DOI: 10.1002/cmdc.201500220

Full Papers

Influence of the Multivalency of Ultrashort Arg-Trp-Based Antimicrobial Peptides (AMP) on Their Antibacterial Activity Barbara C. Hoffknecht,[a] Dennis J. Worm,[a] Sandra Bobersky,[a] Pascal Prochnow,[b] Julia E. Bandow,[b] and Nils Metzler-Nolte*[a] Peptide dendrimers are a class of molecules of high interest in the search for new antibiotics. We used microwave-assisted, copper(I)-catalyzed alkyne–azide cycloaddition (CuAAC; “click” chemistry) for the simple and versatile synthesis of a new class of multivalent antimicrobial peptides (AMPs) containing solely arginine and tryptophan residues. To investigate the influence of multivalency on antibacterial activity, short solid-phasesynthesized azide-modified Arg-Trp-containing peptides were “clicked” to three different alkyne-modified benzene scaffolds to access scaffolds with one, two, or three peptides. The antibacterial activity of 15 new AMPs was investigated by minimal inhibitory concentration (MIC) assays on five different bacterial

strains, including a multidrug-resistant Staphylococcus aureus (MRSA) strain. With ultrashort (2–3 residues) peptides, a clear synergistic effect of the trivalent display was observed, whereas this effect was not apparent with longer peptides. The best candidates showed activities in the low-micromolar range against Gram-positive MRSA. Surprisingly, the best activity against Gram-negative Acinetobacter baumannii was observed with an ultrashort dipeptide on the trivalent scaffold (MIC: 7.5 mm). The hemolytic activity was explored for the three most active peptides. At concentrations ten times the MIC values, < 1 % hemolysis of red blood cells was observed.

Introduction Many bacterial infections have been treated successfully since the discovery of salvarsan, sulfonamides, and penicillins, but antimicrobial resistance against antibiotics in current use has been a growing threat to the effective treatment of infections caused by bacteria.[1] For example, a recent report by the World Health Organization (WHO) shows that the ongoing use of antibiotics is likely to lead to further increases in resistance, and there is increasing concern that many antibacterial agents may lose their potency in the near future. This threatens the achievements of modern medicine, especially in surgery.[1] At the same time, the global development pipeline for new antibiotics has narrowed.[2] Because of this, there is an urgent need to identify new molecules with antibacterial activity, preferably with new mechanisms of action. Virtually all classes of higher organisms (plants, invertebrates, and animals) produce host defense peptides.[3] These antimicrobial peptides (AMPs) have been considered as templates for possible new antibiotics because they have remained effective weapons despite their long history.[3] AMPs are cationic amphipathic peptides associated with membrane activity.[4] However, there are [a] B. C. Hoffknecht, D. J. Worm, S. Bobersky, Prof. Dr. N. Metzler-Nolte Chair of Inorganic Chemistry I—Bioinorganic Chemistry Ruhr University Bochum, Universit•tsstraße 150, 44801 Bochum (Germany) E-mail: [email protected] [b] P. Prochnow, Prof. Dr. J. E. Bandow Applied Microbiology Ruhr University Bochum, Universit•tsstraße 150, 44801 Bochum (Germany) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201500220.

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a number of limitations to the clinical application of natural AMPs, including their toxicity and susceptibility to proteases.[2] Furthermore, production or isolation of natural AMPs is associated with high costs.[5] Therefore, numerous approaches have been made to find smaller synthetic AMPs (synAMPs) that have no natural counterparts and have lower minimal inhibitory concentrations (MIC). An advantage of synAMPs is the possibility to introduce chemical modifications that are not readily available through natural biosynthetic pathways. The addition of metal complexes,[6] peptides containing d-amino acids,[7] and lipidated peptides[8] are only a few examples of synthetic molecules with antibacterial activity. Out of the group of synAMPs, those based on arginine (Arg, R) as the charged moiety and tryptophan (Trp, W) as lipophilic bulk are of special interest. Such peptides are among the smallest candidates that still present significant antimicrobial activity,[9] and thus provide a solution to the issue of production costs, for example. Various modifications of these short RW peptides were shown to further increase antibacterial activity.[8–10] Another approach to overcome some of the disadvantages of natural AMPs are peptide dendrimers. In these molecules, several short peptides are linked to a dendrimeric core to form a larger, branched structure.[10] A similar approach was used in this study to generate a new class of synthetic, multivalent RW peptide conjugates with antibacterial activity. After introducing a C-terminal lysine (Lys, K) residue into short RW peptides, the lysine side chain could be converted into an azide group. In this way, the synthesized linear peptides can be attached to an acetylene-modified benzene ring via copper(I)-catalyzed

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Full Papers alkyne–azide cycloaddition (CuAAC),[11] also referred to as the prototypical “click” reaction.[12] This reaction yields 1,2,3-triazoles as connectors between the (multivalent) scaffold and the peptide. To investigate the influence of such a multivalent display of peptides we choose three benzene scaffolds with one, two, or three alkynes (Figure 1) to obtain monovalent (1 a), divalent

Figure 1. Scaffolds with acetylene moieties to yield monovalent (1 a), divalent (1 b), or trivalent (1 c) peptide conjugates.

Synthesis of multivalent peptide conjugates The modified RW peptides 3–7 (Scheme 1) were used as building blocks for the click reaction with modified benzene scaffolds. To investigate the influence of multivalency, three different scaffolds were used (Figure 1); mono-, di-, and trivalent compounds were expected as reaction products. CuAAC was

Scheme 1. Synthesis of synAMPs 3–7 with azide functionality.

(1 b), and trivalent (1 c) peptide conjugates. By comparing the antibacterial activity of the peptide constructs of these three scaffolds, the impact of multivalency up to three can be explored.

Results and Discussion Synthesis of short modified RW peptides For conjugation of the synAMPs to the acetylene-containing benzene cores, the peptides had to be functionalized with an azide moiety. To this end, the lysine side chain amino group was transformed into an azide with the diazo-transfer reagent imidazol-1-sulfonyl azide hydrochloride[13] to yield the Fmocprotected 2-amino-6-azidohexanoic acid 2. A series of four ultrashort peptides 3–6 (2–3 amino acids (AA), not counting the connecting Lys derivative 2) and one longer peptide 7 (6 AA) containing Arg and Trp were synthesized by solid-phase peptide synthesis (SPPS, Scheme 2 below)[14] using a Rink amide resin that yields a C-terminal amido group after cleavage. The Fmoc-protected 2-amino-6-azidohexanoic acid 2 (referred to as Lys(N3)) was introduced as the C-terminal residue. Coupling of amino acids by SPPS was carried out by using a mixture of O-(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate (TBTU)/1-hydroxybenzotriazole (HOBt) and N,N-diisopropylethylamine (DiPEA). Removal of the Fmoc protecting groups was performed by treatment with 20 % piperidine in N,N-dimethylformamide (DMF). The used amino acids were side-chain protected with tert-butyloxycarbonyl (Boc) groups in the case of Trp, and 2,2,5,6,7-pentamethyldihydrobenzofuran5-sulfonyl (Pbf) groups in the case of Arg. The protecting groups were cleaved from the peptide during cleavage of the peptide from the resin by treatment with 95 % trifluoroacetic acid (TFA), 2.5 % triisopropylsilane (TIS), and 2.5 % water. The synthesized peptide sequences are shown in Scheme 1. ChemMedChem 2015, 10, 1564 – 1569

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Scheme 2. Synthesis of mono- to trivalent peptide conjugates 3 a–7 c. Reagents and conditions: a) H-peptide-K(N3)-NH2 3–7, (EtO)3PCuI, TBTA, DiPEA, DMF, microwave (60 8C, 200 W max., 2000 kPa), 4.5 h.

used as the click reaction, forming 1,2,3-triazole links between the peptides and benzene scaffolds (Scheme 2). In this case, CuAAC was carried out in DMF at 60 8C for a total of 1.5 h per peptide arm under microwave irradiation. (EtO)3PCuI[15] (1 equiv per arm) was used as catalyst to prevent oxidation or disproportionation of the catalytically active copper(I) species, and tris((1-benzyl-1H-1,2,3 triazolyl)methyl)amine (TBTA)[16] (1 equiv per arm) was added to the reaction as stabilizer. A sterically hindered base, in this case N,N-diisopropylethylamine (DiPEA), is required according to the reaction mechanism. After microwave treatment, the mixture was concentrated, and the products were separated by preparative HPLC to > 95 % purity. All products were characterized by analytical HPLC, and their

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Full Papers composition was confirmed by mass spectrometry (see Supporting Information). Antibacterial activity The antibacterial activity of the clicked compounds 3 a–7 c was assessed by determining their minimal inhibitory concentration (MIC) in a microdilution assay as described previously.[8] The MIC value represents the lowest concentration of a given compound that is needed to inhibit the growth of bacteria.[17] In this work, two Gram-negative and three Gram-positive bacterial strains were used (Table 1). A compound is usually designated as antimicrobial if the MIC value is in the low-micromolar range. In general, the investigated compounds are more active against Gram-positive than against Gram-negative bacteria, although in the case of the small peptides 3 b, 3 c, 5 b, 5 c, and 6 c, as well as the longer peptide conjugates 7 a–c, the difference in activity against Gram-positive and -negative bacteria is not very pronounced. At first we compared the compounds built from the smaller peptides 3–6. Here the monovalent compounds show no activity against Gram-negative bacteria and are at best poorly active against Gram-positive bacteria. The divalent peptide conjugates show moderate-to-good activity against Gram-positive bacteria, while the trivalent conjugates show even better activity against Gram-positive bacteria, with the only exception being 6 c. The compound with the best MIC values is compound 5 c, which is not only active against Gram-positive bacteria (5.4– 0.6 mm) but also very active against Gram-negative bacteria (10–1.3 mm). This might be due to the high number of positively charged Arg residues. Overall, a clear synergistic effect is apparent upon comparing the monovalent (a) through divalent (b) to trivalent compounds (c). Clearly, trivalency of smaller peptides (3–4 AA) increases their activity. However, in comparing the conjugates built from the longer peptide 7, it is evident that even the monovalent compound 7 a shows good

activity (9–0.6 mm). This was expected, as good activity was previously reported for the linear hexapeptide H-RWRWRWNH2, which was our lead structure for this study (5.3–1.3 mm).[8] This hexapeptide and compound 7 a only differ by an additional hydrophobic portion, namely the benzyl ring. Comparing 7 a with the divalent or even trivalent derivatives 7 b and 7 c reveals no further increase in activity. Clearly, no synergistic effect can be obtained for the longer peptides. This may be attributed to the size of the compounds, and could imply that there is an optimal number of residues (i.e., molecular size) for antibacterial activity. With molecular weights of the compounds in this study ranging from ~ 650 Da (for 3 a) to 3836 Da (for 7 c) and thus well outside the “Lipinski range”, it seems that the optimal hydrophobicity and number of positive charges is a more relevant parameter for activity. However, many antibiotic compounds (as is the case for many biologics in general) do not adhere to this guideline, while still being orally active. Furthermore, compounds in the RW class of AMPs usually do not show better MIC values when they exceed their optimal length of ~ 6–8 AA. The trivalent derivatives of the smaller peptides studied here could therefore be mimicking an optimum-length peptide. This would imply that the total number of amino acids is more important than their spatial arrangement.

Hemolytic activity Hemolysis was determined for compounds 5 a–c, which showed the lowest MIC values overall. Solutions of 5 a (0.62 mm), 5 b (0.32 mm), or 5 c (0.12 mm) were applied to freshly isolated human red blood cells. These concentrations are 10- to 20-fold greater than the highest MIC value against the Gram-positive bacteria for the respective compounds. All three compounds showed < 1 % hemolysis, so it is safe to say that these compounds are not hemolytic.

Table 1. Minimal inhibitory concentrations of multivalent peptide compounds 2 a–6 c. Compd

3a 3b 3c 4a 4b 4c 5a 5b 5c 6a 6b 6c 7a 7b 7c

Sequence

Gram-negative E. coli DSM 30083 A. baumannii DSM 30007

RWK RWK RWK WRK WRK WRK RWRK RWRK RWRK WRWK WRWK WRWK RWRWRWK RWRWRWK RWRWRWK

NA 44 NA NA NA NA NA 16 2.7 NA NA 47 19 9 6.5

NA 44 7.5 NA NA NA NA 16 10 NA 35 11 4.7 10 26

MIC [mm][a]

B. subtilis 168

Gram-positive S. aureus DSM 20231

S. aureus ATCC 43300

84 11 3.7 NA 22 7.6 31 2.0 0.6 33 1.1 6 1.2 0.6 3.3

NA 88 52 NA 44 60 NA 16 5.4 67 17 47 9 10 13

NA 44 13 NA 44 30 62 16 2.7 67 4.4 11 4.7 5 6.5

[a] For MIC value calculations, molecular weights of the peptides together with one TFA counter-ion for each basic amino acid residue were used. Data listed are the lowest of two replicates; see Experimental Section for details. NA: not active (MIC > 100 mm).

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Full Papers Conclusions The synthesis of a new class of multivalent RW-containing AMPs by copper(I)-catalyzed alkyne–azide cycloaddition (CuAAC) is described. Fifteen different compounds, from mono- to trivalent, were synthesized in high purity and acceptable yields, using three different scaffolds. The influence of multivalency was investigated by comparing mono- to trivalent compounds with each other. All compounds were tested for their antimicrobial activity by MIC assays. The results clearly show a synergistic effect in the case of the smaller peptides (2–3 AA) and no effect in the case of the longer hexapeptides, which were studied for comparison. Interestingly, the trivalent compound 3 c is fairly active, and is only about two- to fivefold weaker than compound 7 a, which contains the same number of Arg and Trp residues, but has a slightly higher molecular weight; 1332 Da for 7 a vs. 1782 Da for 3 c). This leads to the conclusion that the presence of a certain number of Arg and Trp residues is more important than their chemical linkage. In line with previous findings,[18] peptides with more than nine residues do not show increased activity, but instead lower activity. We could show that by multivalent display, otherwise inactive ultrashort peptides (only 2–3 AA long) can become very active against bacteria. In this sense we propose the addition of multivalency to the “antibacterial toolbox”. In conjunction with established methods, multivalency can easily be used to modulate the activity of AMPs, and to help identify AMP conjugates with high activity and other suitable properties that may ultimately find clinical applications.

Experimental Section General: ESI mass spectra were measured on an Esquire 6000 instrument (Bruker Daltonics, Bremerhaven, Germany). MALDI-TOF mass spectra were measured on an Ultraflex III (Bruker Daltonics). NMR data were collected on a DPX 200 instrument (Bruker); chemical shift values (d) are given in ppm relative to tetramethylsilane as an external standard, and coupling constants (J) are given as absolute values in Hz. Analytical HPLC was performed on an automated Knauer instrument (Karlsruhe, Germany). To elute the compounds a gradient of 100 % buffer A (95 % H2O, 5 % CH3CN, 0.1 % TFA) to 100 % buffer B (95 % CH3CN, 5 % H2O, 0.1 % TFA) at a flow rate of 1 mL min¢1 over 40 min was used. Preparative HPLC was performed on a Varian Pro Star instrument. To elute the compounds a gradient of 100 % buffer A to 100 % buffer B at a flow rate of 20 mL min¢1 over 60 min was used. Column chromatography was performed on silica gel 60 (particle size: 0.036–0.2 mm) purchased from Merck. Only l-amino acids were used in this work; they were purchased from IRIS Biotec (Germany). Other chemicals were purchased from IRIS Biotech, Aldrich, and Fluka, and were used without further purification. All reactions were carried out using commercial-grade solvents purchased from Roth, Baker, Fischer, and Biosolve. Imidazol-1-sulfonyl azide hydrochloride and Fmoc-protected 2-amino-6azidohexanoic acid 2 were synthesized as described in earlier.[13] Solid-phase peptide synthesis: Peptides were synthesized manually at room temperature by using an Fmoc-protecting-group strategy and a Rink amide resin. Reactions were carried out in polypropylene syringes on a mechanical shaker. The resin was swollen ChemMedChem 2015, 10, 1564 – 1569

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in DMF before use. Fmoc deprotection: 20 % piperidine in DMF was added to the resin for 2 Õ 10 min. Washing: After each coupling and deprotection step, the resin was thoroughly washed with DMF, CH2Cl2, and DMF. Coupling: Fmoc-protected amino acids (4 equiv) were pre-activated with TBTU (4 equiv), HOBt (4 equiv), and DiPEA (8 equiv) in DMF for 1 min, mixed with the resin and shaken for 1 h. Final cleavage: Final cleavage from the resin and side chain deprotection of the peptides was carried out by using a mixture of 95 % TFA, 2.5 % TIS, and 2.5 % H2O for 3 h at room temperature. Cleaved peptides were precipitated with ice-cold n-hexane/Et2O (1:1), centrifuged (16450 g, 5 min, room temperature) and washed two times with n-hexane/Et2O. The peptides were lyophilized and purified by preparative HPLC. Click chemistry: CuAAC reactions were performed in a microwave reactor at 60 8C, with a maximal power of 200 W and a maximal pressure of 2000 kPa. The peptides were dissolved in degased DMF. One half of the peptide solution was transferred to a microwave reaction tube. The alkyne-containing benzene derivatives were added, as well as TBTA (1 equiv), (EtO)3PCuI (1 equiv), and DiPEA (2 equiv). The reaction was then performed in the microwave for 3 h. The other half of the peptide solution was then added to the reaction mixture as well as TBTA (1 equiv) and (EtO)3PCuI (1 equiv), and the reaction was again carried out in the microwave for an additional 1.5 h. After reaction completion, the solvent was evaporated. The residue was purified by preparative HPLC using the following gradient: 0–5 min 0 % buffer B, 5–45 min up to 100 % buffer B, 45–50 min 100 % buffer B, 50–55 min down to 0 % buffer B, 55–60 min 0 % buffer B. H-RWK(N3)-NH2 3: Synthesized by SPPS as described above. Yield: 52 %; MS (ESI, m/z): 514.0 (calcd 514.3 for [M + H] + ); HPLC (tR, min): 15.5. H-WRK(N3)-NH2 4: Synthesized by SPPS as described above. Yield: 48 %; MS (ESI, m/z): 514.1 (calcd 514.3 for [M + H] + ), 257.5 (calcd 257.6 for [M + 2H]2 + ); HPLC (tR, min): 15.7. H-RWRK(N3)-NH2 5: Synthesized by SPPS as described above. Yield: 42 %; MS (ESI, m/z): 784.0, 670.2 (calcd 670.4 for [M + H] + ), 426.0, 335.6 (calcd 335.7 for [M + 2H]2 + ), 302.8; HPLC (tR, min): 15.5. H-WRWK(N3)-NH2 6: Synthesized by SPPS as described above. Yield: 54 %; MS (ESI, m/z): 700.2 (calcd 700.4 for [M + H] + ), 350.6 (calcd 350.7 for [M + 2H]2 + ); HPLC (tR, min): 17.7. H-RWRWRWK(N3)-NH2 7: Synthesized by SPPS as described above. Yield: 51 %; MS (ESI, m/z): 1198.2 (calcd 1198.7 for [M + H] + ), 599.8 (calcd 599.9 for [M + 2H]2 + ); HPLC (tR, min): 17.5. 1-(H-RWK(1,2,3-triazol-4-methoxy)-NH2)benzene 3 a: The peptide H-RWK(N3)-NH2 (10 mg, 19 mmol, 1 equiv) was clicked with 1-(prop2-ynyloxy)benzene (4.5 mL, 38 mmol, 2 equiv) as described in the general experimental procedure. Yield: 59 % (7.2 mg, 11.2 mmol); MS (ESI, m/z): 646.1 (calcd 646.4 for [M + H] + ), 323.5 (calcd 323.7 for [M + 2H]2 + ); HPLC (tR, min): 17.4. 1-(H-WRK(1,2,3-triazol-4-methoxy)-NH2)benzene 4 a: The peptide H-WRK(N3)-NH2 (10 mg, 19 mmol, 1 equiv) was clicked with 1-(prop2-ynyloxy)benzene (4.5 mL, 38 mmol, 2 equiv) as described in the general experimental procedure. Yield: 51 % (7.2 mg, 11.2 mmol); MS (ESI, m/z): 646.1 (calcd 646.4 for [M + H] + ), 323.5 (calcd 323.7 for [M + 2H]2 + ); HPLC (tR, min): 17.4. 1-(H-RWRK(1,2,3-triazol-4-methoxy)-NH2)-benzene 5 a: The peptide H-RWRK(N3)-NH2 (10 mg, 15 mmol, 1 equiv) was clicked with 1(prop-2-ynyloxy)benzene (3.6 mL, 30 mmol, 2 equiv) as described in the general experimental procedure. Yield: 54 % (6.5 mg, 9.7 mmol);

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Full Papers MS (ESI, m/z): 802.2 (calcd 802.5 for [M + H] + ), 401.6 (calcd 401.8 for [M + 2H]2 + ); HPLC (tR, min): 17.0. 1-(H-WRWK(1,2,3-triazol-4-methoxy)-NH2)-benzene 6 a: The peptide H-WRWK(N3)-NH2 (10 mg, 14 mmol, 1 equiv) was clicked with 1(prop-2-ynyloxy)benzene (3.3 mL, 28 mmol, 2 equiv) as described in the general experimental procedure. Yield: 49 % (5.7 mg, 6.7 mmol); MS (ESI, m/z): 832.2 (calcd 832.4 for [M + H] + ), 416.6 (calcd 416.7 for [M + 2H]2 + ); HPLC (tR, min): 18.7. 1-(H-RWRWRWK(1,2,3-triazol-4-methoxy)-NH2)benzene 7 a: The peptide H-RWRWRWK(N3)-NH2 (10 mg, 14 mmol, 1 equiv) was clicked with 1-(prop-2-ynyloxy)benzene (3.3 mL, 28 mmol, 2 equiv) as described in the general experimental procedure. Yield: 42 % (4.5 mg, 3.4 mmol); MS (ESI, m/z): 666.1 (calcd 666.3 for [M + 2H]2 + ), 444.4 (calcd 444.5 for [M + 3H]3 + ); HPLC (tR, min): 18.4. 1,3-Bis-(H-RWK(1,2,3-triazol-4-methoxy)-NH2)benzene 3 b: The peptide H-RWK(N3)-NH2 (15 mg, 29 mmol, 3 equiv) was clicked with 1,3-bis-(prop-2-ynyloxy)benzene (1.6 mL, 9.6 mmol, 1 equiv) as described in the general experimental procedure. Yield: 42 % (4.5 mg, 3.4 mmol); MS (ESI, m/z): 652.1, 607.2 (calcd 607.4 for [M + 2H]2 + ), 514.1 (calcd 514.3 for [H-RWK(N3)-NH2 + H] + , 405.1 (calcd 405.2 for [M + 3H]3 + ); HPLC (tR, min): 16.4. 1,3-Bis-(H-WRK(1,2,3-triazol-4-methoxy)-NH2)benzene 4 b: The peptide H-WRK(N3)-NH2 (15 mg, 29 mmol, 3 equiv) was clicked with 1,3-bis-(prop-2-ynyloxy)benzene (1.6 mL, 9.6 mmol, 1 equiv) as described in the general experimental procedure. Yield: 44 % (5.1 mg, 4.2 mmol); MS (ESI, m/z): 607.1 (calcd 607.4 for [M + 2H]2 + ), 514.1 (calcd 514.3 for [H-WRK(N3)-NH2 + H] + , 405.1 (calcd 405.2 for [M + 3H]3 + ); HPLC (tR, min): 16.5. 1,3-Bis-(H-RWRK(1,2,3-triazol-4-methoxy)-NH2)benzene 5 b: The peptide H-RWRK(N3)-NH2 (15 mg, 22 mmol, 3 equiv) was clicked with 1,3-bis-(prop-2-ynyloxy)benzene (1.2 mL, 7.3 mmol, 1 equiv) as described in the general experimental procedure. Yield: 32 % (3.6 mg, 2.3 mmol); MS (ESI, m/z): 763.2 (calcd 763.5 for [M + H]2 + ), 509.2 (calcd 509.3 for [M + H]3 + ), 382.1 (calcd 382.2 for [M + 4H]4 + ); HPLC (tR, min): 14.7. 1,3-Bis-(H-WRWK(1,2,3-triazol-4-methoxy)-NH2)benzene 6 b: The peptide H-WRWK(N3)-NH2 (15 mg, 21 mmol, 3 equiv) was clicked with 1,3-bis-(prop-2-ynyloxy)benzene (1.1 mL, 7.0 mmol, 1 equiv) as described in the general experimental procedure. Yield: 41 % (4.5 mg, 2.9 mmol); MS (ESI, m/z): 793.5 (calcd 793.5 for [M + 2H]2 + ), 529.3 (calcd 529.3 for [M + 3H]3 + ); HPLC (tR, min): 17.2. 1,3-Bis-(H-RWRWRWK(1,2,3-triazol-4-methoxy)-NH2)benzene 7 b: The peptide H-RWRWRWK(N3)-NH2 (30 mg, 25 mmol, 3 equiv) was clicked with 1,3-bis-(prop-2-ynyloxy)benzene (1.3 mL, 8.3 mmol, 1 equiv) as described in the general experimental procedure. Yield: 35 % (7.0 mg, 2.9 mmol); MS (MALDI, m/z): 2583.1 (calcd 2582.9 for [M]),2084.9, 1292.1 (calcd 1292.5 for [M + 2H]2 + ), 1198.6 (calcd 1198.7 for [H-RWRWRWK(N3)-NH2 + H] + ); HPLC (tR, min): 17.5. 1,3,5-Tris-(H-RWK(1,2,3-triazol-4-methoxy)-NH2)benzene 3 c: The peptide H-RWK(N3)-NH2 (15 mg, 29 mmol, 4 equiv) was clicked with 1,3,5-tris-(prop-2-ynyloxy)benzene (1.7 mg, 7.3 mmol, 1 equiv) as described in the general experimental procedure. Yield: 36 % (4.7 mg, 2.6 mmol); MS (ESI, m/z): 947.8 (calcd947.9 for [M + 2H + TFA]2 + ), 890.8 (calcd 890.9 for [M + 2H]2 + ), 678.1, 632.5 (calcd 632.3 for [M + 3H + TFA]3 + ), 594.5 (calcd 594.3 for [M + 3H]3 + ); HPLC (tR, min): 16.7. 1,3,5-Tris-(H-WRK(1,2,3-triazol-4-methoxy)-NH2)benzene 4 c: The peptide H-WRK(N3)-NH2 (15 mg, 29 mmol, 4 equiv) was clicked with 1,3,5-tris-(prop-2-ynyloxy)benzene (1.7 mg, 7.3 mmol, 1 equiv) as

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described in the general experimental procedure. Yield: 34 % (4.4 mg, 2.5 mmol); MS (ESI, m/z): 947.7, 891.2 (calcd 891.1 for [M + 2H]2 + ), 615.2, 594.4 (calcd 594.4 for [M + 3H]3 + ), 524.1, 446.0 (calcd 446.0 for [M + 4H]4 + ); MS (MALDI, m/z): 1784.3 (calcd 1781.1 for [M + H] + ), 1232.6; HPLC (tR, min): 17.8. 1,3,5-Tris-(H-RWRK(1,2,3-triazol-4-methoxy)-NH2)benzene 5 c: The peptide H-RWRK(N3)-NH2 (15 mg, 22 mmol, 4 equiv) was clicked with 1,3,5-tris-(prop-2-ynyloxy)benzene (1.3 mg, 5.5 mmol, 1 equiv) as described in the general experimental procedure. Yield: 23 % (2.8 mg, 1.3 mmol); MS (ESI, m/z): 1353.0, 750.5 (calcd 750.5 for [M + 3H]3 + ), 563.1 (calcd 563.1 for [M + 4H]4 + ); MS (MALDI, m/z): 2249.9 (calcd 2249.4 for [M + H] + ); HPLC (tR, min): 14.3. 1,3,5-Tris-(H-WRWK(1,2,3-triazol-4-methoxy)-NH2)benzene 6 c: The peptide H-WRWK(N3)-NH2 (15 mg, 21 mmol, 4 equiv) was clicked with 1,3,5-tris-(prop-2-ynyloxy)benzene (1.2 mg, 5.3 mmol, 1 equiv) as described in the general experimental procedure. Yield: 32 % (3.9 mg, 1.7 mmol); MS (ESI, m/z): 1912.2, 1170.2 (calcd 1170.2 for [M + 2H]2 + ), 801.2, 780.6 (calcd 780.5 for [M + 3H]3 + ), 732.5, 655.2, 585.7 (calcd 585.6 for [M + 4H]4 + ); MS (MALDI, m/z): 2563.7, 2339.7 (calcd 2339.4 for [M + H] + ), 2209.6, 1602.5, 1170.4 (calcd 1170.2 for [M + 2H]2 + ); HPLC (tR, min): 16.6. 1,3,5-Tris-(H-RWRWRWK(1,2,3-triazol-4-methoxy)-NH2)benzene 7 c: The peptide H-RWRWRWK(N3)-NH2 (40 mg, 33 mmol, 4 equiv) was clicked with 1,3,5-tris-(prop-2-ynyloxy)benzene (1.6 mg, 6.5 mmol, 1 equiv) as described in the general experimental procedure. Yield: 21 % (5.2 mg, 1.4 mmol); MS (MALDI, m/z): 3835.6 (calcd 3836.4 for [M + H] + ); HPLC (tR, min): 17.3. Antibacterial activity: Compounds were tested against Escherichia coli DSM 30083, Acinetobacter baumannii DSM 30007, Pseudomonas aeruginosa DSM 50071, Bacillus subtilis 168, Staphylococcus aureus DSM 20231, and Staphylococcus aureus ATCC 43300 (MRSA) in a microtiter plate assay according to CLSI guidelines, as described in ref. [8]. E. coli, A. baumannii, S. aureus, and B. subtilis were grown in Mueller–Hinton broth, and P. aeruginosa was grown in cation-adjusted Mueller–Hinton II. Compounds were dissolved in DMSO to yield stock solutions of 1.25, 2.5, 5, or 10 mg mL¢1. Serial dilutions in culture media were prepared with a Tecan Freedom Evo 75 liquid-handling workstation (Tecan, M•nnedorf, Switzerland). Dilutions, starting from a 10 mg mL¢1 stock solution, covered a range from 512 to 0.5 mg mL¢1. Compound dilutions were inoculated with 5 Õ 105 bacteria per mL from late-exponential cultures grown in the same media. Assay volumes were 200 mL per well. Cells were incubated for 16–18 h at 37 8C. Two biological replicates were measured. In each case, only the highest compound concentration inhibiting bacterial growth visibly was quoted as the MIC. Hemolytic activity: The peptides were tested for their hemolytic activities against human red blood cells (hRBCs). After drawing whole blood into anticoagulant-containing tubes (2.7 mL BD Vacutainer, SNC 0.129M, REF 363079, LOT 1283201), its fractionation was executed with one volume whole blood adding nine volumes sterile 0.9 % NaCl (Sigma) and sedimentation by centrifuge (800 g, 10 min, 4 8C). The lowest fraction containing all hRBCs was then washed twice with nine volumes 1 Õ PBS (Gibco), triturating carefully. The concentrated hRBCs were re-suspended with 1 Õ PBS to an erythrocyte concentration of 5 % v/v. Peptides, dissolved in 1 Õ PBS containing 1 % DMSO (for solubility reasons), were mixed each with 100 mL hRBC (5 % v/v) by adding 100 mL peptide dilution and incubated with agitation on a flat shaker (120 rpm, 30 min, 37 8C) resulting in 0.5 % DMSO in every well. After sedimenting all probes (800 g, 10 min, 4 8C), supernatant from each well (100 mL) was transferred into a fresh 96-well plate. Release of hemoglobin was

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Full Papers monitored by measuring the absorbance of all supernatants at l = 550 nm. Controls for 0 and 100 % hemolysis consisted of hRBC 5 % v/v suspended in 1 Õ PBS, 0.5 % DMSO in 1 Õ PBS, and 1 % TritonX100 in 1 Õ PBS, respectively.

Acknowledgements

[7]

This work was supported financially by the State of North-Rhine Westphalia (NRW), Germany, under the grant “Translation of Innovative Antibiotics from NRW (TinA)” to N.M.-N. and J.E.B.; N.M.N. and J.E.B. are also members of the Research Department Interfacial Systems Chemistry of Ruhr University Bochum (Germany), help from which is gratefully acknowledged. This work is also supported by the Cluster of Excellence RESOLV (EXC 1069) funded by the Deutsche Forschungsgemeinschaft. Keywords: antibiotics · antimicrobial peptides chemistry · multivalency · solid-phase synthesis

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Received: May 18, 2015 Published online on July 6, 2015

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Influence of the Multivalency of Ultrashort Arg-Trp-Based Antimicrobial Peptides (AMP) on Their Antibacterial Activity.

Peptide dendrimers are a class of molecules of high interest in the search for new antibiotics. We used microwave-assisted, copper(I)-catalyzed alkyne...
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