinium ion, wherein p is an integer from 1-10.

13. The polymer of claim 9, wherein both —C(═O)O— groups in (II) occupy the exo-stereo position.

14. The polymer of claim 12, wherein R1 is a linear or branched C1-C6 alkyl group; and
p is an integer from 1-5.

15. The polymer of claim 9, wherein X is C═R3; R1 is a linear or branched C1-C12 alkyl group; R2 is




or a guanidinium ion, wherein p is an integer from 1-10.

16. The polymer of claim 15, wherein R3 is a C1-C12 alkyl.

17. The polymer of claim 9, wherein n is an integer from about 1 to about 1,000.

18. The polymer of claim 9, wherein the polymer is a co-polymer.

19. The co-polymer of claim 18, wherein the co-polymer comprises the structural monomer units of:



wherein
each of X′ and X′′ is C═R3, O or S, wherein R3 is a C1-C12 alkyl or alkoxy group;
each of R1′ and R1′′ is a hydrophobic group and each of R2′ and R2′′ is a hydrophilic group, provided that R1′ and R1′′ are not the same, R2′ and R2′′ are the same, or X′ and X′′ are not the same; and
each of m and n is an integer from about 1 to about 1,000.

20. The polymer of claim 19, wherein the hydrophilic group comprises a group selected from ammonium ions, sulfonium ions, guanidinium ions, phosphonium ions, and mono- and di-alkyl substituted amino groups.

21. The polymer of claim 19, wherein all —C(═O)O— groups in (III) and (IV) occupy the exo-stereo position.

22. The co-polymer of claim 19, wherein
each of R1′ and R1′′ is independently a linear or branched C1-C12 alkyl group; and
each of R2′ and R2′′ is independently





or a guanidinium ion, wherein p is an integer from 1-10.

23. A polymer prepared by ring-opening metathesis polymerization, the polymer comprising hydrophobic and hydrophilic groups attached to the polymeric backbone such that, within a monomeric structural unit, the hydrophobic and hydrophilic groups are attached to the polymeric backbond at adjacent atoms.

24. The polymer of claim 23, wherein the hydrophobic and hydrophilic groups are attached to the polymeric backbond via ester linkages.

25. The polymer of claim 23, wherein the
hydrophobic group comprises a linear or branched C1-C12 alkyl group; and
the hydrophilic group comprises a —NH3+ group.

26. The polymer of claim 25, wherein the hydrophilic group comprises




or a guanidinium ion, wherein p is an integer from 1-10.

27. The polymer of claim 23, wherein the polymer is a co-polymer.