Communication Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Synthesis of Antimicrobial Poly(guanylurea)s Md Salauddin Ahmed,† Thirunavukkarasu Annamalai,† Xuerong Li,† Ahmed Seddek,† Peng Teng,‡ Yuk-Ching Tse-Dinh,† and Joong Ho Moon*,† †
Department of Chemistry and Biochemistry, Biomolecular Sciences Institute, Florida International University, 11200 SW 8th Street, Miami, Florida 33199, United States ‡ Department of Chemistry, University of South Florida, 4202 East Fowler Avenue, Tampa, Florida 33620, United States S Supporting Information *
ABSTRACT: Bacterial infections are serious health threats. Emerging drug resistance in bacteria further poses serious challenges to the treatment options involving traditional antibiotics. Antimicrobial polymers disrupt the physical cell membrane integrity of bacteria to address the drug resistance problems. Here, we introduce a conceptually new class of antimicrobial polymers containing positively charged guanylurea backbones for enhanced antimicrobial effects. The initial structure−activity relationship studies demonstrate that poly(guanylurea piperazine)s (PGU-Ps) exhibit excellent antimicrobial activity against different types of bacteria with high selectivity. The new design concept of using a positively charged guanylurea backbone will contribute to the development of future biocompatible, specific, and selective antimicrobial polymers.
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Here, we designed a new class of linear polymers containing positively charged guanylurea backbones (Scheme 1). Compared with most antimicrobial polymers with positively charged side chains, the new polymer design introduces both positive charge and hydrophobicity into the flexible polymer backbones. The guanylurea functional group is a fusion product of
ntimicrobial drug resistance has emerged rapidly and thwarted treatment options, leading to prolonged illness, disability, and death.1 Despite considerable efforts to develop new antimicrobial drugs,2 many bacterial infections remain difficult to treat due to acquired drug resistance. Compared to small molecular antibiotics designed to interrupt the bacterial intracellular biochemical processes, antimicrobial polymers disrupt the membrane integrity, offering a promising strategy to overcome drug resistance.3−5 Bacteria have little chance of developing resistance mechanism against the physical disruption, which often leads to cell death. The design principle of antimicrobial polymers has been primarily focused on balancing positive charges and the hydrophobicity in the pendant side chains in order to achieve selective disruption of the bacterial membrane over the mammalian cell membranes.6−10 By polymerizing various monomers containing positively charged hydrophobic side chains, many polymers with different functionalities and architectures have been developed and demonstrated for antimicrobial activities against a broad spectrum of bacterial species.11 Although current synthetic approaches have proven the capability of developing highly selective antimicrobial polymers, a simple synthetic method for developing antimicrobial polymers with improved biocompatibility, selectivity, and specificity is urgently needed.12 Using well-established polycondensation reactions, Zhang et al. recently demonstrated economical synthesis of positively charged amine-containing polyesters, polyamides, polyureas, and polyguanidines exhibiting antimicrobial activities.13 © XXXX American Chemical Society
Scheme 1. Synthesis of Poly(guanylurea)s (PGUs)
Received: January 22, 2018 Revised: March 7, 2018
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DOI: 10.1021/acs.bioconjchem.8b00057 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Communication
Bioconjugate Chemistry Table 1. Antimicrobial and Hemolytic Activities of Poly(guanylurea)s (PGUs) MIC (μg/mL)
selectivity (HC50/MIC)
PGU
M. smegmatis
S. f lexneri
MRSA
S. aureus
HC50(μg/mL)
M. smegmatis
S. f lexneri
MRSA
S. aureus
P-14K P-8K P-3K E-11K E-7K
6.5 3.1−6.5 102 25 50−99
25.5 25.5 >203 25−50 198
25.5 13 >203 99 198
13 13 >203 99 198
504 394 >2000 232 257
77.5 60.6−126 >19.6 4.6−9.0 2.6−5.0
19.8 15.4 >9.9 6.0 1.3
19.8 30.3 >9.9 2.3 1.3
38.8 30.3 >9.9 2.3 1.3
guanidine and urea. Low molecular weight synthetic compounds and naturally occurring peptides containing guanylurea derivatives have shown certain activities against bacteria, fungi, and viruses.14−16 With the increased positive charge density per repeating unit, various hydrophobic/hydrophilic units can be introduced to balance the amphiphilicity of the polymer. The initial structure−activity relationship studies demonstrate that poly(guanylurea piperazine)s (PGU-Ps) exhibit excellent antimicrobial activity against different types of bacteria with high selectivity. Prototype PGU-Ps were synthesized by reacting piperazine with a monomer containing tert-butyloxycarbonyl (Boc)protected guanidine groups at the end of short ethylene oxide side chains (Monomer A, Scheme 1). This monomer has been used by our group for synthesis of poly(phenyleneethynylene)s.17 We used the monomer without further modification because the aryl iodide group provides the hydrophobicity and the reaction sites for future structure− activity relationship studies. Piperazine was selected to create the rigidity between two guanylurea groups. It is known that the backbone rigidity and planarity of synthetic mimics of antimicrobial peptides (AMPs) play important roles in antimicrobial activities because of favorable membrane interaction.18,19 A noncyclic ethylenediamine was also used to synthesize control poly(guanylurea ethylenediamine)s (PGUEs) in order to examine the effects of the backbone chemical structures at the charged guanylurea on the antimicrobial activities. Positively charged PGUs were obtained as white solids after reacting with corresponding monomers at ∼70 °C for overnight followed by Boc deprotection (Supporting Information). Polymerization via guanylurea bond formation was confirmed by gel permeation chromatography (GPC) and the appearance and disappearance of 1H NMR peaks at ∼12.2 ppm for an amide proton of guanylurea and ∼1.45 ppm for Boc protons, respectively (Supporting Information). Typical molecular weight and polydispersity index of Boc-protected PGUs were 14,000 g/mol and 1.9, respectively. Positively charged PGUs were not water-soluble but were highly soluble in DMSO. When a portion of PGU solution in DMSO was mixed with an excess amount of bacteria culture medium [e.g., Mueller-Hinton Broth (MHB) and 7H9], nanoparticles (NPs) with narrow size distributions were formed. For example, the average hydrodynamic diameters (HDs) of PGU-P and PGU-E with molecular weights of 8,000 (i.e., PGU-P-8K) and 7,000 g/ mol (i.e., PGU-E-7K) in 7H9 medium were determined as 87 ± 2.6 and 103 ± 8.1 nm, respectively, by nanoparticle tracking analysis (Supporting Information). NPs in both culture media surprisingly exhibit the zeta potentials (ζ) in the range of −14 to −18 mV, implying that positively charged PGUs form ionic complexes with negatively charged components (e.g., proteins) in the medium (Supporting Information).
Two assays were conducted to evaluate potential toxicity toward mammalian cells: MTT and hemolysis assays. The MTT assay measures the metabolic activity of live cells using the color change of a tetrazolium dye [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide]. PGUs with different molecular weights exhibited ignorable cell viability inhibition when human cervical carcinoma cells (HeLa) were incubated with various amounts of polymers overnight (Supporting Information). Using human red blood cells (RBCs), the hemolytic activities of PGUs were also evaluated. The polymer concentrations required for 50% of hemolysis (HC50) were determined by averaging the amounts of hemoglobin released from RBCs due to the membrane damage (Supporting Information). As shown in Table 1, all PGUs are relatively nontoxic to RBC. PGU-Ps exhibit substantially lower hemolytic activity than PGU-Es, implying that the chemical identity along the backbone is an important structural factor affecting the hemolytic activity. It is worth noting that the subtle conformational difference in hydrophobic units of binary antimicrobial polyamide copolymers substantially influences the eukaryotic cytotoxicity.20 Antimicrobial activities of the PGUs with different molecular weights against Mycobacterium smegmatis (a mycobacterium), Staphylococcus aureus (a Gram-positive bacterium), methicillinresistant Staphylococcus aureus (MRSA), and Shigella f lexneri (a Gram-negative bacterium) were evaluated by measuring minimal inhibitory concentrations (MIC) (Table 1). PGU-Ps function well against the tested model bacteria. Particularly, PGU-P-8K exhibits the highest efficiency against M. smegmatis (3.1−6.5 μg/mL), implying that the mechanism of PGU-Ps could be associated with the membrane disruption. One of the hallmarks of the host defense AMPs and their synthetic mimics is nonspecific antimicrobial activity against a broad spectrum of bacterial species due to the nonspecific membrane disruption mechanism.21 While the MIC values of PGU-P-8K and -14K were relatively comparable, low molecular weight (3,000 g/ mol) PGU-P-3K exhibits poor antimicrobial activity. The active species responsible for membrane disruption have not been studied well, especially for amphiphilic polymers forming loosely aggregated polymer NPs in an aqueous solution. Depending on the integrity and chemical composition of NPs, either NPs9 or disintegrated polymer chains10 can be interacting with the membranes. The poor antimicrobial activity of PGU-P-3K, despite exhibiting similar HD (97 ± 0.9) and ζ (−16 ± 2.6 in 7H9) to those of PGU-P-8K NPs, implies that the active membrane disrupting species are not NPs but dissociated oligomer chains. The greatly reduced electrostatic interactions between the low molecular weight oligomers and the negatively charged membranes are responsible for the poor antimicrobial activity. To examine the effects of the backbone structures on antimicrobial activities, PGU-Es with comparable molecular weights (e.g., 11,000 and 7000 g/mol) to those of PGU-Ps B
DOI: 10.1021/acs.bioconjchem.8b00057 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Communication
Bioconjugate Chemistry
(circles) and PGU-E-11K (triangles) were substantially increased (Figure 1), supporting the possible membrane disruption mechanism of PGUs. After 2 h incubation, PGUP-8K induced higher membrane permeability (likely due to membrane damage) than PGU-E-11K, determined by the relative fluorescent intensities. Similar fluorescence patterns were observed from Gram-negative Shigella f lexneri incubated with PGU-P-8K and PGU-E-11K, respectively, although the level of membrane damage determined by the fluorescence intensity within 2 h of incubation was lower than that of M. smegmatis (Supporting Information). Even though PGU-P-8K exhibited better antimicrobial efficiency (MIC 13 μg/mL) against MRSA than a widely used antibiotic Ciprofloxacin (MIC 20 μg/mL), the EB assay result obtained within 2 h of incubation was not informative as no similar patterns of fluorescence intensity changes were correlated to the amounts and types of PGUs. Transmission electron microscopic (TEM) imaging of bacteria cells treated with NPs confirms that the antibiotic mechanism is associated with the membrane disruption. Compared with the intact bacteria cells (Figure 2A), M. smegmatis cells treated with PGU-P-8K at the MIC (i.e., 3 μg/ mL) are severely damaged (Figure 2A).
were prepared, and their MIC and HC50 were measured under the tested condition. As shown in Table 1, the MIC values of PGU-Es increased sharply, and the HC50 values increased by ∼2-fold when the two guanylurea groups along the backbone were coupled with the ethylene group. This initial structure− activity relationship suggests that the rigid connection of two positively charged planar guanylurea groups plays an important role in selective disruption of the bacteria over mammalian membranes. In a series of aromatic urea-containing oligomers, limited conformational flexibility along the backbone contributes to increased antimicrobial activities.22 In general, PGU-Ps exhibited a good selectivity (defined as the ratios of HC50/ MIC) across the tested bacteria when compared to PGU-Es. Specifically, the effects of rigidity on antimicrobial activity and selectivity were more pronounced on M. smegmatis. The antimicrobial activity and selectivity of PGU-P-8K against M. smegmatis were enhanced ∼16- and ∼14-fold, respectively, when compared to the similarly sized PGU-E-7K. PGU-E-11K exhibits moderate activity against M. smegmatis and no activity against other types of bacteria. The backbone of PGU-Es is more flexible and capable of additional H-bonding within NPs, which can contribute to the better NP integrity, resulting in fewer isolated polymer chains at the membrane interface. To gain insight into the antimicrobial mechanism, a membrane permeability assay using ethidium bromide (EB) was conducted.23 Nonfluorescent EB becomes fluorescent upon permeation through the membranes and the subsequent intercalation into intracellular nucleic acids. Due to tight regulation of the membrane permeability and the efflux pumps, the fluorescence intensity of normal bacteria cells treated with EB is generally low. However, if the membranes are damaged (i.e., allowing increased intracellular diffusion of EB) and/or the efflux pumps are inhibited (i.e., reducing the pump drainage efficiency), the fluorescence intensity of bacterial cells can be increased. Fluorescence intensities of bacteria cells treated with PGUs at the MIC values were monitored for 2 h. As shown in Figure 1, the fluorescence intensity of control M. smegmatis was slightly increased in the initial 20 min of EB incubation and then reached a plateau background (rectangles), indicating that M. smegmatis tightly controlled the amount of intracellular EB by balancing the influx and outflow of EB. At the MICs, fluorescence intensities of M. smegmatis treated with PGU-P-8K
Figure 2. TEM image of M. smegmatis. (A) intact cells and (B) cells treated with PGU-P-8K NPs. Scale bars: 500 nm.
In conclusion, we have introduced novel antimicrobial polymers containing positively charged guanylurea backbones. Unlike existing antimicrobial polymers with charged side chains, the newly developed PGUs simplify the polymer architecture by linearly combining the key functional units (i.e., positive charge, H-bonding, and the lipophilicity) along the backbone. The initial structure−activity relationship studies indicate that the backbone chemical structures, especially at the charged sites, play an important role in antimicrobial and hemolytic activities. PGUs synthesized with piperazine exhibit broader antimicrobial activities against different types of bacteria. The simplicity of polymerization without requiring any catalysts or specialized conditions, the availability of numerous commercial amines, and the biocompatible guanylurea functional group are clear advantages over the existing materials. The new design concept will provide a promising method toward the realization of potent antimicrobial polymers exhibiting high selectivity over mammalian cells and/or high specificity toward a target microorganism.
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ASSOCIATED CONTENT
S Supporting Information *
Figure 1. Kinetics of EB membrane permeation and nucleic acids intercalation. Control cells tightly regulate the amount of EB (rectangles), while PGU-treated cells (circles and triangles) allow high EB internalization caused by the membrane disruption.
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00057. C
DOI: 10.1021/acs.bioconjchem.8b00057 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Communication
Bioconjugate Chemistry
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Synthetic procedures, characterizations (1H NMR, NTA, Zeta potential, and TEM), and biochemical assays (MTT, hemolysis, and EB penetration assays) (PDF)
(13) Zhang, M. S., Teo, J. J., Liu, S. Q., Liang, Z. C., Ding, X., Ono, R. J., Breyta, G., Engler, A. C., Coady, D. J., Garcia, J., et al. (2016) Simple and cost-effective polycondensation routes to antimicrobial consumer products. Polym. Chem. 7, 3923−3932. (14) Sanguinetti, M., Sanfilippo, S., Castagnolo, D., Sanglard, D., Posteraro, B., Donzellini, G., and Botta, M. (2013) Novel macrocyclic amidinoureas: Potent non-zzole antifungals active against wild-type and resistant Candida species. ACS Med. Chem. Lett. 4, 852−857. (15) Manetti, F., Castagnolo, D., Raffi, F., Zizzari, A. T., Rajamaki, S., D’Arezzo, S., Visca, P., Cona, A., Fracasso, M. E., Doria, D., et al. (2009) Synthesis of new linear guanidines and macrocyclic amidinourea derivatives endowed with high antifungal activity against Candida spp. and Aspergillus spp. J. Med. Chem. 52, 7376−7379. (16) Williams, R. M., Yuan, C. G., Lee, V. J., and Chamberland, S. (1998) Synthesis and antimicrobial evaluation of TAN-1057A/B analogs. J. Antibiot. 51, 189−201. (17) Vokata, T., Twomey, M., Mendez, E., and Moon, J. H. (2015) Synthesis of biodegradable conjugated polymers with controlled backbone flexibility. J. Polym. Sci., Part A: Polym. Chem. 53, 1403− 1412. (18) Gabriel, G. J., and Tew, G. N. (2008) Conformationally rigid proteomimetics: A case study in designing antimicrobial aryl oligomers. Org. Biomol. Chem. 6, 417−423. (19) Ishitsuka, Y., Arnt, L., Majewski, J., Frey, S., Ratajczek, M., Kjaer, K., Tew, G. N., and Lee, K. Y. C. (2006) Amphiphilic poly(phenyleneethynylene)s can mimic antimicrobial peptide membrane disordering effect by membrane insertion. J. Am. Chem. Soc. 128, 13123−13129. (20) Liu, R. H., Chen, X. Y., Chakraborty, S., Lemke, J. J., Hayouka, Z., Chow, C., Welch, R. A., Weisblum, B., Masters, K. S., and Gellman, S. H. (2014) Tuning the biological activity profile of antibacterial polymers via subunit substitution pattern. J. Am. Chem. Soc. 136, 4410−4418. (21) Brogden, K. A. (2005) Antimicrobial peptides: Pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol. 3, 238−250. (22) Tang, H. Z., Doerksen, R. J., and Tew, G. N. (2005) Synthesis of urea oligomers and their antibacterial activity. Chem. Commun. 12, 1537−1539. (23) Gupta, K., Singh, S., and van Hoek, M. L. (2015) Short, synthetic cationic peptides have antibacterial activity against Mycobacterium smegmatis by forming pores in membrane and synergizing with antibiotics. Antibiotics 4, 358−378.
AUTHOR INFORMATION
Corresponding Author
*E-mail: jmoon@fiu.edu. ORCID
Joong Ho Moon: 0000-0001-6178-4628 Author Contributions
M.S.A. and T.A. contributed equally. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS J.H.M. acknowledges the generous support of the National Science Foundation (DMR1352317). REFERENCES
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DOI: 10.1021/acs.bioconjchem.8b00057 Bioconjugate Chem. XXXX, XXX, XXX−XXX
