All chemicals and solvents for peptide synthesis were from Iris Biotech (Germany). The column for lipopeptide RP-HPLC purification (Nucleodur C8e/c, 100 Å, 5 μm, 250 × 10 mm) was from Macherey-Nagel (Germany). The analytical column for peptide characterization (Chromolith Performance RP-18e, 100 × 4.6 mm) was from Merck Millipore (Germany). The gradient grade acetonitrile was from Scharlau (Spain). Deionized water (18.2 M'Ω) was obtained using a Direct-Q 3 UV-R Ultrapure Water System, Merck-Millipore, (Germany). The ion exchange device for TFA removal was a VariPure IPE (Ion Pair Extraction), Agilent (USA).

All lipopeptides were synthesized using the Fmoc/tBu procedure on Fmoc-Rink Amide AM resin (0.59 mmol/g) (Table 1). The Fmoc protecting group of amino acid was detached by 20% piperidine in DMF. Peptide bond formation was carried out with a 3-fold excess of Fmoc-protected amino acid dissolved in a DMF/DCM mixture (1:1 v/v) in the presence of 1% of Triton X-100 using DIC and HOBt as coupling reagents. Removal of the side chain protecting group and cleavage from the resin was achieved via 95% TFA with 2.5% of TIS and 2.5% of water for 1 h (Chan and White, 2000). The cleaved lipopeptides were precipitated and lyophilized. They were then purified by semi-preparative reverse-phase high performance liquid chromatography (RP-HPLC) with a Macharey Nagel C8 e/c column and eluted with a 20–60% linear gradient of phase B (phase A—0.1% TFA in water, phase B—0.1% TFA in ACN), at a flow rate of 3 mL/min, and analyzed at 214 nm.

The lipopeptides were analyzed by RP-HPLC on a Chromolith Performance monolithic column with a linear gradient 2–98% phase B (where phase A was 0.1% TFA in water and phase B was 0.1% TFA in ACN), at a flow rate of 2 mL/min, and analyzed at 214 nm. Fractions with a purity greater than 95% were collected and freeze-dried on an Alpha 2–4 LD plus system (Christ, Germany). Lipopeptides were further characterized by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF, Bruker, Germany).

The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) were determined according to the procedure recommended by the Clinical Laboratory Standards Institute (CLSI). The following reference strains were tested: Gram-negative E. coli (ATCC 25922), K. pneumoniae (ATCC 700603), P. vulgaris (PCM 2668), P. aeruginosa (ATCC 9027), Gram-positive S. aureus (ATCC 25923), S. epidermidis (PCM 2118), B. subtilis (ATCC 6633), E. faecalis (ATCC 29212) and fungi C. albicans (ATCC 10231), C. tropicalis (PCM 2681), and A. brasiliensis (ATCC 16404). All the microorganisms were from the Polish Collection of Microorganisms (Wroclaw, Poland). MIC was determined using a microbroth dilution method with Mueller-Hinton (MH) broth for bacteria and Sabouraud Dextrose broth for fungi (Becton Dickinson, Le Pont de Claix, France). An initial inoculum of 10⁵ cfu/mL was used for bacteria and 10³ cfu/mL for fungi. Polypropylene 96-well plates (Kartell, Noviglio, Italy) were incubated for 18 h at 37°C for bacteria and for 48 h at 25°C for the tested fungi. MIC (μg/mL) was taken as the lowest concentration of the lipopeptide, at which the observable growth was inhibited. All the MIC wells which did not reveal turbidity were cultured on solid media: a Mueller-Hinton II Agar for bacteria and a Sabouraud 2% Glucose Agar for fungi. The lowest concentrations of lipopeptides that did not show any visible growth on the plates after 24 h of incubation at 37°C and 48 h of incubation at 25°C were recorded as MBCs and MFCs, respectively. The experiments were performed in triplicate on three different days.

The hemolytic activity of the lipopeptides was determined by exposing human red blood cells (4% v/v) to tested compounds at graded concentrations. Red blood cells were obtained from a healthy donor. EDTA was used to prevent the blood clotting. The plasma was removed via centrifugation and the erythrocytes were washed three times with PBS. The red blood cells were suspended in PBS and incubated with different concentrations of lipopeptides at 37°C for 1 h and centrifuged for 5 min at 1000 × g. The supernatants were transferred to a sterile 96-well plate and hemoglobin release was measured with an Epoch microplate spectrophotometer (BioTek, USA) by recording the absorbance at 550 nm. A 0.1% Triton X-100 solution was used as the positive control (100% of hemolysis) and pure PBS as the negative (0% of hemolysis). Minimum hemolytic concentration (MHC) was taken as the lowest concentration which induced 10% of hemolysis.

Removal of trifluoroacetate counter ions was performed on VariPure IPE exchange columns. Before applying lipopeptides, ion exchange devices were conditioned as follows: 2 × 30 mL MeOH, 2 × 30 mL ACN, and 2 × 30 mL purified water. Previously lyophilized lipopeptides were dissolved in 10% acetonitrile and applied to ion exchange columns. Lipopeptides were eluted from the ion-exchange resin using a clean matrix. Thus obtained counter ion-free eluates were diluted with purified water and then subjected to a freeze-drying process. In order to confirm the absence of trifluoroacetate counter ions in the synthesized cationic lipopeptides, infrared spectroscopic studies were carried out. Infrared spectra were recorded on a Jasco FT/IR-410 spectrophotometer (Labor and Datentechnik GmbH, Germany) using the KBr tablet method. Tablets were prepared from 100 mg of potassium bromide (KBr) and 1.5 mg of the tested lipopeptide.

The antimicrobial tests indicated that lipopeptides containing hexadecanoic acid residue reveal high antibacterial activity toward Gram positives and Gram negatives; however, this activity is higher toward Gram positive bacteria (Tables 2, 3). The most potent bacteriostatic effect was observed with analogs containing more than one basic amino acid, that is C₁₆-KK-NH₂, C₁₆-KKK-NH₂, C₁₆-KKKK-NH₂, C₁₆-KGK-NH₂, and C₁₆-KGKG-NH₂. It was demonstrated that lipopeptides inhibit the growth of G+ and G− in the concentration range of 4–16 μg/mL. The exception is the P. vulgaris strain, which is resistant to the most active palmitate analogs and its MIC value reaches 512 μg/mL for all lipopeptide analogs. The lowest growth inhibition of G− was recorded for lipopeptides containing one basic amino acid: C₁₆-K-NH₂ and C₁₆-KG-NH₂. Results obtained for lipopeptides modified with tetradecanoic acid varied depending on the tested strain. The compounds exhibited relatively high antibacterial activity toward B. subtilis and S. epidermidis. The strongest bacteriostatic effect, from 4 to 8 μg/mL, was observed for the analogs C₁₄-KK-NH₂ and C₁₄-KKK-NH₂. Moderate bacteriostatic activity against the remaining Gram positive bacteria and Gram negatives was found (Tables 2, 3). However the activity toward gram-positive strains was higher in comparison to gram-negative strains. Lipopeptides modified with dodecanoic acid revealed rather poor bacteriostatic activity against tested G+ and G− strains (Tables 2, 3). Among all of the dodecanoic analogs, the highest growth inhibitory activity was perceived for lipopeptides containing one basic amino acid, i.e., C₁₂-K-NH₂ or C₁₂-KG-NH₂. MIC values determined for these compounds toward G+ and G− are in the range of 128–256 μg/mL, but for the strain P. vulgaris MIC was found at 512 μg/mL. Lipopeptides with two and three lysine residues (C₁₂-KK-NH₂, C₁₂-KKK-NH₂) are characterized by irregular activity. They are bacteriostatic to S. epidermidis and B. subtillis at a concentration of 64 μg/mL, but to S. aureus at a concentration of 2 and 1 mg/mL, respectively. The compounds are practically inactive against strains G−. Lipopeptides modified with decanoic acid are characterized by either poor bacteriostatic activity against G+ and G− strains or a complete lack thereof (Tables 2, 3). Among these lipopeptides, some higher bacteriostatic effect against G+ was observed for analogs C₁₀-KKK-NH₂ and C₁₀-KKKK-NH₂. They are also moderately active toward S. epidermidis and B. subtillis, while S. aureus and E. fecalis strains revealed complete resistance to them. None of the obtained lipopeptides modified with decanoic acid inhibited the growth of tested Gram negatives. Only the C₁₀-K-NH₂ analog exhibited very poor activity toward the P. aeruginosa strain; MIC value was observed at 512 μg/mL. Lipopeptides with attached octanoic fatty acid are generally inactive against gram positives as well as gram negatives. Among the 7 synthesized compounds, bacteriostatic activity at the concentration of 256 μg/mL toward S. epidermidis was noticed for C₈-KKK-NH₂ and C₈-KGK-NH₂. Additionally, C₈-KGK-NH₂ was bacteriostatic to B. subtillis at the concentration of 512 μg/mL.

The highest bactericidal activity was revealed for lipopeptides modified with hexadecanoic acid. C₁₆-KK-NH₂, C₁₆-KKK-NH₂, C₁₆-KKKK-NH₂, C₁₆-KGK-NH₂, and C₁₆-KGKG-NH₂ are bactericidal to G+ and G− in a concentration range similar to MIC, i.e., from 4 to 32 μg/mL. The bactericidal activity of the analogs containing one lysine residue (C₁₆-K-NH₂ or C₁₆-KG-NH₂) is distinctly lower. Although, S. epidermidis, B. subtillis and E. fecalis are already sensitive to these at a concentration of 4–64 μg/mL; unlike S. aureus, which is sensitive only at 512 μg/mL. For G− bacteria, much weaker bactericidal activity was observed. MBC values of compounds where the total net charge of the molecule is +1 reach 256–1·10³ μg/mL. Selected myristates as well as palmitates exhibit good bactericidal activity. The obtained MBC values were usually as much as twice as high as the MICs. The strongest bactericidal effect was proven for analogs of C₁₄-KK-NH₂ and C₁₄-KKK-NH₂. The most sensitive strains are B. subtillis and S. epidermidis. MBC determined for these organisms is within the range of 4–8 μg/mL. MBC of the lipopeptides C₁₂-K-NH₂ and C₁₂-KG-NH₂ is only the same as MIC for S. epidermidis and E. fecalis. For strains of S. aureus and B. subtillis, MBC is twice as high as MIC. Lipopeptides containing two or three lysine residues (C₁₂-KK-NH₂, C₁₂-KKK-NH₂) are weakly bactericidal to all of the studied gram negatives. Most susceptible to C₁₂-KKK-NH₂ was P. aeruginosa; MBC was observed at 512 μg/mL. None of the decanoic analogs have any bactericidal activity against S. aureus and E. fecalis. Strains of S. epidermidis and B. subtillis are more sensitive, in particular to C₁₀-KKK-NH₂ and C₁₀-KKKK-NH₂, and MBC values determined for these compounds range 128–256 μg/mL. From MBC values it can be concluded that decanoic modified lipopeptides are not bactericidal toward the tested strains. The MBC test demonstrated that only C₈-KKK-NH₂ was bactericidal to S. epidermidis at the concentration of 512 μg/mL.

It was found that palmitic acid derived peptides are weakly active against Candida species and Aspergillus molds (Table 4). This was confirmed by MIC and MFC determination. Of the seven analogs, the best fungistatic properties were confirmed for C₁₆-KK-NH₂, C₁₆-KKK-NH₂, and C₁₆-KKKK-NH₂. Both the MIC and the MFC are 128 μg/mL. Analogs containing glycine in the sequence (i.e., C₁₆-KG-NH₂, C₁₆-KGK-NH₂, and C₁₆-KGKG-NH₂) show weaker activity against C. tropicalis and A. brasiliensis. The lowest antifungal activity among all hexadecanoic analogs was revealed for lipopeptides containing one lysine: C₁₆-K-NH₂. Lipopeptides modified with tetradecanoic acid revealed some fungistatic and fungicidal activity against Candida and Aspergillus species. The lowest MIC and MBC value, i.e., 64 μg/mL, toward A. brasiliensis was noted in two cases: C₁₄-KKK-NH₂ and C₁₄-KKKK-NH₂. These compounds inhibit the growth of C. albicans and C. tropicalis slightly less effectively, because they require a concentration twice as high—128 μg/mL (Table 4). It was found that, at the same concentrations, all the tetradecanoic lipopeptides are fungistatic and fungicidal to the tested strains of C. albicans, C. tropicalis and A. brasiliensis. It was observed that dodecanoic lipopeptides weakly inhibits the growth of yeasts of Candida and A. brasiliensis (Table 4). The most bacteriostatically efficient compound proved to be that containing four lysines—C₁₂-KKKK-NH₂; MIC of this compound is in the range 128–256 μg/mL. Nearly identical properties were demonstrated by C₁₂-K-NH₂. Other analogs did not show significant bacteriostatic activity toward the tested fungi species. The best MFC results were observed for C₁₂-KKKK-NH₂. Analogs C₁₂-K-NH₂ and C₁₂-KG-NH₂ presented the same fungicidal activity—512 μg/mL. Generally, decanoic lipopeptides revealed no fungistatic activity toward Candida species and A. brasiliensis (Table 4). Some very weak fungistatic effect at the concentration of 512 μg/mL was noticed for C₁₀-KGK-NH₂ and C₁₀-KGKG-NH₂ toward C. tropicalis and for C₁₀-K-NH₂ toward A. brasiliensis. All tested fungi were totally resistant to octanoic analogs, as can be seen from the high MIC and MFC values. Results of antimicrobial studies are presented in Tables 2–4.

The most promising compounds, i.e., lipopeptides containing palmitic and myristic fatty acid residues, were tested after removal of counter ions to determine whether this influences their antimicrobial activity. We noticed some differences in the activity of compounds toward various strains. The determined antimicrobial activity was usually either the same for compounds with and without TFA or 2-fold higher/ lower, depending on the compounds and strain tested. For S. aureus, palmitic analogs without TFA (with the exception of C₁₆-K-NH₂) were twice as active as compounds containing TFA in both MIC and MBC assays. MICs obtained for C₁₄-KK-NH₂ and C₁₄-KKK-NH₂ were also twice as low as those obtained for lipopeptides after TFA removal. For the compounds C₁₄-KGK-NH₂ and C₁₄-KGKG-NH₂ the same difference was observed, but for the MBC values. Similar results were obtained with the E. faecalis strain. For almost all palmitic analogs without counter ions, the bacteriostatic and bactericidal concentrations were twice as low as peptides containing TFA. For myristic analogs, we did not observe any discrepancies in the activity of compounds both before and after removal of the counter ion. Bactericidal activity of the majority of palmitic analogs without TFA against E. coli was twice as high as peptides with a counter ion. MIC values were different for palmitic analogs containing glycine and more than one lysine residues. Bacteriostatic activity was also higher for myrisitic analogs: C₁₄-K-NH₂, C₁₄-KK-NH₂, and C₁₄-KKK-NH₂. For K. pneumoniae, differences between the activity of the compounds after counter ion removal were noticed only for a few compounds. Obtained MBCs were lower for lipopeptides without TFA, i.e., C₁₆-K-NH₂, C₁₆-KKKK-NH₂, and C₁₄-KKKK-NH₂, while for compounds C₁₆-KGK-NH₂, C₁₆-KGKG-NH₂, and C₁₄-KG-NH₂ we observed a decrease in antibacterial activity after removal of TFA. A decrease in the activity of compounds without counter ions was observed for the majority of the tested myristic analogs in MIC and MBC tests with C. albicans.

Lytic activity against human erythrocytes was measured to assess the toxicity of lipopeptides to eukaryotic cells. Lipopeptides containing a hexadecanoic chain displayed strong hemolysis. The strongest toxicity was demonstrated by palmitic derivatives containing more than one lysine residue: C₁₆-KK-NH₂, C₁₆-KKKNH₂, and C₁₆-KKKK-NH₂ (MHC 16–32 μg/mL). Compounds with the same cationic charge additionally containing a glycine residue showed slightly reduced lytic activity (MHC 64 μg/mL). Among the group of lipopeptides with a tetradecanoic acid, two analogs—C₁₄-KK-NH₂ and C₁₄-KGK-NH₂—were established as moderately hemolytic. These lead to lysis of red blood cells at the concentration of 128 μg/mL. Compounds containing 3 or 4 basic amino acid destroy erythrocytes at a higher concentration, i.e., only at 256 μg/mL. The rest of the synthesized lipopeptides are very weakly hemolytic or display no hemolysis at all within the range of the tested concentrations. The study shows that the hemolytic activity of lipopeptides increases with the length of the attached hydrophobic chain. Among the synthesized lipopeptides, the greatest hemolytic activity was observed for hexadecanoic modified lipopeptides. For those lipopeptides comprising a fragment of octanoic acid, hemolysis was not observed, even at a concentration of 4·10³ μg/mL. Furthermore, within the group of lipopeptides with a hexadecanoic fatty acid a strong dependence of the hemolytic activity on amino acid composition of the hydrophilic portion of the molecule was observed. Compounds endowed with a greater positive charge were more toxic to human erythrocytes.

Since the most significant antimicrobial activities were observed for lipopeptides containing hexadecanoic and tetradecanoic hydrocarbon chains, only those two groups of compounds were subjected to TFA counter ion removal to test the influence of TFA presence on the antimicrobial activity of lipopeptides themselves. Obtained FTIR spectra confirmed the absence of TFA counter ions, thus proving that the process of removing the counter ions via a VariPure IPE ion-exchange column ran properly. Figure 1 presents IR spectra of lipopeptides before (green line) and after (blue line) TFA removal. There are visible bands derived from the stretching vibration of CF₂ and CF₃ groups within the range of 1350–1120 cm⁻¹ (green line). However, there is no such band in the sample subjected to removal of the counter ion.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.