Patent Publication Number: US-2017362283-A1

Title: Nisin-based compounds and use thereof in the treatment of bacterial infections

Description:
FIELD OF THE INVENTION 
     The invention relates to the field of medicine. More in particular, the invention relates to antimicrobial compounds based on nisin and the use thereof as medicaments. 
     BACKGROUND OF THE INVENTION 
     Nisin is a polycyclic antibacterial peptide produced by the bacterium  Lactococcus lactis . Because of its antibacterial activity it is often used as an additive in food, like processed cheese, meats, and milk. In its original form nisin has 34 amino acids, including the uncommon lanthionine (Lan), methyllanthionine (MeLan), didehydroalanine (Dha) and didehydroaminobutyric acid (Dhb) that are introduced during posttranslational modifications of the originating 57-aa precursor peptide. Nisin is a member of the class of molecules referred to as ‘lantibiotics’. Other members of this class are subtilin and epidermin. Nisin was already approved for use in food in the late 1960s. Its E number is E234. Because of its antibacterial properties it has also been envisioned as an antibiotic. However, one of the major disadvantages of using it as a medicinal antibiotic in humans is that it metabolizes relatively readily in the human stomach and in human blood. 
     Variations to nisin were reported in literature. WO 2007/103548 discloses a 12 amino acid containing structure herein further referred to as “nisin [1-12]” that is being connected through a linker to an antibiotic moiety, in particular to vancomycin. WO 2014/085637 describes a 5-ring nisin-based lantibiotic wherein some of the amino acids can be replaced and which can further comprise a hydrocarbon substituent. In WO 2010/058238 a 4-ring lantibiotic comprising a wide range of substituents, e.g. C 1 -C 20  alkyl, is disclosed. Further nisin derivatives having at least one amino acid substituted in the peptide sequence were disclosed in WO 2009/13545. Most if not all of these known compounds are large and therefore degraded rapidly through the action of proteolytic enzymes. It goes without saying that there is a continuous need for new antibiotics that act against a wide variety of microbes, and that do not become degraded rapidly once present in circulation. 
     SUMMARY OF THE INVENTION 
     The present invention pertains to an antimicrobial compound according to Formula (1), 
     
       
         
         
             
             
         
       
     
     wherein:
         Z is selected from any one of the substituents NHR 1 , NR 1 R 2 , OR 1  and SR 1 , and Y is selected from any one of the substituents NHR 3 , NR 3 R 4 , NHCR 3 R 4 , NHCOR 3 , NHCSR 3 , NHOR 3  and NHC(NR 3 NHR 4 ), wherein R 1 , R 2 , R 3  and/or R 4  is a substituted or non-substituted substituent selected from: alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and polyaryl, wherein said substituent comprises at least 2, 4 or 6 carbon atoms and at most 30, 40 or 50 carbon atoms;   A 1  and A 3  are independently  D -Alanine or  D -Aminobutyric acid;   A 2  and A 4  are  L -alanine;   A 1 +A 2  and A 3 +A 4  independently form a (2S,6R)-lanthionine or (2S,3S,6R)-methyllanthionine linkage; and   X 1  to X 8  are each independently selected from natural or non-natural amino acids.       

     In a preferred embodiment, Y and Z each have a molecular weight of less than 1200 Dalton. In a further preferred embodiment, and in relation to all different antimicrobial compounds as disclosed herein, R 1 , R 2 , R 3  and R 4  are each a substituted or unsubstituted substituent, independently selected from: C 4  to C 50  alkyl, C 2  to C 50  alkenyl, C 2  to C 50  alkynyl, cycloalkyl, aryl and polyaryl. In another preferred embodiment, both R 1  and R 2  and/or both R 3  and R 4  are substituted or non-substituted substituents selected from: C 5  to C 40  alkyl, C 4  to C 40  alkenyl, C 4  to C 40  alkynyl, cycloalkyl, aryl and polyaryl. 
     In another preferred embodiment, X 8  is an amino acid that carries no charge on the side-chain. More preferably, X 8  is lysine which is acylated on the side-chain. Even more preferably, X 8  is lysine which is acetylated on the side-chain. In a highly preferred aspect of the invention the structures of the present invention have an antimicrobial activity exceeding the activity of the unsubstituted nisin [1-12] structure known in the art. 
    
    
     DETAILED DESCRIPTION 
     The objective of the present invention is to provide novel antimicrobial compounds. The nisin-derived compounds of the present invention exhibit antimicrobial activity, in particular antibacterial activity. Moreover, the compounds of the present invention generally are capable of killing drug-resistant strains, in particular drug-resistant strains of Gram-positive bacteria. The inventors have surprisingly found that the bactericidal mechanism is different from that of nisin. The compounds of the invention are capable to bind to the pyrophosphate of lipid II in the bacterial cell wall, similar to nisin. Nisin additionally causes pores to form in the cell wall, whereas the compounds of the present invention do not cause such pore formation. It is further noted that the nisin [1-12] structure known in the art, wherein Z is OH (R 1  is hydrogen), and Y is NH 2  (R 3  and R 4  are hydrogen), generally does not have significant antimicrobial activity. Therefore, it was surprising that the compound of the present invention exhibits considerable antimicrobial activity, and appeared generally active against a wide variety of bacterial strains. A further advantage appeared to be the improved stability of the compounds of the present invention in human serum compared to nisin. 
     The present invention relates to an antimicrobial compound according to Formula (1), 
     
       
         
         
             
             
         
       
     
     wherein Z is selected from any one of the substituents NHR 1 , NR 1 R 2 , OR 1  and SR 1 , and Y is selected from any one of the substituents NHR 3 , NR 3 R 4 , NHCR 3 R 4 , NHCOR 3 , NHCSR 3 , NHOR 3  and NHC(NR 3 NHR 4 ), wherein R 1 , R 2 , R 3  and/or R 4  is a substituted or non-substituted substituent selected from: alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and polyaryl, wherein said substituent comprises at least 2, 4 or 6 carbon atoms and at most 30, 40 or 50 carbon atoms; A 1  and A 3  are independently  D -Alanine or  D -Aminobutyric acid; A 2  and A 4  are  L -alanine; A 1 +A 2  and A 3 +A 4  independently form a (2S,6R)-lanthionine or (2S,3S,6R)-methyllanthionine linkage; and X 1  to X 8  are each independently selected from natural or non-natural amino acids. 
     In a preferred embodiment, Y and Z each have a molecular weight of less than 1200 Dalton. In another preferred embodiment, R 1 , R 2 , R 3  and/or R 4  is a substituted or unsubstituted substituent, independently selected from: C 4  to C 50  alkyl, C 2  to C 50  alkenyl, C 2  to C 50  alkynyl, cycloalkyl, aryl and polyaryl. In an especially preferred embodiment, both R 1  and R 2  and/or both R 3  and R 4  are substituted or non-substituted substituents selected from: C 5  to C 40  alkyl, C 4  to C 40  alkenyl, C 4  to C 40  alkynyl, cycloalkyl, aryl and polyaryl. In a further preferred embodiment, X 8  is an amino acid that carries no charge on the side-chain, preferably lysine that is acylated or acetylated on the side-chain. Preferably, it is acetylated. 
     In a particular aspect the invention relates to an antimicrobial compound according to the invention, wherein Z has a molecular weight of less than 1000 Dalton, preferably less than 800 Dalton, and more preferably less than 600 Dalton; and/or Y has a molecular weight of less than 1000 Dalton, preferably less than 800 Dalton, and more preferably less than 600 Dalton. In one particular aspect, Y is not NH 2 , and Z is not OH or NH—CH 3 , because it was found that a compound according to Formula (1) with these Y and Z groups did not exceed the antimicrobial activity of the unsubstituted nisin [1-12] structure. Hence, in a highly preferred aspect, the invention relates to an antimicrobial compound according to the present invention, wherein said compound has an antimicrobial activity exceeding the activity of the unsubstituted nisin [1-12] structure. Preferably, the amide-form of Z independently lacks antimicrobial activity. 
     In another preferred aspect, the compound of the present invention exhibits an MIC value below 100 μg/ml, preferably below 70 μg/ml, 50 μg/ml, 20 μg/ml, and most preferably below 10 μg/ml. 
     The invention furthermore relates to an antimicrobial compound according to the invention, for use in the treatment of a bacterial infection. The invention also relates to a pharmaceutical composition comprising the antimicrobial compound according to the invention, and a pharmaceutically acceptable diluent and/or carrier. 
     The invention furthermore relates to a use of an antimicrobial compound according to the invention for the manufacture of a medicament for use in the treatment of an infection, preferably a bacterial infection. 
     In yet another embodiment, the invention relates to a method of treating a subject suffering from a bacterial infection, comprising administering an antimicrobial compound according to the invention, or a pharmaceutical composition according to the invention, to said subject. 
     Preferred compounds as disclosed herein are compounds (6), (10), (12) and (20). Especially preferred are compounds (10), (12) and (20). Also preferred is compound (24) carrying R 1  structure (e) as disclosed herein. A very highly preferred compound is compound (12). 
     Preferred amino acids used in the compounds of the invention are those derived from known type A lantibiotics, in particular nisin, subtilin, gallidermin and epidermin. Specific compounds of the invention and their amino acid sequences are exemplified in Table 1. In a preferred embodiment of the invention, the compound according to Formula (1) wherein X 6  is proline and X 7  is glycine, and A 3  is  D -aminobutyric acid and A 4  is  L -alanine. 
     In a preferred embodiment of the invention, in the compound according to Formula (1), X 6  is proline, X 7  is glycine, A 3  is  D -aminobutyric acid and A 4  is  L -alanine. The remaining amino acids can be any known natural or non-natural amino acid. They can be incorporated by general methods known to the person skilled in the art. An example of such a method can for instance be gleaned in Rink et al. (in Appl. Environ. Microbiol., September 2007, pp. 5809-5816). 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Examples of X and A amino acid residues within the nisin-derived compounds according 
               
               
                 to the present invention. The left column shows the lantibiotics from which the indicated 
               
               
                 amino acids (given here with their respective 3-letter notation) are chosen. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 X 1   
                 X 2   
                 X 3   
                 X 4   
                 X 5   
                 X 6   
                 X 7   
                 X 8   
                 A 1   
                 A 2   
                 A 3   
                 A 4   
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Nisin 
                 Ile 
                 Dhb 
                 Ile 
                 Dha 
                 Leu 
                 Pro 
                 Gly 
                 Lys 
                 D-Ala 
                 L-Ala 
                 D-Abu 
                 L-Ala 
               
               
                 Subtilin 
                 Trp 
                 Lys 
                 Glu 
                 Dha 
                 Leu 
                 Pro 
                 Gly 
                 Val 
                 D-Ala 
                 L-Ala 
                 D-Abu 
                 L-Ala 
               
               
                 Subtilin 
                 Trp 
                 Lys 
                 Glu 
                 Dha 
                 Leu 
                 Pro 
                 Gly 
                 Ala 
                 D-Ala 
                 L-Ala 
                 D-Abu 
                 L-Ala 
               
               
                 Gallidermin 
                 Ile 
                 Ala 
                 Lys 
                 Phe 
                 Leu 
                 Pro 
                 Gly 
                 Ala-Lys 
                 D-Ala 
                 L-Ala 
                 D-Abu 
                 L-Ala 
               
               
                 Epidermin 
                 Ile 
                 Ala 
                 Lys 
                 Phe 
                 Ile 
                 Pro 
                 Gly 
                 Ala-Lys 
                 D-Ala 
                 L-Ala 
                 D-Abu 
                 L-Ala 
               
               
                   
               
            
           
         
       
     
     In one particular embodiment, the invention relates to a nisin-derived antimicrobial compound having a Minimum Inhibitory Concentration (MIC) value below 100 μg/ml. Preferably, the compound has a MIC value below 70 μg/ml, more preferably below 50 μg/ml, even more preferably below 20 μg/ml, and most preferably below 10 μg/ml. Determining the MIC value is a standard technique well known to the skilled person, in particular the MIC value can be measured using method M07-A9 (CLSI standard, January 2012, Vol. 32, No. 2, “Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically”). 
     The compound of the invention comprises a Y and a Z substituent which may be the same or different. The precursor of the substituent Z and/or Y, preferably Z, of the inventive compounds of Formula (1) generally lack antimicrobial activity themselves, which means that Z taken individually lacks antimicrobial activity. Examples of such precursors of the Z substituent include H 2 NR 1 , HNR 1 R 2 , HOR 1  and HSR 1 . Examples of such precursors for the Y substituent include R 3 HCO R 3 R 4 CO, R 3 COOH, R 3 CSOH and R 3 —I. The precursors can generally be used in the process of preparing the antimicrobial compound of the invention. The term “lack antimicrobial activity” means that no relevant antimicrobial or antibacterial activity could be measured using conventional techniques known in the art. The substituent is thus not another antibacterial compound or a derivative thereof. The antimicrobial compounds disclosed in WO 2007/103548 (e.g. Z being vancomycin) are not considered to be part of an antimicrobial compound according to the present invention. 
     The compounds according to the present invention comprise a substituent Z having a molecular weight of less than 1200 Dalton. Preferably, the molecular weight is less than 1000 Dalton, more preferably less than 800 Dalton, and most preferably less than 600 Dalton. The compound of the invention further comprises a substituent Y having a molecular weight of less than 1200 Dalton. Preferably, the molecular weight is less than 1000 Dalton, more preferably less than 800 Dalton, and most preferably less than 600 Dalton. An advantage of such relatively small substituents is that the preparation of the compounds of the invention is relatively simple and can be kept economically and commercially interesting. 
     The antimicrobial compound of the present invention is based on the original nisin [1-12] structure and preferably comprises a substituent Y (having an R 3  and/or R 4 ) and/or Z, preferably Z, (having an R 1  and/or R 2 ), which is a substituted or unsubstituted substituent independently selected from C 4  to C 50  alkyl, C 2  to C 50  alkenyl, C 2  to C 50  alkynyl, cycloalkyl, aryl and polyaryl. In the context of the present invention the wording “substituted” refers to the substitution of the substituent with a further substituent and/or the modification of the substituent with a hetero atom like O, S, or N for example. Preferably, both R 1  and R 2 , and/or both R 3  and R 4 , even more preferably both R 1  and R 2 , are independently substituted or non-substituted substituents selected from C 5  to C 40  alkyl, C 4  to C 40  alkenyl, C 4  to C 40  alkynyl, cycloalkyl, aryl and polyaryl. Even more preferred are substituted or non-substituted substituents selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl or polyaryl, wherein the substituent comprises at least 4 carbon atoms, more preferably at least 5 carbon atoms and most preferably at least 6 carbon atoms, and at most 50 carbon atoms, more preferably at most 40 carbon atoms and most preferably at most 30 carbon atoms. 
     The inventors have found that, as an exception to the other compounds of the present invention, a compound according to Formula (1) wherein Y is NH 2 , and Z is OH or NH—CH 3  does not provide antimicrobial activity exceeding the antimicrobial activity of the nisin [1-12] structure. 
     In a preferred aspect of the present invention, the antimicrobial compounds according to the invention are as shown in Table 2 and according to basic Formula (1) as given above, wherein X 1 =Ile, X 2 =Dhb, X 3 =Ile, X 4 =Dha, X 5 =Leu, X 6 =Pro, X 7 =Gly; A, = D -Ala, A 2   =L -Ala, A 3   =D -Abu and A 4   =L -Ala, wherein Z is a substituent of the type NHR 1 , wherein Y is a substituent of the type NHR 3 , and wherein X 8 , R 1  and R 3  are selected from the structures as provided in Table 2, leading to compounds referred to as Formulas (2) to (23). 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Preferred antimicrobial compounds with Formulas (2) to (23)-as indicated in the 
               
               
                 right column-based on Formula (1) with different X 8 , R 1  and R 3  groups as indicated. 
               
            
           
           
               
               
               
               
            
               
                 X 8   
                 R 1   
                 R 3   
                   
               
               
                   
               
               
                 Lys 
                 —C 6 H 13   
                 H 
                  (2) 
               
               
                 Lys 
                 —C 7 H 15   
                 H 
                  (3) 
               
               
                 Lys 
                 —C 8 H 17   
                 H 
                  (4) 
               
               
                 Lys 
                 —C 9 H 19   
                 H 
                  (5) 
               
               
                 Lys 
                 —C 10 H 21   
                 H 
                  (6) 
               
               
                 Lys 
                 —C 11 H 23   
                 H 
                  (7) 
               
               
                 Lys 
                 —C 12 H 25   
                 H 
                  (8) 
               
               
                 Lys 
                 —C 13 H 27   
                 H 
                  (9) 
               
               
                 Lys 
                 —C 14 H 29   
                 H 
                 (10) 
               
               
                 Lys 
                 —C 15 H 31   
                 H 
                 (11) 
               
               
                   
               
               
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
                 (12) 
               
               
                   
               
               
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 (13) 
               
               
                   
               
               
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 (14) 
               
               
                   
               
               
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 (15) 
               
               
                   
               
               
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 (16) 
               
               
                   
               
               
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 (17) 
               
               
                   
               
               
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 (18) 
               
               
                   
               
               
                 Lys((C═O)C 8 H 17 ) 
                 —CH 3   
                 H 
                 (19) 
               
               
                 Acetyllysine 
                 —C 12 H 25   
                 H 
                 (20) 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 —C 12 H 25   
                 H 
                 (21) 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 —C 12 H 25   
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 (22) 
               
               
                   
               
               
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 (23) 
               
               
                   
               
            
           
         
       
     
     In yet another preferred embodiment, the Z substituent comprises an R 1  group selected from the ones indicated in Table 2, and the Y substituent is NHR 3  wherein R 3  is hydrogen. 
     A precursor to the antimicrobial compounds in accordance with the invention is comparative compound E; according to Formula (1) wherein R 1  has the following structure (a): 
     
       
         
         
             
             
         
       
     
     The invention also relates to an antimicrobial compound according to Formula (24), 
     
       
         
         
             
             
         
       
     
     wherein: 
     Y is selected from any one of the substituents NHR 3 , NR 3 R 4 , NHCOR 3 , NHCSR 3 , NHOR 3  and NHC(NR 3 NHR 4 ), wherein R 1 , R 3  and R 4  are independently selected substituents as disclosed herein; A 1 , A 3  are independently  D -Alanine or  D -Aminobutyric acid; A 2  and A 4  are L-alanine; A 1 +A 2  and A 3 +A 4  independently form a (2S,6R)-lanthionine or (2S,3S,6R)-methyllanthionine linkage; and X 1  to X 8  are each independently selected from natural or non-natural amino acids. 
     In a preferred embodiment, Y and R 1  each have a molecular weight of less than 1200 Dalton. In another preferred embodiment, X 8  is an amino acid that carries no charge on the side-chain. More preferably, X 8  is lysine which is acylated on the side-chain, even more preferably, X 8  is lysine which is acetylated on the side-chain. 
     The invention further pertains to the antimicrobial compounds according to Formula (24) comprising an R 1  group selected from the following four structures (b), (c), (d), and (e) (see also Table 4 below): 
     
       
         
         
             
             
         
       
     
     In one preferred embodiment, the present invention relates to an antimicrobial compound according to Formula (24), wherein the R 1  group is selected from the four structures (b), (c), (d) and (e), and the Y substituent is NHR 3  wherein R 3  is hydrogen. In a further preferred embodiment, the present invention relates to an antimicrobial compound according to Formula (24), wherein the R 1  group is structure (e), and the Y substituent is NH 2 . 
     The antimicrobial compounds of the present invention according to Formulae (1) to (24), with their indicated R groups, preferably all exhibit antimicrobial activity exceeding the activity of the unsubstituted nisin [1-12] structure. Especially preferred antimicrobial compounds of the present invention are those according to Formulas (6), (7), (8), (9), (10), (12), (13), (14), (15), (16), (17), (18), (20) and the compound according to Formula (24) carrying structure (e) as the R1 group. These compounds particularly provide for an even higher antimicrobial activity and/or stability in human serum and/or lower hemolytic activity than the other compounds of the invention. Highly preferred is an antimicrobial compound according to Formula (12). 
     The compounds of the invention can be prepared by starting with the basic nisin [1-12] structure which is then substituted at the C-terminus side by coupling with a nucleophile precursor or an alkyne precursor at the Z substituent forming a covalent connection. The basic nisin [1-12] structure may be prepared by treating nisin with an enzyme capable of cutting nisin at position 12. An example of such an enzyme is Trypsin. Materials and methods are provided in the accompanying examples. 
     The present invention further pertains to a combination of an antimicrobial compound of the invention and an active pharmaceutical ingredient. The active pharmaceutical compound can be any such compound known to the skilled person. Preferably, the active pharmaceutical ingredient is a second antimicrobial agent. In the context of the present application the term “combination” refers to a composition comprising both the antimicrobial compound of the invention and an active pharmaceutical ingredient, or to a plurality of pharmaceutical compositions comprising both the antimicrobial compound and the pharmaceutical ingredient in two or more different compositions. The present invention therefore also pertains to a kit-of-parts comprising an antimicrobial compound and an active pharmaceutical ingredient, in particular a pharmaceutical ingredient being a second antimicrobial agent. The plurality of compositions of the invention may be administered to a patient simultaneously and/or consecutively. 
     Examples of such antimicrobial agents include aminoglycosides such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin and spectinomycin; ansamycins like rifaximin, geldanamycin and herbimycin; carbapenems such as ertapenem, doripenem and meropenem; cephalosporins like cefadroxil, cefazolin, cefalotin, cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, cefibuten, ceftizoxime, ceftriaxone, cefepime, ceftaroline fosamil and ceftobiprole; glycopeptides such as teicoplanin, vancomycin, oritavancin, telavancin, dalbavancin and ramoplanin; lincosamides like clindamycin and lincomycin; lipopeptides such as daptomycin; macrolides like azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, and spiramycin; monobactams such as aztreonam; nitrofurans like furazolidone, nifrofurantoin; oxazolidinones such as linezolid, posizolid, radezolid and torezolid; penicillins such as amoxicillin, ampicillin, aziocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, meziocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, temocillin, ticarcillin; penicillin combinations such as amoxicillin/clavulanate, ampicillin/sulbactam, piperacillin/tazobactam and ticarcillin/clavulanate; polypeptides such as bacitracin, colistin and polymyxin B; quinolones such as ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, levofloxacin, lomefloxacin, moxifloxacin, nilidixic acid, norfloxacin, trovafloxacin, grepafloxacin, sparfloxacin and temafloxacin; sulfonamides like mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole (co-trimoxazole), sulfonamidochrysoidine; tetracyclines like demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline; drugs against mycobacteria such as ciofazimine, dapsone, capreomycin, cycloserine, ethambuto, ethionamide, isoniazid, pyrazinamide, rifampcin, rifabutin, rifapentine and streptomycin; and arsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, metronidazole, mupirocin, platensimycin, quinupristin/dalfopristin, thiamphenicol, tigecycline, tinidazole and trimethoprim. 
     In one embodiment of the invention, the molar ratio between the antimicrobial compound and the active pharmaceutical ingredient is at most 10:1, preferably at most 5:1, and most preferably at most 2:1, and generally at least 1:20, preferably at least 1:10, more preferably at least 1:5, and most preferably at least 1:2. 
     The invention further pertains to a pharmaceutical composition comprising the antimicrobial compound or the combination of the invention, and a pharmaceutically acceptable diluent or carrier. The term “pharmaceutical composition” or “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. A “pharmaceutically acceptable diluent or carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable diluent or carrier includes, but is not limited to, water, a buffer, excipient, stabilizer, or preservative. 
     In another embodiment of the invention, the antimicrobial compound or the combination of the invention is divided over two or more pharmaceutical compositions, wherein the antimicrobial compound of the invention is comprised in one pharmaceutical composition and the active pharmaceutical ingredient is comprised in a second pharmaceutical composition. In this way the antimicrobial compound and the active pharmaceutical ingredient can be administered to a patient consecutively. It is also envisaged to provide compositions comprising part of the total amount of the antimicrobial compound and/or the active pharmaceutical ingredient. 
     In one embodiment, the invention pertains to the use of the antimicrobial compound, the combination of the invention or the pharmaceutical composition of the invention as a medicament. In yet another embodiment, the invention pertains to the use of the antimicrobial compound, the combination of the invention or the pharmaceutical composition of the invention in the treatment of infections, preferably bacterial infections. 
     The term “infection” as used herein refers to diseases caused by microorganisms, such as bacteria or a virus, to which the human or animal body reacts, generally causing an inflammatory reaction. The antimicrobial compounds of the present invention are particularly effective against bacteria. Such bacteria may be Gram-negative and Gram-positive bacteria. Of particular interest are bacterial strains which comprise a cell wall of which the precursor is lipid II. Examples of Gram-negative bacteria include Coccobacilli such as  Hemophilus influenzae, B. pertussis, Brucella  spp.,  F. tularensis, P. multocida , and  Legionella pneumophila ; Cocci such as  Neisseria gonorrhoeae, Neisseria meningitidis  and  Moraxella catarrhalis ; Bacilli like  Klebsiella pneumoniae, Pseudomonos aeruginosa, Proteus mirabilis, Enterobacter cloacae, Heliobacter pylori, Serratia marcescens, Salmonella enteritidis, Salmonella typhi ; and  Acinetobacter baumannii . Examples of Gram-positive bacteria include  Staphylococcus  like  Staphylococcus aureus, Staphylococcus epidermidis  and  Staphylococcus saprophyticus; Streptococcus  such as  Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Viridans mutans, Enterococcus faecalis , and  Enterococcus faecium ; Micrococcaceae such as  Micrococcus luteus; Corynebacterium, Mycobacterium, Firmicutes, Streptomyces, Clostridium, Listeria  and  Bacillus.    
     The antimicrobial compounds of the invention are generally active against drug-resistant bacteria. The invention therefore also pertains to the use of the antimicrobial compounds in the treatment of drug-resistant bacteria. With the wording “drug-resistant” it is meant that a resistance towards one or more existing drugs exists. Additionally, pharmaceutical compositions and the combinations comprising the antimicrobial compound of the invention can also be used in the treatment of drug-resistant bacteria. 
     In one embodiment, the drug-resistant bacteria are resistant to at least one drug selected from the group consisting of penicillin, beta-lactam, vancomycin, linezolid, fluoroquinolone, clindamycin, carbapenem, isoniazid, rifampin, tetracycline, cyphalosporin, aminoglycoside, methicillin, ampicillin and daptomycin. Examples of the drug-resistant bacteria include methicillin-resitant  Staphylococcus aureus  (MRSA), vancomycin-resistant  Staphylococcus aureus  (VRSA), penecillin-resistant  Streptococcus pyogenes , macrolide-resistant  Streptococcus pyogenes , penicillin-resistant  Streptococcus pneumonia , beta-lactam-resistant  Streptococcus pneumonia , penicillin-resistant  Enterococcus faecalis , vancomycin-resistant  Enterococcus faecalis , linezolid-resistant  Enterococcus faecalis , penicillin-resistant  Enterococcus faecium , vancomycin-resistant  Enterococcus faecium , linezolid-resistant  Enterococcus faecium, Pseudomonas aeruginosa , fluoroquinolone-resistant  Clostridium difficile , clindamycin-resistant  Clostridium difficile , fluoroquinolone-resistant  Escherichia coli, Salmonella, Acinetobacter baumannii  (MRAB), carbapenem-resistant  Klebsiella pneumoniae, Mycobacterium tuberculosis  (XDR TB), isoniazid-resistant  Mycobacterium tuberculosis , rifampin-resistant  Mycobacterium tuberculosis , tetracycline-resistant  Neisseria gonorrhoeae , aminoglycoside-resistant  Neisseria gonorrhoeae , cephalosporin-resistant  Neisseria gonorrhoeae , and penicillin-resistant  Neisseria gonorrhoeae.    
     Specific examples of drug-resistant bacteria include  Streptococcus mutans  (ATCC 700610),  Streptococcus mutans  (strain Xc),  Streptococcus sobrinus  (ATCC 33478),  Streptococcus uberis  (strain 1978),  Streptococcus uberis  (strain 1979),  Streptococcus uberis  (strain 1980),  Streptococcus uberis  (strain 1981),  Streptococcus pyogenes  (strain 5448),  Streptococcus pyogenes  (strain JRS4),  Streptococcus pyogenes  (ATCC BAA-595),  Streptococcus. Pneumoniae, Streptococcus mitis, Streptococcus sanguis, Streptococcus bovis, Streptococcus salivarius, Streptococcus intermedius, Streptococcus viridans, Streptococcus oralis, Streptococcus salivarus, Staphylococcus lugdunensis, Staphylococcus aureus  (ATCC BAA-1717),  Staphylococcus aureus  (ATCC 25904),  Staphylococcus aureus  (strain MRSA-16),  Staphylococcus aureus  (strain Cowan),  Staphylococcus capiticus  (strain V19),  Staphylococcus epidermidis  (strain 1587),  Staphylococcus hominis  (strain V27),  Staphylococcus warneri  (strain V64),  Staphylococcus saprofyticus  (strain NCTC 7292),  Staphylococcus haemolyticus  (strain V8/1),  Salmonella typhimurium, Eneterococcus faecium  (ATCC 700221),  Eneterococcus faecium  (daptomycin resistant strain),  Eneterococcus faecium  (linezolid resistant strain),  Eneterococcus faecium  (ampicillin resistant strain),  Enterococcus faecium  (vancomycin-resistant strains E0013, E0072, E0300, E0321, E0333, E0338, E0341, E0506, E0745, E1130, E1441, E1679, E1763, E2297, E2359, E2365, E2373, E6016, E7312, E7314, E7319, E7329, E7401, E7403, E7413, E7424, E7464, E8218, E8235, E8237),  Eneterococcus faecalis  (ATCC 700802),  Eneterococcus faecalis  (strain JH2-2),  Eneterococcus faecalis  (strain MMH594),  Eneterococcus faecalis  (ATCC 29212),  Eneterococcus faecalis  (ATCC 47077),  Eneterococcus hirae, Eneterococcus casseliflavus, Eneterococcus gallinarum, Eneterococcus. Raffinosus, Eneterococcus avium, Eneterococcus cecorum, Eneterococcus saccharominimus, Eneterococcus columbae, Eneterococcus durans, Klebsiellsa pneumoniae, Lactobacillus paracasei, Clostridium tetani, Clostridium botulinum, Clostridium perfringes, Clostridium difficile, Bacillus anthracis  and  Listeria moncytogenes . The above drug-resistant strains were identified and known at the Utrecht Medical Center. 
     As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual or animal being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. 
     An “effective amount” of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. 
     “Natural or non-natural amino acids” refers to any of the common naturally occurring amino acids as well as modified, derivatized, enantiomeric, rare and/or unusual amino acids which may be synthetically obtained or originating from a natural source. Examples of naturally occurring amino acids includes alanine (Ala, A), cysteine (Cys, C), aspartic acid (Asp, D), glutamic acid (Glu, E), phenylalanine (Phe, F), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), lysine (Lys, K), leucine (Leu, L), methionine (Met, M), asparagine (Asn, N), proline (Pro, P), glutamine (Gln, Q), arginine (Arg, R), selenocysteine (Sec, U), serine (Ser, S), threonine (Thr, T), valine (Val, V), tryptophan (Trp, W), and tyrosine (Tyr, Y). Examples of the modified amino acids include hydroxyproline, hydroxylysine, actetyllysine, desmosine, isodesmosine, ε-N-methyllysine, ε-N-trimethyllysine, methylhistidine, dehydrobutyrine (Dhb), dehydroalanine (Dha), α-aminobutyric acid (Abu), 2,3-diaminopropioninc acid, β-alanine, γ-aminobutyric acid, homocysteine, homoserine, citrulline and ornithine. 
     The invention is exemplified in the following non-limiting examples. 
     EXAMPLES 
     Example 1. Preparation of Nisin-Derived Antimicrobial Compounds 
     All reagents employed were of American Chemical Society (ACS) grade or finer and were used without further purification unless otherwise stated. Flash chromatography was performed using Merck type 60, 230-400 mesh silica gel. Peptides were purified on a Reprospher 100 C8-Aqua column (10 250×20 mm) at a flow rate of 12 mL·min −1 . High resolution mass spectrometry (HRMS) analysis was performed using an ESI-TOF LC/MS instrument  1 H NMR spectra were recorded at 400 MHz with chemical shifts reported in parts per million (ppm) downfield relative to tetramethylsilane (TMS).  1 H NMR data are reported in the following order: multiplicity (s, single; d, doublet; t, triplet, q, quartet; qn, quintet and m, multiplet), number of protons, coupling constant (J) in Hertz (Hz). When appropriate, the multiplicity is preceded by br, indicating that the signal was broad.  13 C NMR spectra were recorded at 100 MHz with chemical shifts reported relative to the residual carbon resonance of the solvent used. All literature compounds had  1 H NMR, and mass spectra consistent with the assigned structures. 
     Preparation of the Nisin [1-12] Structure (Comparative Compound A) 
     Nisin (600 mg, 0.18 mmol) was dissolved in 250 mL Tris buffer (25 mmol NaOAc, 5 mmol Tris acetate, 5 mmol CaCl 2 , pH 7) and the solution was cooled on ice for 15 min. Trypsin (50 mg) was added and the mixture was stirred at RT for 15 min. The mixture was then heated to 30° C. for 16 hours and an aliquot was analyzed by HPLC. Another 50 mg of trypsin was added and after an additional 24 hours the reaction was complete, as evidenced by HPLC. The reaction was acidified with HCl (1 N) to a pH of 4 and solvents were removed in vacuo. The nisin [1-12] structure was isolated from the mixture by preparative HPLC. Product fractions were lyophilized to obtain a white powder (80 mg, 39%). 
     Preparation of the Farnesyl-Amine (According to G M Coppola and M Prashad,  Synthetic Communications  23, No. 4 (1993): 535-41) 
     Lithium bis(trimethylsilyl) amide (7.7 mL; 1.0 M in THF) was added to trans,trans-farnesyl bromide (6.7 mmol, 1.9 g) under a blanket of argon and the mixture was stirred for 16 hours, followed by quenching with a saturated ammonium chloride solution. The mixture was extracted twice with MTBE and the organic phases were combined and dried over Na 2 SO 4 . To this oil was added 31 mL MeOH and 4 mL CH 2 Cl 2  and the resulting solution was stirred at room temperature for 16 hours. Solvents were removed under vacuum to give a brown solid as product (1.5 g, quant.). 
       1 H NMR (400 MHz, CDCl 3 ): δ 5.28-5.24 (m, 1H), 5.12-5.07 (m, 2H), 3.29-3.27 (d, 2H, J=7.0 Hz), 2.10-1.95 (m, 8H), 1.67-1.59 (m, 12H), 1.12 (s, 2H);  13 C NMR (100 MHz, CDCl 3 ): δ□137.8, 135.2, 131.3, 125.4, 124.3, 123.9, 39.7, 39.5, 26.7, 26.4, 25.7, 17.6, 16.1, 16.0. 
     Preparation of 2,5-dioxopyrrolidin-1-yl pent-4-ynoate 
     4-Pentynoic acid (2.00 g, 20.36 mmol) was dissolved in DMF (40 mL) and EDCl (5.84 g, 29.84 mmol, 1.5 equiv.) and NHS (4.68 g, 40.76 mmol, 2.0 equiv.) were added. The mixture was stirred for 16 hours at RT. After evaporation of DMF the residue was diluted in EtOAc (120 mL) and washed two times with NH 4 Cl (1 M, 120 mL) and two times with saturated NaHCO 3  (120 mL). The organic layer was dried with Na 2 SO 4  and the product was purified with flash column chromatography (EtOAc: PE) to obtain the desired activated ester as a white powder (3.3 g, 83%). 
       1 H NMR (400 MHz, CDCl 3 ): δ 2.89-2.83 (m, 4H), 2.63-2.59 (m, 4H), 1.55 (bs, 1H);  13 C NMR (100 MHz, CDCl 3 ): δ 168.8, 167.0, 80.8, 70.0, 30.3, 25.5, 14.1. 
     Preparation of Didecyl-Alkyne 
     Prepared via procedure 3 (p3) using didecylamine. 
     Yield: 105 mg, 30% 
       1 H NMR (400 MHz, CDCl 3 ): δ 3.31-3.27 (m, 2H), 3.22-3.18 (m, 2H), 2.54 (m, 4H), 1.95 (2, 1H), 1.56-1.48 (m, 4H), 1.27-1.25 (m, 28H), 0.90-0.85 (m, 6H);  13 C NMR (100 MHz, CDCl 3 ): δ 170.0, 83.8, 68.5, 47.8, 46.1, 32.1, 31.9, 29.6, 29.5, 29.3, 22.7, 14.6, 14.1; HRMS calculated for C 25 H 48 NO [M+H] + : 378.3736, found 378.3743. 
     Preparation of Octadecyl-Alkyne 
     Prepared via procedure 3 (p3) using octadecylamine. 
     Yield: 560 mg, 52% 
       1 H NMR (400 MHz, CDCl 3 ): δ 5.61 (bs, 1H), 3.27-3.21 (m, 2H), 2.53-2.50 (m, 2H), 2.39-2.34 (m, 2H), 1.99-1.97 (m, 1H), 1.47-1.47 (m, 3H), 1.27-1.24 (m, 32H), 0.88-0.84 (m, 3H);  13 C NMR (100 MHz, CDCl 3 ): δ 170.7, 83.0, 69.2, 39.6, 35.4, 31.9, 29.7, 29.6, 29.5, 29.3, 26.9, 22.7, 15.0, 14.1; HRMS calculated for C 23 H 44 NO [M+H] + : 350.3423, found 350.3430. 
     Preparation of Farnesyl-Alkyne 
     Prepared via procedure 3 (p3) using farnesyl-amine. 
     Yield: 540 mg, 60% 
       1 H NMR (400 MHz, CDCl 3 ): δ 5.69 (bs, 1H), 5.19-5.15 (m, 1H), 5.06-5.04 (m, 2H), 4.12-4.08 (m, 2H), 3.85-3.82 (m, 2H), 2.78 (s, 1H), 2.50-2.48 (m, 4H), 2.38-2.34 (m, 4H), 1.64 (s, 9H), 1.57 (s, 3H), 1.25-1.21 (m, 2H);  13 C NMR (100 MHz, CDCl 3 ): δ 170.5, 140.2, 135.4, 131.4, 124.2, 123.7, 119.7, 83.0, 69.2, 39.7, 39.5, 37.6, 35.4, 26.7, 26.3, 25.7, 17.7, 16.3, 16.0, 14.9; HRMS calculated for C 20 H 32 NO [M+H] + : 302.2484, found 302.2474. 
     Preparation of Terphenyl-Alkyne 
     A mixture of terphenyl carboxylic acid (250 mg, 1.0 equiv.) and thionyl chloride (10 mL, per mmol carboxylic acid) was refluxed until all solid was dissolved followed by additional heating for 16 hours. After evaporation of excess thionyl chloride at reduced pressure the obtained acid chloride was dried in vacuo. The acid chloride was dissolved in 15 mL DCM and propargylamine HCL (183 mg, 2.0 equiv.) was added. Upon addition of TEA (558 μL, 4.0 equiv.) the solution turned into a thick white suspension. 5 mL of DCM was added to facilitate stirring. TLC showed little conversion after 3 hours. Pyridine (790 μL, 10 equiv.) was added and the mixture was heated to reflux. After 2 hours the reaction was complete. Solvents were removed under vacuum and the residue suspended in CHCl 3 . The precipitate were collected by filtration, washed with MeOH and dried under vacuum. Yield: 189 mg, 63%. 
       1 H NMR (400 MHz, DMSO-d6): δ 8.98 (s, 1H), 7.97-7.95 (m, 2H), 7.84-7.71 (m, 4H), 7.48-7.37 (m, 3H), 4.07 (s, 1H), 3.31 (s, 6H);  13 C NMR (100 MHz, DMSO-d6): δ 166.0, 142.8, 140.2, 139.9, 138.6, 133.1, 129.5, 128.5, 127.8, 127.7, 127.1, 126.9, 81.8, 73.3, 29.0; HRMS calculated for C 22 H 18 NO [M+H] + : 312.1388, found 312.1361. 
     Preparation of Boc-Nisin [1-11]Lys(Boc)-OH (Comparative Compound F) 
     Boc 2 O (50 mg, 229 μmol) and DIPEA (51 μL, 293 μmol) were added to a solution of nisin [1-12] (100 mg, 86.9 μmol) in dry MeOH (30 mL) and the mixture was stirred for 4.5 hours. The reaction mixture was concentrated, redissolved in H 2 O/MeCN/TFA (70/30/0.1) and purified by preparative HPLC using a C18 Maisch 250×22 mm to yield 68.9 mg (51.0 μmol) of white powder (57% yield). ESI-MS: calcd for C 61 H 99 ON 13 O 17 S 2  [M+H] +  1350.6796, found 1350.6818. 
     Preparation of ((S)-2-amino-N-decyl-3-(1H-indol-3-yl)propanamide) 
     Prepared via procedure 7 (p7) using Boc-Trp-OH. Yield=88%  1 H NMR (CD 3 OD): δ 7.55 (d, 7.88 Hz, 1H), 7.29 (d, 8.12 Hz, 1H), 7.04 (t, 7.50 Hz, 2H), 6.96 (t, 7.42 Hz, 1H), 3.55 (t, 6.72 Hz, 1H), 3.14-2.91 (m, 4H), 1.32-1.00 (m, 16H), 0.90-0.82 (t, 6.64 Hz, 3H).  13 C NMR (CD 3 OD): δ 176.70, 138.1, 128.8, 124.7, 122.4, 119.8, 119.5, 112.3, 111.1, 57.0, 40.4, 33.0, 32.3, 30.5, 30.1, 27.9, 23.7, 18.2, 14.5. ESI-MS: calcd for C 21 H 33 N 3 O [M+H] +  344.26, found 344.15. 
     Preparation of ((S)-2-amino-N-decyl-3-(4-hydroxyphenyl)propanamide) 
     Prepared via procedure 7 (p7) using Boc-Tyr-OH. Yield=31%  1 H NMR (400 MHz; CD 3 OD): δ 8.17-8.10 (m, 1H), 7.04 (d, J=8.0 Hz, 2H), 6.74 (d, J=7.6 Hz, 2H), 3.90 (t, J=7.0 Hz, 1H), 3.25-3.11 (m, 2H), 3.08-2.86 (m, 2H), 1.39-1.36 (m, 2H), 1.35-1.09 (m, 14H), 0.87 (t, J=6.4 Hz, 3H).  13 C NMR (100 MHz; CD 3 OD): 6168.0, 156.8, 130.1, 124.6, 115.3, 54.6, 39.2, 36.6, 31.6, 29.3, 29.2, 29.0, 28.9 28.7, 26.5, 22.3, 13.0. ESI-MS: calcd for C 19 H 32 N 2 O 2 [M+H] + : 321.2537, found 321.75. 
     Preparation of ((S)-2-amino-N-decyl-3-phenylpropanamide) 
     Prepared via procedure 7 (p7) using Boc-Phe-OH. Yield=3%  1 H NMR (CD 3 OD): δ 7.39-7.23 (m, 5H), 3.99 (t, J=7.48 Hz, 1H), 3.24-3.01 (m, 4H), 1.43-1.13 (m, 16H), 0.90 (t, J=6.86 Hz, 3H).  13 C NMR (CD 3 OD): δ 135.7, 130.5, 130.0, 128.8, 55.9, 40.6, 38.8, 33.1, 30.5, 30.1, 27.9, 23.7, 14.4. ESI-MS: calcd for C 19 H 32 N 2 O [M+H] +  305.25, found 305.15. 
     Preparation of ((S)-2-amino-N-decyl-3-(1H-imidazol-4-yl)propanamide) 
     Prepared via procedure 7 (p7) using Boc-His-OH. Yield=quant.  1 H NMR (CD 3 OD): δ 8.84 (d, J=1.2 Hz, 1H), 8.24 (bs, 1H), 7.07 (bs, 1H), 4.18-4.12 (m, 1H), 3.31-3.27 (m, 2H), 3.18 (t, J=7.2 Hz, 2H), 1.31-1.23 (m, 16H), 0.87 (t, 3H).  13 C NMR (100 MHz; CD 3 OD): δ 143.9, 140.8, 118.1, 52.3, 39.4, 31.6, 29.2, 29.0, 28.9, 28.8, 28.7, 26.5, 22.3, 13.0. ESI-MS: calcd for C 16 H 30 N 4 O [M+H] + : 295.2492, found 295.20. 
     Preparation of (tert-butyl ((S)-1-(((S)-1-(decylamino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)carbamate) 
     A solution of H-Trp-C10 (1.0 g, 2.91 mmol), Boc-Trp-OH (1.1 eq.) BOP (1.1 eq.) and DIPEA (3.3 eq) in CH 2 Cl 2  was stirred for 1 hour at room temperature. The reaction mixture was concentrated, taken up in EtOAc and washed with 1M KHSO 4  and sat.NaHCO 3 . Drying with Na 2 SO 4  and concentrating yielded Boc-Trp-Trp-C10 as yellow solid foam (1.45 g, 2.30 mmol, yield: 79%). This material (0.5 g, 0.79 mmol) was treated with TFA/CH 2 Cl 2  in the presence of TiS (0.195 mL, 0.95 mmol) for 1 hour at room temperature. After concentration, the residue was taken up in EtOAc and washed with sat. NaHCO 3 . Drying with Na 2 SO 4  and concentrating yielded H-Trp-Trp-C10 as oily substance (quantitative yield). MS analysis confirmed removal of the Boc group and the material was used without further purification. Boc-Trp-Trp-C10  1 H NMR: (CDCl 3 ): δ 8.73 (s, 1H), 8.43 (1H), 7.63 (d, J=7.6 Hz, 1H), 7.42 (d, J=8.0 Hz, 1H), 7.29-7.00 (m, 6H), 6.73-6.63 (m, 3H), 6.40 (s, 1H), 6.33 (d, J=7.6 Hz, 1H), 4.90 (d, J=5.2 Hz, 1H), 4.66 (m, 1H), 4.26 (m, 1H), 3.45-3.33 (m, 2H), 3.13-3.08 (m, 2H), 2.99-2.96 (m, 1H), 2.58-2.54 (m, 1H), 1.46-1.02 (m, 25H), 0.87 (t, J=6.8 Hz).  13 C NMR: (100 MHz; CDCl 3 ): δ 171.9, 171.1, 155.9, 136.7, 136.3, 127.6, 127.4, 123.6, 123.5, 122.8, 22.3, 120.1, 119.7, 119.0, 118.4, 111.7, 111.4, 109.8, 55.7, 53.8, 39.9, 32.0, 29.7, 29.6, 29.4, 29.4, 29.2, 27.9, 26.9, 22.8, 14.2. MS: [M+H] +  calcd 630.4014; measured 629.89. H-Trp-Trp-C10: MS [M+H] +  calcd 530.3490; measured 530.10. 
     Preparation of (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl 3-((tert-butoxycarbonyl) amino) propanoate) 
     DCC (5.5 g, 26.6 mmol) in EtOAc (10 mL) was added to a mixture of Boc-β-Ala-OH (5.0 g, 26.4 mmol) and NHS (3.1 g, 26.9 mmol) in EtOAc (100 mL). The white suspension was stirred overnight at room temperature followed by filtration over celite. The clear filtrate was concentrated and recrystallized from MTBE/hexanes to yield white crystals (6.05 g, 21.1 mmol). 
     Yield: 80%  1 H NMR: (CDCl 3 ): δ 5.14 (br, 1H), 3.48-3.46 (m, 2H), 2.81-2.79 (m, 6H), 1.40 (s, 9H).  13 C NMR: (100 MHz; CDCl 3 ): δ 169.2, 167.6, 155.8, 79.7, 36.2, 32.2, 28.4, 25.6. MS: [M-tBu+H] +  calcd 229.0455; measured 230.71. 
     Procedure 1 (p1): Amide Coupled Lipid-Nisin 
     The resulting powder of the nisin [1-12] structure was dissolved in DMF or THF (240 μl) and the corresponding lipid-amine (59 equivalents), BOP (2 equivalents) and DiPEA (4 equivalents) were added. The reaction was stirred for 20 min and subsequently quenched with 4 mL buffer A (H 2 O:MeCN, 95:5+0.1% TFA). The solution was centrifuged for 5 min at 5000 rpm to remove any insoluble material and the supernatant was purified via preparative HPLC. Product fractions were lyophilized to obtain the final product. 
     Procedure 2 (p2): Clicked Lipid-Nisin 
     A 10× stock solution of copper sulfate (16.2 μmol, 2.59 mg in 1 mL H 2 O), a 10× stock solution of sodium ascorbate (32.4 μmol, 6.42 mg in 1 mL H 2 O), and a 10× stock solution of TBTA (4.1 μmol, 2.18 mg in 1 mL DMF) were prepared. Nisin [1-12]-azide was prepared using procedure 1 (p1) as indicated in Table 3 (Comparative compound E). Nisin [1-12]-azide (8.1 μmol, 10 mg) was dissolved in DMF (200 μL). Lipid-alkyne was added to the μW vessel. The nisin [1-12]-azide solution was added along with 100 μL of the TBTA stock solution, 100 μL of the sodium ascorbate stock solution and 100 μL of the copper sulfate stock solution. The vessel was put in the microwave and reacted at 80° C. for 20 min. After completion, the reaction mixture was quenched with 4 mL buffer B (H 2 O:MeCN, 5:95+0.1% TFA) and purified via preparative HPLC. 
     Procedure 3 (p3): Lipid-Alkynes 
     The lipid-amine (3 mmol) was dissolved in DMF (20 mL) and 2,5-dioxopyrrolidin-1-yl pent-4-ynoate (2.0 mmol, 390 mg) was added while stirring and the reaction was allowed to run for 16 hours. After evaporation of DMF the product was purified with flash column chromatography (EtOAc:PE, 1:4) to obtain the final product. 
     Procedure 4 (p4): Amine Coupling to Boc-Protected Nisin [1-12] 
     The lipid-amine (1.2 eq), BOP (1.2 eq) and DiPEA (3 eq) were added to a solution of Boc-Nisin [1-11]Lys(Boc)-OH (1 eq) in dry CH 2 Cl 2  (2 μmol/mL). A few drops of DMF aided in the solution of the compounds. The mixture was stirred for 45 min, concentrated and the residue treated with TFA/TiS/H 2 O (95/2.5/2.5) for 1 hour and precipitated in MTBE/hexanes (1:1), centrifuged (5 min at 4.500 rpm). The pellet was dissolved in H 2 O/t-BuOH (1:1) and lyophilized. The lyophilized powder was dissolved in 4 mL buffer B (H 2 O:MeCN, 5:95+0.1% TFA) and purified via preparative HPLC. 
     Procedure 5 (p5): Lys 12  Acylated Compounds 
     Nisin [1-12] was dissolved in DMF/THF (1/1) and 4 eq of DiPEA was added. Dropwise addition of a solution of 1 eq. of the carboxylic acid activated ester dissolved in THF resulted in preferred acylation of the lysine side chain. The addition of more than 1 equivalent of activated ester results in the acylation of both the N-terminus and the Lys 12  side chain After 1.5 hours the reaction mixture was concentrated and purified by preparative HPLC using a Maisch Reprospher 100 C8-Aqua, 250 mm×20 mm. The lipid-amine (1.2 eq), BOP (1.2 eq) and DiPEA (3 eq) were added to a solution of the acylated Nisin [1-12] (1 eq) in DMF/THF (2 μmol/mL). The mixture was stirred for 45 min, concentrated, precipitated in MTBE/hexanes (1:1) and centrifuged (5 min at 4.500 rpm). The pellet was dissolved in H 2 O/t-BuOH (1:1) and lyophilized. The lyophilized powder was dissolved in 4 mL buffer B (H 2 O:MeCN, 5:95+0.1% TFA) and purified via preparative HPLC. Note: the mono and bis β-Ala acylated variants of Nisin [1-12]-C12 were obtained by treating the respective Boc protected precursors with TFA/TIS/H 2 O (95/2.5/2.5) followed by precipitation in MTBE/hexanes and preperative HPLC purification as described before. 
     Procedure 6 (p6): Amino-Acid-Lipids 
     A Boc-protected amino acid (Boc-AA-OH) was dissolved in CH 2 Cl 2  and cooled at 0° C. EDC (2.5 eq.), HOBT (2.5 eq), decylamine (1.5 eq) and triethylamine (1.5 eq.) were added and the mixture was stirred overnight while warming to room temperature. The reaction mixture was washed with H 2 O, 1M NaOH and 1M HCl. Purification via recrystallization (hexanes/EtOAc) or silica gel column chromatography (petroleum ether/EtOAc) yielded the Boc-AA-decylamine intermediates. The Boc-amino acid-decylamine compound was dissolved in CH 2 Cl 2  and TiS (2 eq.) and TFA were added to reach a ratio of CH 2 Cl 2 :TFA (2:1) and the mixture was stirred for 1 hour. The reaction mixture was concentrated and the deprotected amino acid-C10 was optionally taken up in EtOAc and washed with sat. NaHCO 3 . Concentrating yielded the lipidated amino acids as oily substances. 
     The compounds used for further testing were prepared as indicated in Table 3 and 4. In the Tables the procedure used as well as the specific substances used in the procedure are shown. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Preparation of antimicrobial compounds according to Formula (1) 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 Z = NHR 1   
                 Y = NHR 3   
                   
                   
               
               
                 Compound 
                 X 8   
                 R 1  = 
                 R 3  = 
                 Procedure used 
                 Comments 
               
               
                   
               
               
                 A 
                 Lys 
                 Z = —OH 
                 H 
                 p1 
                   
               
               
                 D 
                 Lys 
                 —CH 3   
                 H 
                 p1 
                 with methylamine and using THF as solvent 
               
               
                   
               
               
                 E 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 p1 
                 With 3-azidopropan-1-amine and using DMF as solvent 
               
               
                   
               
               
                 F 
                 Lys(Boc) 
                 Z = OH 
                 Boc 
                   
                   
               
               
                  (2) 
                 Lys 
                 —C 6 H 13   
                 H 
                 p1 
                 with hexylamine and using DMF as solvent 
               
               
                  (3) 
                 Lys 
                 —C 7 H 15   
                 H 
                 p1 
                 With heptylamine and using DMF as 
               
               
                   
                   
                   
                   
                   
                 solvent 
               
               
                  (4) 
                 Lys 
                 —C 8 H 17   
                 H 
                 p1 
                 With octylamine and using DMF as solvent 
               
               
                  (5) 
                 Lys 
                 —C 9 H 19   
                 H 
                 p1 
                 With nonylamine and using DMF as solvent 
               
               
                  (6) 
                 Lys 
                 —C 10 H 21   
                 H 
                 p1 
                 with decylamine and using DMF as solvent 
               
               
                  (7) 
                 Lys 
                 —C 11 H 23   
                 H 
                 p1 
                 With undecylamine and using DMF as 
               
               
                   
                   
                   
                   
                   
                 solvent 
               
               
                  (8) 
                 Lys 
                 —C 12 H 25   
                 H 
                 p1 
                 With dodecylamine and using DMF as 
               
               
                   
                   
                   
                   
                   
                 solvent 
               
               
                  (9) 
                 Lys 
                 —C 13 H 27   
                 H 
                 p1 
                 With tridecylamine and using DMF as 
               
               
                   
                   
                   
                   
                   
                 solvent 
               
               
                 (10) 
                 Lys 
                 —C 14 H 29   
                 H 
                 p1 
                 with tetradecylamine and using THF as 
               
               
                   
                   
                   
                   
                   
                 solvent 
               
               
                 (11) 
                 Lys 
                 —C 15 H 31   
                 H 
                 p1 
                 With pentadecylamine and using THF as 
               
               
                   
                   
                   
                   
                   
                 solvent 
               
               
                   
               
               
                 (12) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 p1 
                 with farnesylamine and using DMF as solvent 
               
               
                   
               
               
                 (13) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 p4 
                 With (4′-chloro-[1,1′-biphenyl]-4- yl)methanamine and using CH 2 Cl 2  as solvent 
               
               
                   
               
               
                 (14) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 p1 
                 With N-(3-aminopropyl)-N,N- dimethyldecan-1-aminium 2,2,2- trifluoroacetate and using DMF as solvent 
               
               
                   
               
               
                 (15) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 p4 
                 With (S)-2-amino-N-decyl-3-(1H-indol-3- yl)propanamide and using CH 2 Cl 2  as solvent 
               
               
                   
               
               
                 (16) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 p4 
                 With (S)-2-amino-N-decyl-3- phenylpropanamide and using CH 2 Cl 2  as solvent 
               
               
                   
               
               
                 (17) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 p4 
                 With (S)-2-amino-N-decyl-3-(1H-imidazol-4- yl)propanamide and using CH 2 Cl 2  as solvent 
               
               
                   
               
               
                 (18) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 p4 
                 With (S)-2-amino-N-decyl-3-(4- hydroxyphenyl)propanamide and using CH 2 Cl 2  as solvent 
               
               
                   
               
               
                 (19) 
                 Lys((C═O)C 8 H 17 ) 
                 —CH 3   
                 H 
                 p5 
                 With 2,5-dioxopyrrolidin-1-yl nonanoate, 
               
               
                   
                   
                   
                   
                   
                 methylamine and using DMF as solvent 
               
               
                 (20) 
                 Acetyllysine 
                 —C 12 H 25   
                 H 
                 p5 
                 With 2,5-dioxopyrrolidin-1-yl acetate, 
               
               
                   
                   
                   
                   
                   
                 dodecylamine and using MeOH, then DMF 
               
               
                   
                   
                   
                   
                   
                 as solvents. 
               
               
                   
               
               
                 (21) 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 —C 12 H 25   
                 H 
                 p5 
                 With Boc-β-alanine-OSu and dodecylamine, using DMF as solvent 
               
               
                   
               
               
                 (22) 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 —C 12 H 25   
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 p5 
                 With Boc-β-alanine-OSu and dodecylamine, using DMF as solvent 
               
               
                   
               
               
                 (23) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 p1 
                 With (S)-2-amino-N-((S)-1-(decylamino)-3- (1H-indol-3-yl)-1-oxopropan-2-yl)-3-(1H- indol-3-yl)propanamide and using DMF as solvent 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Preparation of antimicrobial compounds based on Formula (24) with four different R 1  structures. 
               
            
           
           
               
               
               
               
               
               
            
               
                 R 1   
                   
                   
                 Y = NHR 3   
                 Procedure 
                   
               
               
                 structure 
                 X 8  = 
                 R 1  = 
                 R 3  = 
                 used 
                 Comments 
               
               
                   
               
               
                 (b) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 p2 
                 with didecylalkyne 
               
               
                   
               
               
                 (c) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 p2 
                 With octadecyl alkyne 
               
               
                   
               
               
                 (d) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 p2 
                 With farnesyl alkyne 
               
               
                   
               
               
                 (e) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 p2 
                 With terphenyl alkyne 
               
               
                   
               
            
           
         
       
     
     The analysis of the prepared compounds are shown in Tables 5 and 6. The retention times (R t ) were measured using a Dr. Maisch C8 column (250×4.6 mm, 300 Å, 10 μm) using a flow rate of 1.0 mL/min and the following gradients: (a) 5-60% MeCN (0.1% TFA) in 40 min; (b) 5-95% MeCN (0.1% TFA) in 40 min, and (c) 5-95% MeCN (0.1% TFA) in 60 min; or using a Dr. Maisch C18 column (250×4.6 mm, 300 Å, 10 μm) using a flow rate of 1.0 mL/min and the following gradients: (d) 5-95% MeCN (0.1% TFA) in 40 min; (e) 5-95% MeCN (0.1% TFA) in 60 min. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Analysis of the compounds (2) to (23) according to Formula (1), left column. Compounds A, D, E, and F are comparative examples. 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                 Z = NHR 1   
                 Y = NHR 3   
                   
                 Yield 
                 Calculated 
                 Measured 
                 R t   
               
               
                   
                 X 8  = 
                 R 1  = 
                 R 3  = 
                 Formula 
                 (%) 
                 MW 
                 MW 
                 (min) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 A 
                 Lys 
                 Z = OH 
                 H 
                 C 51 H 83 N 13 O 13 S 2   
                 37 
                 1150.5753 
                 1150.5718 
                 17.8 (b)   
               
               
                 D 
                 Lys 
                 —CH 3   
                 H 
                 C 52 H 86 N 14 O 12 S 2   
                 27 
                 1163.61 
                 1163.63 
                 12.2 (b)   
               
               
                   
               
               
                 E 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 C 54 H 89 N 17 O 12 S 2   
                 65 
                 1232.6396 
                 1232.6387 
                 18.2 (a)   
               
               
                   
               
               
                 F 
                 Lys(Boc) 
                 Z = OH 
                 Boc 
                 C 61 H 99 N 13 O 17 S 2   
                 57 
                 1350.6796 
                 1350.6818 
                 37.93 (c)   
               
               
                  (2) 
                 Lys 
                 —C 6 H 13   
                 H 
                 C 57 H 96 N 14 O 12 S 2   
                 30 
                 1233.6852 
                 1233.6873 
                 21.9 (c)   
               
               
                  (3) 
                 Lys 
                 —C 7 H 15   
                 H 
                 C 58 H 98 N 14 O 12 S 2   
                 39 
                 1247.7003 
                 1247.7064 
                 30.47 (e)   
               
               
                  (4) 
                 Lys 
                 —C 8 H 17   
                 H 
                 C 59 H 100 N 14 O 12 S 2   
                 18 
                 1261.7159 
                 1261.7184 
                 31.01 (e)   
               
               
                  (5) 
                 Lys 
                 —C 9 H 19   
                 H 
                 C 60 H 102 N 14 O 12 S 2   
                 33 
                 1275.7316 
                 1275.7339 
                 32.38 (e)   
               
               
                  (6) 
                 Lys 
                 —C 10 H 21   
                 H 
                 C 61 H 104 N 14 O 12 S 2   
                 45 
                 1289.7478 
                 1289.7565 
                 21.9 (a)   
               
               
                  (7) 
                 Lys 
                 —C 11 H 23   
                 H 
                 C 62 H 106 N 14 O 12 S 2   
                 32 
                 1303.7629 
                 1303.7623 
                 35.17 (e)   
               
               
                  (8) 
                 Lys 
                 —C 12 H 25   
                 H 
                 C 63 H 108 N 14 O 12 S 2   
                 31 
                 1317.7785 
                 1317.7802 
                 36.03 (e)   
               
               
                  (9) 
                 Lys 
                 —C 13 H 27   
                 H 
                 C 64 H 110 N 14 O 12 S 2   
                 26 
                 1331.7942 
                 1331.7927 
                 38.53 (e)   
               
               
                 (10) 
                 Lys 
                 —C 14 H 29   
                 H 
                 C 65 H 112 N 14 O 12 S 2   
                 31 
                 1345.8104 
                 1345.8109 
                 28.4 (c)   
               
               
                 (11) 
                 Lys 
                 —C 18 H 31   
                 H 
                 C 66 H 114 N 14 O 12 S 2   
                 27 
                 1359.8255 
                 1359.8226 
                 42.58 (e)   
               
               
                   
               
               
                 (12) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 C 66 H 108 N 14 O 12 S 2   
                 45 
                 1353.7713 
                 1353.7821 
                 21.5 (b)   
               
               
                   
               
               
                 (13) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 C 64 H 93 ClN 14 O 12 S 2   
                 23 
                 1349.6300 
                 1349.6308 
                 31.68 (e)   
               
               
                   
               
               
                 (14) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 C 66 H 116 N 15 O 12 S 2   +   
                 9 
                 1374.8364 
                 1374.8357 
                 31.20 (e)   
               
               
                   
               
               
                 (15) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 C 72 H 114 N 16 O 13 S 2   
                 21 
                 1475.8265 
                 1474.37 
                 36.37 (e)   
               
               
                   
               
               
                 (16) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 C 70 H 113 N 15 O 13 S 2   
                 13 
                 1436.8156 
                 1437.25 
                 36.50 (e)   
               
               
                   
               
               
                 (17) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 C 67 H 111 N 17 O 13 S 2   
                 26 
                 1426.8061 
                 1426.90 
                 31.10 (e)   
               
               
                   
               
               
                 (18) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 C 70 H 113 N 15 O 14 S 2   
                 51 
                 1452.8106 
                 1452.90 
                 34.55 (e)   
               
               
                   
               
               
                 (19) 
                 Lys((C═O)C 8 H 17 ) 
                 —CH 3   
                 H 
                 C 61 H 102 N 14 O 13 S 2   
                 48 
                 1303.7265 
                 1304.15 
                   
               
               
                 (20) 
                 Acetyllysine 
                 —C 12 H 25   
                 H 
                 C 65 H 110 N 14 O 13 S 2   
                 36 
                 1359.7891 
                 1359.65 
                 29.87 (e)   
               
               
                   
               
               
                 (21) 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 —C 12 H 25   
                 H 
                 C 66 H 113 N 15 O 13 S 2   
                 6 
                 1388.8156 
                 1387.98 
                 30.03 (e)   
               
               
                   
               
               
                 (22) 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 —C 12 H 25   
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 C 69 H 118 N 16 O 14 S 2   
                 12 
                 1459.8528 
                 1458.88 
                 28.88 (e)   
               
               
                   
               
               
                 (23) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 C 83 H 124 N 18 O 14 S 2   
                 6 
                 1661.9059 
                 1663.45 
                 38.41 (e)   
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Analysis of antimicrobial compounds based on Formula (24) with four different R 1  structures (b), (c), (d) and (e). 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 Calculated 
                 Measured 
                   
               
               
                 struc- 
                   
                   
                 Y = NHR 3   
                   
                 Yield 
                 MW 
                 MW 
                 R t   
               
               
                 ture 
                 X 8  = 
                 R 1  = 
                 R 3  = 
                 Formula 
                 (%) 
                 [M + H] +   
                 [M + H] +   
                 (min) 
               
               
                   
               
               
                 (b) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 C 79 H 136 N 18 O 13 S 2   
                 13 
                 1610.0054 
                 1610.0011 
                 25.2 (a)   
               
               
                   
               
               
                 (c) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 C 75 H 132 N 18 O 13 S 2   
                 24 
                 1581.9741 
                 1581.9741 
                 30.9 (a)   
               
               
                   
               
               
                 (d) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 C 74 H 120 N 18 O 13 S 2   
                 60 
                 1533.8802 
                 1533.8789 
                 19.6 (b)   
               
               
                   
               
               
                 (e) 
                 Lys 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 H 
                 C 76 H 106 N 18 O 13 S 2   
                 20 
                 1543.7706 
                 1543.7703 
                 22.2 (a)   
               
               
                   
               
            
           
         
       
     
     Example 2. Analysis of Antimicrobial Compounds in a MIC Assay 
     Different microorganisms (from glycerol stock) were plated out on blood agar and incubated at 37° C. for 24 hours. A colony was selected and 2×5 mL of TSB was inoculated. The samples and a sterile control were cultured for 16-20 hours at 37° C. Comparative compound B is nisin acting as a control. Comparative compound C is vancomycin, also acting as a control. Comparative compounds A, D and E are also control compounds not part of the present invention. 
     100 μL of the compound (2) to (23), and compound (24) with R 1 -groups (b) to (e), and Comparative compounds A, D and E (128 μg/mL, 2% DMSO in TSB), 100 μL of the positive controls of nisin (Comparative compound B) and vancomycin (Comparative compound C) (2 μg/mL, 2% DMSO in TSB), and 100 μL of a negative control (2% DMSO in TSB), were added to the top of the row of a 96-well plate, 50 μL of TSB to the rest of the wells and the compounds were diluted serially. The overnight cultures were diluted to 0.5×10 6  CFU in TSB. 50 μL of the bacterial solution was added to each well and the plates were sealed with an adhesive membrane and incubated at 37° C. for 16 hours. The next day, the plates were visually inspected for bacterial growth. 
     The results of the MIC assays for various bacteria are shown in Table 7. Table 8 shows the activity of compound (10) against a large number of different VRE strains, allowing for the determination of a MIC 50  and MIC 90 , which were 4 and 8, respectively. The same values were found for comparative compound B (nisin), illustrating the potency of the new compounds. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 MIC values a  (measured in μg/mL). All data stem from duplicate 
               
               
                 experiments. Where appropriate, values are represented as a range. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 MRSA 
                 MRSA 
               
               
                 Compound 
                 
                   B. subtilis 
                 
                 
                   S. aureus 
                 
                 
                   E. coli 
                 
                 
                   M. luteus 
                 
                 VRE 155 
                 WKZ2 
                 USA300 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 A 
                 &gt;128 
                 &gt;128 
                 &gt;128 
                  8-16 
                 &gt;128 
                 &gt;128  
                 &gt;128 
               
               
                 B 
                 0.625-2.5  
                 10 
                 &gt;10 
                 0.02-0.03 
                 5 
                 10 
                 10 
               
               
                 C 
                 0.03-0.06 
                 0.25-0.31 
                 &gt;10 
                   0.03 
                 &gt;10 
                    0.625 
                 0.625 
               
               
                 D 
                 &gt;128 
                 &gt;128 
                 &gt;128 
                 — 
                 — 
                 — 
                 — 
               
               
                 E 
                 &gt;128 
                 &gt;128 
                 &gt;128 
                 — 
                 — 
                 — 
                 — 
               
               
                  (2) 
                 32 
                 &gt;64 
                 &gt;64 
                 2 
                 — 
                 — 
                 — 
               
               
                  (3) 
                 16 
                 64 
                 &gt;64 
                 — 
                 8 
                 — 
                 64 
               
               
                  (4) 
                 — 
                 — 
                 — 
                 — 
                 16 
                 — 
                 64 
               
               
                  (5) 
                 8 
                 16 
                 64 
                 — 
                  8-16 
                 — 
                 32 
               
               
                  (6) 
                 4-8 
                 16 
                 64 
                 2 
                 8 
                 16 
                 16 
               
               
                  (7) 
                 4 
                 8 
                 &gt;64 
                 — 
                 4 
                 — 
                 16 
               
               
                  (8) 
                 4 
                 4-8 
                 &gt;64 
                 — 
                 2 
                 — 
                 4 
               
               
                  (9) 
                 4 
                 8 
                 &gt;64 
                 — 
                 2 
                 — 
                 4 
               
               
                 (10) 
                 4 
                 16 
                 &gt;64 
                 1-2 
                 4 
                 32 
                 64 
               
               
                 (11) 
                 8 
                 32 
                 &gt;64 
                 — 
                 8 
                 — 
                 64 
               
               
                 (12) 
                 4 
                 8 
                 &gt;64 
                 2 
                 8 
                 16 
                 16 
               
               
                 (13) 
                 2-4 
                 — 
                 — 
                 — 
                 4 
                 — 
                 8 
               
               
                 (14) 
                 4 
                 4 
                 &gt;64 
                 — 
                 4 
                 — 
                 8 
               
               
                 (15) 
                 4 
                 8 
                 &gt;64 
                 — 
                 2 
                 — 
                 8 
               
               
                 (16) 
                 4 
                 8 
                 &gt;64 
                 — 
                 2 
                 — 
                 8 
               
               
                 (17) 
                 — 
                 — 
                 — 
                 — 
                 4 
                 — 
                 8 
               
               
                 (18) 
                 4 
                 4-8 
                 &gt;64 
                 — 
                 2 
                 — 
                 8 
               
               
                 (19) 
                 — 
                 — 
                 — 
                 — 
                 8 
                 — 
                 32 
               
               
                 (20) 
                 — 
                 — 
                 — 
                 — 
                 4 
                 — 
                 &gt;64 
               
               
                 (21) 
                 — 
                 — 
                 — 
                 — 
                 4 
                 — 
                 8 
               
               
                 (22) 
                 — 
                 — 
                 — 
                 — 
                 8 
                 — 
                 16 
               
               
                 (23) 
                 — 
                 — 
                 — 
                 — 
                 4-8 
                 — 
                 16 
               
               
                 (24) with 
                 &gt;64 
                 &gt;64 
                 &gt;64 
                 8 
                 — 
                 — 
                 — 
               
               
                 R 1- group 
               
               
                 (b) 
               
               
                 (24) with 
                 &gt;64 
                 &gt;64 
                 &gt;64 
                 8 
                 — 
                 — 
                 — 
               
               
                 R 1- group 
               
               
                 (c) 
               
               
                 (24) with 
                 32 
                 64 
                 &gt;64 
                 2-4 
                 — 
                 — 
                 — 
               
               
                 R 1- group 
               
               
                 (d) 
               
               
                 (24) with 
                 4 
                 8 
                 &gt;64 
                 2 
                 8 
                  8 
                 8 
               
               
                 R 1- group 
               
               
                 (e) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 MIC 50  and MIC 90  determination for compound (10) and 
               
               
                 Comparative compounds B and C against thirty VRE strains 
               
               
                 (MIC measured in μg/mL). All data stems from duplicate experiments. 
               
               
                 Where appropriate, values are represented as a range. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Comparative 
                 Comparative 
               
               
                 Strain 
                   
                 van 
                 compound 
                 compound B 
                 compound C 
               
               
                 ID a   
                 Country 
                 gene 
                 (10) 
                 (nisin) 
                 (vancomycin) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 E0013 
                 GBR 
                 vanA 
                 4 
                 8 
                 32 
               
               
                 E0072 
                 NLD 
                 vanA 
                 2 
                 2 
                 8 
               
               
                 E0300 
                 USA 
                 vanA 
                  8-16 
                 8 
                 &gt;128 
               
               
                 E0321 
                 FRA 
                 vanA 
                 2 
                 2 
                 &gt;128 
               
               
                 E0333 
                 ISR 
                 vanA 
                 4 
                 4-8 
                 &gt;128 
               
               
                 E0338 
                 ITA 
                 vanA 
                 4 
                 8 
                 &gt;128 
               
               
                 E0341 
                 GBR 
                 vanA 
                 4 
                 4 
                 128 
               
               
                 E0506 
                 AUS 
                 vanA 
                 8 
                 4-8 
                 &gt;128 
               
               
                 E0745 
                 NLD 
                 vanA 
                 4 
                 4 
                 64 
               
               
                 E1130 
                 USA 
                 vanA 
                 4 
                 4-8 
                 &gt;128 
               
               
                 E1441 
                 GRC 
                 vanA 
                 4 
                 4 
                 128 
               
               
                 E1679 
                 BRA 
                 vanA 
                 4 
                 4 
                 &gt;128 
               
               
                 E1763 
                 BEL 
                 vanA 
                 8 
                 8 
                 &gt;128 
               
               
                 E2297 
                 USA 
                 vanA 
                 2 
                 4 
                 128 
               
               
                 E2359 
                 SGP 
                 vanB 
                 2 
                 8 
                 &lt;1-1 
               
               
                 E2365 
                 HUN 
                 vanB 
                 1 
                 2-4 
                 &lt;1 
               
               
                 E2373 
                 SRB 
                 vanA 
                 4 
                 4 
                 &gt;128 
               
               
                 E6016 
                 LVA 
                 vanA 
                 2-4 
                 4 
                 128 
               
               
                 E7312 
                 NLD 
                 vanA 
                 2 
                 4 
                 64 
               
               
                 E7314 
                 NLD 
                 vanB 
                 4 
                 4-8 
                 &gt;128 
               
               
                 E7319 
                 NLD 
                 vanA 
                 2 
                 4 
                 &gt;128 
               
               
                 E7329 
                 NLD 
                 vanA 
                 2 
                 4 
                 &lt;1 
               
               
                 E7401 
                 NLD 
                 vanB 
                 4 
                 8 
                 16 
               
               
                 E7403 
                 NLD 
                 vanB 
                 1 
                 4 
                 &lt;1 
               
               
                 E7413 
                 NLD 
                 vanA 
                 4 
                 4 
                 &gt;128 
               
               
                 E7424 
                 NLD 
                 vanB 
                 1-2 
                 4 
                 &gt;128 
               
               
                 E7464 
                 NLD 
                 vanB 
                 4 
                 4 
                 2 
               
               
                 E8218 
                 NLD 
                 vanB 
                 2 
                 4 
                 8 
               
               
                 E8235 
                 NLD 
                 vanB 
                 4 
                 4 
                 8 
               
               
                 E8237 
                 NLD 
                 vanA 
                 8 
                 8 
                 128 
               
               
                   
                   
                   
                 MIC 90  = 8 
                 MIC 90  = 8 
                 MIC 90  = &gt;128 
               
               
                   
                   
                   
                 MIC 50  = 4 
                 MIC 50  = 4 
                 MIC 50  = 128 
               
               
                   
               
               
                   a See http://www.nationsonline.org/oneworld/countrycodes.htm for country codes. 
               
            
           
         
       
     
     Example 3. Lipid II-Binding in Model Membranes 
     Large unilamellar vesicles (LUVs), composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) spiked with 0.2% lipid II, were loaded with carboxyfluorescein (CF). The CF efflux was monitored by measuring the increase in fluorescence intensity at 515 nm, with excitation at 492 nm. In a cuvette, a solution (1 mL) of CF-loaded vesicles (20 μM final concentration) in buffer (Tris.HCl, pH 7.0 containing 100 mM NaCl) was prepared and the relevant final concentration of compounds (6), (10), and (12), and also compound (24) with R 1 -group (e), were added and the mixture was stirred for 1 min and the fluorescence was recorded (A 0 ). After ca. 10 seconds, nisin (comparative compound B) was added (5 nM final concentration) and the fluorescence was followed until it stabilized, then recorded (A stable ). Total membrane leakage was induced by the addition of Triton-X100 (final concentration 0.1%) and the fluorescence was recorded (A total ). The percentile values were calculated by: 
     
       
         
           
             
               
                 
                   A 
                   stable 
                 
                 - 
                 
                   A 
                   0 
                 
               
               
                 
                   A 
                   total 
                 
                 - 
                 
                   A 
                   0 
                 
               
             
             × 
             100 
              
             
               % 
               . 
             
           
         
       
     
     Treatment with the compounds (6), (10), and (12) and treatment with compound (24) with R 1 -group (e) did not show any detectable dye leakage, whereas nisin (comparative compound B) at a concentration of 5 nM resulted in leakage of about 50% of the CF dye. The competition assay revealed that each compound effectively antagonized nisin induced membrane leakage when administered at a concentration 10-fold higher than nisin, except for compound 24 with R 1 -group e, which inhibited dye leakage at equimolar concentrations, suggesting a binding affinity for lipid II on par with nisin in this model. 
     Example 4. Serum Stability 
     2 mg/mL peptide solutions were prepared in 26% DMSO in MilliQ. Duplicate samples were prepared with 42 mL peptide solution and 518 mL human serum, making the final DMSO concentration 2%. The samples were incubated at 37° C., and samples were taken at t=0, 1, 2, 4 and 24 hours as follows: to 100 μL serum solution, 200 μL MeOH (containing 0.075 mg/mL ethylparaben as an internal standard) was added to precipitate the proteins. The sample was vortexed briefly and allowed to stand for 10 min at RT. The samples were then centrifuged at 13,000 rpm for 5 min, and the supernatant was taken and stored at −20° C. until analysis. Each sample was analyzed by HPLC on a C4 column. The peaks were integrated and normalized to the internal standard. 
     The stability of compounds (6), (10), and (12) and the stability of compound (24) with R 1 -group (e) in human serum was compared to the stability of nisin [1-12] (compound B). It was found that the stability of the new compounds was well above 50%, and significantly exceeded the stability of compound B, i.e. only 33% of nisin remained intact after 24 hours, whereas 94% of compound (6) remained intact after 24 hours. 
     Example 5. Hemolysis Assay 
     Human whole blood was centrifuged at 600×g for 15 min and levels of plasma and hematocrit were marked on the tube. Plasma was removed and the erythrocytes washed 3× with PBS (centrifuging at 600×g for 15 mins). After discarding the supernatant, the packed cells were stored on ice. 100 μL of the peptides (128 μg/mL in PBS, 2% DMSO) as well as a control solution comprised of 2% DMSO in PBS were added to the top row of a polypropylene, round-bottom 96 well plate and 50 μL of PBS to the rest of the wells. The peptides and the DMSO control solutions were then diluted serially down the rows. 200 μL of the packed cells were added to PBS (10 mL) and 50 μL of this suspension was added to each well. A column with DI water containing 0.1% Triton X-100 was used as the 100% lysis control, and the column containing the serially-diluted PBS (1.0% DMSO) control served as the 0% lysis reference. The cells were incubated at 37° C. for 1 h. After incubation the plates were centrifuged (800×g, 5 min) and 25 μL of the supernatant was added to 100 μL DI water in a flat-bottom plate (polystyrene). The absorption at 414 nm was recorded to measure the amount of free hemoglobin. The hemolysis of compounds (6), (10), (12), and (20) and the hemolysis of compound (24) carrying R 1 -group (e) was compared to Comparative compounds B and C. All new compounds showed a level of hemolysis below 15% at concentrations as high as 32 μg/mL. Comparative compounds B and C show negligible hemolysis at 32 μg/mL. Compound (20) showed no detectable hemolysis up to the highest concentration tested (64 μg/mL), which illustrates the preferable masking of the positive charge on the Lys 12  group to prevent hemolysis. 
     Example 6. BioScreen Growth Assays with  E. faecium    
     A BioScreen C instrument (Oy Growth Curves AB, Helsinki, Finland) was used to monitor effects of the compounds (6), (10), and (12) and the effect of compound (24) with R 1 -group (e), or Comparative compound B (nisin) on  E. faecium  growth (each compound administered at a fixed concentration 5 μM).  E. faecium  strains were inoculated at an initial OD 660  of 0.05 into 300 μl TSB containing 1% DMSO and 1% glucose or into the same medium containing the antibiotic compounds at a final concentration of 5 μM. The cultures were incubated in the Bioscreen C system at 37° C. with continuous shaking, and the absorbance at 600 nm (A 600 ) recorded every 15 min for 15 hours to determine growth/inhibitory effects. 
     The  E. faecium  strains used in these experiments are  E. faecium  E745 (vancomycin-ampicillin resistant hospital outbreak strain),  E. faecium  E980 (vancomycin-ampicillin susceptible human commensal isolate), E.  Faecium  E1133 (vancomycin-ampicillin resistant hospital outbreak strain), and  E. faecium  E1162 (vancomycin-susceptible ampicillin-resistant clinical isolate). The BioScreen growth assays for each strain was determined for the compounds (6), (10), (12) and also for compound (24) with R 1 -group (e) as well as nisin (Comparative compound B) and compared to non-treated strains, the results being shown in the Tables 9 to 12 below. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 BioScreen growth assay for  E. faecium  E745. 
               
            
           
           
               
               
            
               
                   
                 OD 600   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 compound 
               
               
                 Time 
                 No 
                 compound B 
                 compound 
                 compound 
                 compound 
                 (24) with R 1 - 
               
               
                 (h) 
                 treatment 
                 (nisin) 
                 (6) 
                 (10) 
                 (12) 
                 group (e) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 0 
                 0.013667 
                 0.017333 
                 0.012000 
                 0.015333 
                 0.015333 
                 0.015000 
               
               
                 4 
                 0.434333 
                 0.010333 
                 0.069333 
                 0.012000 
                 0.009000 
                 0.013667 
               
               
                 8 
                 0.835000 
                 0.010333 
                 0.575667 
                 0.011667 
                 0.008667 
                 0.017667 
               
               
                 12 
                 0.818333 
                 0.010667 
                 0.685333 
                 0.011667 
                 0.009667 
                 0.022667 
               
               
                 16 
                 0.801667 
                 0.008667 
                 0.679000 
                 0.011333 
                 0.010000 
                 0.031000 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 BioScreen growth assay for  E. faecium  E980. 
               
            
           
           
               
               
            
               
                   
                 OD 600   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 compound 
               
               
                 Time 
                 No 
                 compound B 
                 compound 
                 compound 
                 compound 
                 (24) with R 1 - 
               
               
                 (h) 
                 treatment 
                 (nisin) 
                 (6) 
                 (10) 
                 (12) 
                 group (e) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 0 
                 0.016333 
                 0.016000 
                 0.017333 
                 0.018333 
                 0.017333 
                 0.017333 
               
               
                 4 
                 0.831667 
                 0.011667 
                 0.464000 
                 0.015000 
                 0.012000 
                 0.022333 
               
               
                 8 
                 0.955667 
                 0.011000 
                 0.913667 
                 0.014333 
                 0.013000 
                 0.072000 
               
               
                 12 
                 0.893000 
                 0.011000 
                 0.886000 
                 0.013667 
                 0.016667 
                 0.269667 
               
               
                 16 
                 0.802333 
                 0.010667 
                 0.849667 
                 0.013667 
                 0.027333 
                 0.366667 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 BioScreen growth assay for  E. faecium  E1133 
               
            
           
           
               
               
            
               
                   
                 OD 600   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 compound 
               
               
                 Time 
                 No 
                 compound B 
                 compound 
                 compound 
                 compound 
                 (24) with R 1 - 
               
               
                 (h) 
                 treatment 
                 (nisin) 
                 (6) 
                 (10) 
                 (12) 
                 group (e) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 0 
                 0.014667 
                 0.016000 
                 0.016000 
                 0.017000 
                 0.016667 
                 0.016667 
               
               
                 4 
                 0.596000 
                 0.009667 
                 0.226667 
                 0.012667 
                 0.010333 
                 0.024667 
               
               
                 8 
                 0.703667 
                 0.009333 
                 0.666333 
                 0.011667 
                 0.013667 
                 0.102333 
               
               
                 12 
                 0.689333 
                 0.009333 
                 0.665333 
                 0.011667 
                 0.026667 
                 0.403000 
               
               
                 16 
                 0.676000 
                 0.008667 
                 0.629000 
                 0.011000 
                 0.124333 
                 0.514000 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 BioScreen growth assay for  E. faecium  E1162. 
               
            
           
           
               
               
            
               
                   
                 OD 600   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 compound 
               
               
                 Time 
                 No 
                 compound B 
                 compound 
                 compound 
                 compound 
                 (24) with R 1 - 
               
               
                 (h) 
                 treatment 
                 (nisin) 
                 (6) 
                 (10) 
                 (12) 
                 group (e) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 0 
                 0.015667 
                 0.024000 
                 0.013667 
                 0.019000 
                 0.017000 
                 0.018667 
               
               
                 4 
                 0.719333 
                 0.015333 
                 0.194000 
                 0.013667 
                 0.011000 
                 0.027333 
               
               
                 8 
                 0.842667 
                 0.011333 
                 0.671333 
                 0.013000 
                 0.012333 
                 0.095333 
               
               
                 12 
                 0.827667 
                 0.012000 
                 0.678667 
                 0.012667 
                 0.016667 
                 0.378333 
               
               
                 16 
                 0.814333 
                 0.010333 
                 0.665667 
                 0.012333 
                 0.025667 
                 0.397000 
               
               
                   
               
            
           
         
       
     
     The conclusion is that all compounds that were generated and produced in accordance with the present invention as well as nisin demonstrate a delay as well as an inhibition of the growth of all tested  E. faecium  strains. The compounds (6) and (10) and also compound with Formula (24) carrying R 1 -group (e), and in particular compound (12) showed very good growth inhibition performances.