Ammonium and guanidine carbosilane dendrimers and dendrons as microbicides

Accepted Manuscript Ammonium and guanidine carbosilane dendrimers and dendrons as microbicides Irene Heredero-Bermejo, José M. Hernández-Ros, Leticia Sánchez-García, Marek Maly, Cristina Verdú-Expósito, Juan Soliveri, F. Javier de la Mata, José L. Copa-Patiño, Jorge Pérez-Serrano, Javier Sánchez-Nieves, Rafael Gómez PII: DOI: Reference:

S0014-3057(18)30076-4 https://doi.org/10.1016/j.eurpolymj.2018.02.025 EPJ 8299

To appear in:

European Polymer Journal

Received Date: Revised Date: Accepted Date:

11 January 2018 15 February 2018 18 February 2018

Please cite this article as: Heredero-Bermejo, I., Hernández-Ros, J.M., Sánchez-García, L., Maly, M., VerdúExpósito, C., Soliveri, J., Javier de la Mata, F., Copa-Patiño, J.L., Pérez-Serrano, J., Sánchez-Nieves, J., Gómez, R., Ammonium and guanidine carbosilane dendrimers and dendrons as microbicides, European Polymer Journal (2018), doi: https://doi.org/10.1016/j.eurpolymj.2018.02.025

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AMMONIUM AND GUANIDINE CARBOSILANE DENDRIMERS AND DENDRONS AS MICROBICIDES Irene Heredero-Bermejo,a José M. Hernández-Ros,a Leticia Sánchez-García,b Marek Maly, c Cristina Verdú-Expósito,a Juan Soliveri,a F. Javier de la Mata,b,d José L. CopaPatiño,a Jorge Pérez-Serrano,a Javier Sánchez-Nieves,b,d,* Rafael Gómezb,d,*

a

Department of Biomedicina y Biotecnología. Facultad de Farmacia, Universidad de

Alcalá, E-28805 Alcalá de Henares, Madrid, Spain. b

Department of Química Orgánica y Química Inorgánica, Universidad de Alcalá

(IRYCIS), Campus Universitario; Instituto de Investigación Química "Andrés M. del Río" (IQAR), Universidad de Alcalá; E-28805 Alcalá de Henares (Madrid) Spain. c

Faculty of Science, J. E. Purkinje University, Ceske mladeze 8, 400 96 Usti nad

Labem, Czech Republic. d

Networking Research Centre for Bioengineering, Biomaterials and Nanomedicine

(CIBER-BBN), Madrid, Spain. * Corresponding author: J. Sánchez-Nieves, [email protected]; Rafael Gómez, [email protected].

1

Abstract Carbosilane dendrimers (generations 1-3) and dendrons (generations 2-3) functionalized with guanidine functions (GU) have been synthesized

from

corresponding ammonium (-NH3+) derivatives with good yield. These dendritic molecules were tested as microbicides against Escherichia coli (Gram-negative), Staphylococcus aureus (Gram-positive) and methicillin resistant S. aureus (MRSA) bacteria; and against Acanthomoeba polyphaga in both stages, trophozoites and cysts. The best results were obtained for lower generation dendrimers and dendrons, with no remarkable influence of the peripheral moieties against bacteria but with better amoebicide activity for -NH3+ systems. Moreover, the activity values of first generation dendrimers and second generation dendrons were below their cytotoxicity against eukaryote cells. Molecular dynamics study of interactions between selected dendrimers and anionic phospholipid bilayers indicates possible connections between the antibacterial activity and interaction modes. Keywords:

carbosilane

dendrimers;

amoebicide; molecular dynamics.

2

ammonium;

guanidine;

antibacterial;

1. Introduction Bacteria and protozoa are ubiquitous microorganisms, being the vast majority of terrestrial biomass. However contamination with some strains is a major problem for public health systems [1-3]. Bacteria are prokaryotic whilst protozoa are eukaryotic unicellular microorganisms. Due to this important difference, the treatment of each type of microorganism requires different drugs or drug combinations. A variety of antibiotics have been developed against bacteria, whereas amoebicidal systems are less explored in part as consequence of lower number of sickness caused by these microorganisms in developed countries, although being responsible for severe illness [4-7]. Moreover, the response of different species of same microorganisms towards a specific drug depends on structural characteristics of the microorganisms, as for example the cell walls of bacteria. The widespread use, or misuse, of antibiotics leads to the appearance of resistances in bacteria [8-10], a serious concern mainly in hospital facilities [11]. On the other hand, amoebae have two stages in its life cycle: the mobile and infective stage, trophozoite; and the resistant stage, cyst, which occurs under unfavorable environmental conditions. Therefore, encystment stage causes the main problems in the treatment of amoeba infections since many biocides available are ineffective in killing them due to its high resistance [4,12]. All these considerations make difficult infection prevention in risky environments and therefore, the search of alternative antimicrobial systems have become a priority [13,14]. A good starting point should be the seeking of simple molecules with non-specific activity towards different microorganisms. Polycationic macromolecules are being studied as selective microbicides with low toxicity toward human cells [13,15,16]. Their activity is related with the cationic multivalency of the systems, their main target being the plasmatic membrane in

3

eukaryotic cells and the cytoplasmic membrane in bacteria [17,18]. Dendrimers are one type of these macromolecules. They are highly branched monodisperse macromolecules with well-defined globular shape that are synthesized by controlled step-by-step procedures. The presence of high density of active cationic surface groups at the periphery have attracted attention as potential antibacterial [19-23] or amoebicidal agents [24,25]. The use of dendrons, molecules with analogous characteristics but with cone-shaped structure [26-28], is expected to behave in the same direction. However, these molecules offer the possibility to introduce extra functions at the focal point or to be bound to material surfaces or other biomaterials [29,30]. A drawback of polyammonium macromolecules is their toxicity associated with the polycationic charge. Although this effect could be obviated in some materials, in a human body it should be minimized. In this sense, guanidine functions have proposed as replacement of ammonium functions [31-33]. Biguanide moieties are the active groups of chlorhexidine and polyhexamethylene biguanide, the main drugs for the treatment of Acanthamoeba keratitis, although these drugs show high toxicity. However, guanidine groups are present in antimicrobial peptides as part of arginine amino acid [34,35]. Furthermore, several polymers and dendrimers containing these fragments have shown better biological properties than related ammonium derivatives [31,36]. In particular, a polyguanidine polymethacrylate polymer was active against Gram-positive bacteria whilst the ammonium analogue were inactive, both systems being innocuous against Gram-negative bacteria [37]. We have reported elsewhere the antibacterial [19,38-41] and antiamoebic [24,25] activity of cationic carbosilane dendrimers and dendrons. These studies highlight the wide spectra antimicrobicide behavior of lower generation systems toward Grampositive and Gram-negative bacteria as well as toward Acanthamoeba trophozoites. On

4

the other hand, the activity of related hyperbranched carbosilane polymers was clearly worse than that of dendritic molecules [40,42]. The good activity of these small systems is consequence of an adequate balance between the hydrophilic and hydrophobic parts of the molecules, the ammonium periphery and the carbosilane framework, respectively [19]. Moreover, one of these compounds, decorated with –NH3+ groups, when combined with chlorhexidine digluconate presented synergistic effects against A. polyphaga [43]. Regarding other dendritic macromolecules, their antimicrobicide properties are also related with the size and type of skeleton, as can be inferred for dendrimers like PAMAM (second generation) [44], PPI (fourth generation) [22], or poly(propyleneoxide) amines (first generation) [45]. Taking into account these previous results, herein we reported the microbicide properties of carbosilane dendrimers and dendrons with guanidine (GU) moieties at the periphery. Their activity has been compared with that of their precursors functionalized with ammonium –NH3+ groups. For this purpose, novel dendrimers with these guanidine functions as well as dendrons with –NH3+ and guanidine groups have been synthesized. As models of Gram-positive and Gram-negative bacteria we have chosen Staphylococcus aureus and Escherichia coli, respectively, and resistant S. aureus (MRSA), whilst as model of amoebae Acanthomoeba polyphaga strains were used.

2. Results and discussion 2.1. Dendrimers and dendrons synthesis The nomenclature used in this work for dendrimers is of the type GnO3(S-Y)m) and for dendrons of the type XGn(S-Y)m), which describes the structure of compounds as follow: Gn corresponds with carbosilane framework and generation (n) (see Figure 1, S1 and S2); O3, describes the core of dendrimers (1,3,5-C6H3O3); X, means type of focal

5

point in dendrons; and (S-Y)m indicates the type of peripheral groups (Y), its number (m), and the presence of a sulfur atom close to the surface due to the preparative methodology. Carbosilane dendrimers with ammonium –NH3+ groups (GnO3(S-NH3+)m; n = 1, m = 6 (1); n = 2, m = 12 (2); n = 3, m = 24 (3)) have been previously reported [39]. These ammonium dendrimers are the starting materials to prepare the corresponding guanidine (GU) dendrimers by reaction with 1H-pyrazole-1-carboxamidine hydrochloride at 55ºC in the presence of base (Scheme 1). This simple procedure afforded compounds GnO3(S-GU+)m (n = 1, m = 6 (4);[43] n = 2, m = 12 (5); n = 3, m = 24 (6)) in good yield as water soluble solids (Figure 1 and Figure S1). Compounds 4-6 were characterized by nuclear magnetic resonance (NMR), mass spectrometry (MS) an elemental analysis. The main NMR data that confirms formation of compounds 4-6 are: a) in the 1H NMR spectra the shifting to higher frequency of the resonances belonging to the CH 2N groups (from δ ca. 2.9 in 1-3 to δ ca. 3.3 in 4-6); b) in the 13C NMR spectra the presence of the corresponding carbon resonances (from δ ca. 38.5 in 1-3 to δ ca. 39.0 in 4-6) and the presence of a peak for the new carbon atom N-C(NH2+)-N (at δ ca. 156.5); and c) the shifting of the

15

N resonance belonging to the CH2N group from δ ca. -340.5 in

compounds 1-3 to δ ca. -303.5 in compounds 4-6. MS of these compounds showed peaks corresponding with cationic dendrimers bearing different charges (see experimental section).

Scheme 1. Synthesis of GnO3(S-GU+)m (4-6) from GnO3(S-NH3+)m (1-3). 6

Regarding dendrons synthesis with -NH3+ motifs, the thiol-ene addition was also successful, showing the versatility of this procedure [39,46]. Thus, reaction of HS(CH2)2NH3+ with the vinyl derivatives HOGnVm (n = 2, m = 4; n = 3, m = 8) [46] under UV irradiation led to new dendrons with ammonium groups HOGn(S-NH3+)m (n = 2, m = 4 (7); n = 3, m = 8 (8)) (Scheme 2, Figure 1 and S2). Afterward, addition of 1H-pyrazole-1-carboxamidine hydrochloride to derivatives 7 and 8 afforded new dendrons with charged guanidine groups HOGn(S-GU+)m (n = 2, m = 4 (9); n = 3, m = 8 (10)) (Scheme 2, Figure 1 and S2). Both reactions were quantitative by NMR, although due to the smaller size of second generation dendrons the final yields of these compounds were moderate after purification by dialysis. Again, dendrons 7-10 were obtained as solids soluble in water. Compounds 7-10 were characterized by NMR, MS (9-10) an elemental analysis. The characterization of these dendrons by NMR showed resonances similar to those of their analogous dendrimers (vide supra). Regarding MS, we could not identify peaks corresponding to cationic dendrons [Mn+] in spectra of compounds 7-8, but they were observed in the spectra of cationic guanidium dendrons 9-10 (see experimental).

Scheme 2. Synthesis of dendrons HOGn(S-NH3+)m (n = 2, m = 4 (7); n = 3, m = 8 (8)) and HOGn(S-GU+)m (n = 2, m = 4 (9); n = 3, m = 8 (10)). i) HS(CH2)2NH3+, hν; ii) 1H-pyrazole-1-carboxamidine hydrochloride, 55 ºC.

7

Figure 1. Drawing of carbosilane dendrimers (first generation) and dendrons (second generation) used in this work. Chloride anions are omitted for clarity. 2.2. Antibacterial activity The bactericidal behaviour of cationic dendritic systems was evaluated using S. aureus and E. coli as models of Gram-positive and Gram-negative bacteria, respectively. Figure 1 depicts drawings of some representative dendrimers and dendrons and Table 1 summarizes the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of dendrimers and dendrons discussed in this section. Table 1. Minimum inhibitory (MIC) and bactericidal (MBC) concentrations of dendrimers GnO3(S-NH3+)m (1-3) [39] and GnO3(S-GU+)m (4-6) and dendrons HOGn(S-NH3+)m (7, 8) and HOGn(S-GU+)m (9, 10). Data shown in ppm (mg L-1). 8

S. aureus

E. coli

MRSA

MIC

MBC

MIC

MBC

MIC

MBC

[G1O3(S-NH3)6]6+ (1)

2

2

2

2

1

2

[G2O3(S-NH3)12]12+ (2)

4

4

8

8

-

-

[G3O3(S-NH3)24]24+ (3)

32

32

128

128

-

-

[G1O3(S-GU)6]6+ (4)

2

2

4

4

2

2

[G2O3(S-GU)12]12+ (5)

32

64

64

64

-

-

[G3O3(S-GU)24]24+ (6)

64

64

64

128

-

-

[HOG2(S-NH3)4]4+ (7)

2

2

2

4

2

2

[HOG3(S-NH3)8]8+ (8)

128

128

128

128

-

-

[HOG2(S-GU)4]4+ (9)

2

2

2

2

2

2

[HOG3(S-GU)8]8+ (10)

16

32

16

16

-

-

First of all, the data clearly show better activity of low generation systems, first generation dendrimers (dendrimers 1 and 4) and second generation dendrons (dendrons 7 and 9). The influence of -NH3+ and guanidine groups on the bactericide activity is quite similar in both dendrimers and dendrons, particularly for those compounds with greater activity. As the generation increases, both MIC and MBC values also increase. Regarding the type of bacteria, each of the compounds showed also similar values against both bacteria strains, probably due to their non-specificity. Hence, the best hydrophobic/hydrophilic balance of the compounds 1-10 correspond to dendrimers of first generation and dendrons of second generation, which are the most active compounds [19]. Similar results were observed when measuring the antibacterial behaviour of these compounds using concentration values per ammonium group present in the dendritic structure (Tables S1 and S2). This latter analysis determines the

9

effectiveness of each active function, the relevance of multivalency, in the overall activity of the dendrimers, since lower concentration value means higher activity of this group. With this analysis for compounds 1-10, the value of guanidine dendrimer 4 slightly rises up over the value of ammonium dendrimer 1; although it was similar to second generation dendrons 7 (ammonium groups) and 9 (guanidine groups). The lower generations dendrimers G1O3(S-NH3+)6 (1) and G1O3(S-GU+)6 (4) and dendrons HOG2(S-NH3+)4 (7) and HOG2(S-GU+)4 (9) were chosen to evaluate their activity in multi-resistant S. aureus strains (MRSA) (Table 1 and S3). These four compounds were the best of all studied and comprise different topologies and types of peripheral groups. According to the results, these four compounds kept their activity in MRSA, in a similar way than the carbosilane dendrimer G1O3(S-NMe3+)6 and dendron HOG2(S-NMe3+)4 containing ammonium -NMe3+ groups previously described [19]. Hence, any of these low generation derivatives are broad spectrum antibacterial compounds and are also active in resistant strains. 2.3. Trophocidal properties The IC50 values of dendrimers, dendrons and chlorhexidine (CLX, a reference drug against Acanthamoeba) tested after 4, 24, 48 and 72 h of treatment on A. polyphaga trophozoites are shown in Tables 2, S4 and S5. According to these results, growth inhibition of trophozoites is the most noticeable after 24 h of treatment for all dendritic systems (Table 2). Among all compounds tested, only lower generation systems 1, 4, 7, and 9 showed a similar effect than CLX. Once again, the smallest systems behaved better as microbicides than higher generations. Hence, for these compounds a similar structureactivity relationship was established as that discussed for antibacterial activity (see above) and also observed previously for carbosilane derivatives [19,25,39].

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Table 2. IC50 and of dendrimers GnO3(S-NH3+)m (1-3)[25] and GnO3(S-GU+)m (4-6) and dendrons HOGn(S-NH3+)m (7, 8) and HOGn(S-GU+)m (9, 10) in trophozoites A. polyphaga and HeLa cells after 24 h of treatment. Data shown in ppm (mg L -1). MCC, minimum cystidal concentration. Acanthamoeba

HeLa cells

IC50

MCC

IC50

[G1O3(S-NH3)6]6+ (1)

2.4+0.1

128

6.2+0.8

[G2O3(S-NH3)12]12+ (2)

19.0+0.6

-

8.5+1.1

[G3O3(S-NH3)24]24+ (3)

27.5+0.1

-

10.4+0.7

[G1O3(S-GU)6]6+ (4)

7.6 ± 0.2

256

20.9 ± 2.5

[G2O3(S-GU)12]12+ (5)

73.9 ± 0.6

-

14.1 ± 0.8

[G3O3(S-GU)24]24+ (6)

63.9 ± 0.8

-

16.0 ± 1.7

[HOG2(S-NH3)4]4+ (7)

4.1 ± 0.2

512

11.3 ± 1.1

[HOG3(S-NH3)8]8+ (8)

39.8 ± 1.6

-

17.8 ± 0.7

[HOG2(S-GU)4]4+ (9)

4.6 ± 0.2

256

10.9 ± 0.3

-

12.5 ± 0.7

10

8.2 ± 1.6

[HOG3(S-GU)8]8+ (10) 43.5 ± 0.9 CLX

1.7 ± 0.1

2.4. Cytotoxicity The toxicity (IC50) of all dendrimers and dendrons were tested in HeLa cells after 24 h of incubation (Table 2). Non-cytotoxicity was established when IC50 values were lower for trophozoites than for HeLa cells. Among all compounds tested, only the lower generation dendrimers and dendrons (1, 4, 7 and 9), apart from CLX, were classified as non-cytotoxic dendrimers, and selected to continue the present study. Once again, the

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non-cytotoxic compounds were also the most effective against both amoebae and bacteria. The four dendritic systems 1, 4, 7 and 9 were also tested in murine retinal cell line (MU-PH1), since A. polyphaga infection is responsible of keratitis and hence, toxicity of any potential drug in this type of cells is of interest. Calculation of difference between IC50 values for trophozoites and for MU-PH1 cells showed that the four of them could be still classified as non-cytotoxic compounds (Table S6). Regarding topology and peripheral moieties, small differences are observed, being guanidine dendrimer 4 the less cytotoxic compound (2-fold). 2.6. Cysticidal properties Finally, dendrimers selected as non-cytotoxic (1, 4, 7 and 9) were also tested against cysts, because they are less susceptible to any treatment than trophozoites. Table 2 shows the concentrations needed to avoid the complete reversion of cysts to trophozoites (MCC, minimum cystidal concentration) after treatment and subsequent incubation in fresh culture medium for 24 h. This parameter showed that none of the dendritic compounds neither CLX were completely effective because concentrations that reduce population of viable cyst to 100% were toxic against cells. Nevertheless, we must consider that if excystment is produced when concentrations of these compounds are below MCC but over the toxicity value, the trophozoites will not survive. That is, these compounds could be used as protecting agents against infection. 3. Interaction of dendritic systems with lipid bilayers The antibacterial activity of polycationic compounds is related with their ability to penetrate bacterial membranes and remove the divalent cations that help to maintain the membrane structure [47]. Main component of these membranes is a phospholipid bilayer that acts as an impermeable barrier, although it contains channels for molecular

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transporting. To better understand the interaction of cationic dendrimers with these bilayers, a preliminary study of the interaction of two dendrimers with two different phospholipid bilayers has been carried out using molecular dynamics. Two representative dendrimers, differing significantly in their antibacterial activity, were used for this preliminary study to determine if this difference is reflected in their interaction with selected negatively charged lipid bilayers (considering simulation times in order of hundreds of nanoseconds). As the representative of compounds with good antibacterial behaviour was chosen dendrimer 1 ([G1O3(S-NH3)6]6+) whereas as representative of compounds with bad antibacterial behaviour dendrimer [G0Si(SNH3)4]4+ (11, Figure S7) was selected (MIC = 128 and MBC = 256 for both bacteria strains), which was previously studied by us [19]. Regarding the phospholipid layers, we used here two very simplified membrane models (Figure S7) -i.e. negatively charged phospholipid bilayers composed in the first case of dipalmitoylphosphatidylglycerol (DPPG) and in the second case of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) molecules. DPPG and POPG lipids are quite similar (identical positive head, one tail, being the tail just two carbons longer and containing one double C-C bond in POPG case). However, bilayers composed of those molecules have significantly different transition temperatures and so properties at working temperature (310.15 K). At this temperature, DPPG bilayer is in solid (“gel”) phase, its transition temperature is 314.15 K, whilst the POPG bilayer is in liquid phase, since its transition temperature is significantly lower (271.15 K) due to the presence of the above mentioned C-C double bond and the slightly longer hydrocarbon tail.

13

Figure 2. Computer models of dendrimer 1 (left) and 11 (right) interacting with DPPG bilayer (final configurations). Sphere representation is used for atoms belonging to dendrimers, stick representation for lipids and ball representation for Na+ ions. Colors: C, gray (lipid carbons) or black (dendrimer carbons); H, white; O, red; P, orange; S, yellow; Si, beige; Na, purple.

Figure 3. Computer models of dendrimer 1 (left) and 11 (right) interacting with POPG bilayer (final configurations). Sphere representation is used for atoms belonging to dendrimers, stick representation is used for lipids and ball representation for Na+ ions. Colors: C, gray (lipid carbons) or black (dendrimer carbons); H, white; O, red; P, orange; S, yellow; Si, beige; Na, purple.

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In these conditions, our two simple membrane models are chemically quite similar (identical interaction region) but physically different. The DPPG bilayer is more dense and stiffer (area per lipid of simulated model 47 Å2) than the POPG bilayer (area per lipid of simulated model 68 Å2). The results are clear from Figures 2 and 3 showing final molecular configurations and from Figures 4 and 5 informing us about the dynamics of the binding process. In DPPG case, we can see that the hydrophobic part of dendrimer 1 is embedded into the hydrophobic part of lipids (carbon tails) and that the hydrophilic (charged) dendrimer end units interact with the anionic lipid heads. Moreover, from Figure 4 we can clearly observe that it took more than 250 ns to anchor properly dendrimer 1 in the bilayer. On the other hand, in the final configuration of the interacting system dendrimer 11/DPPG bilayer, this dendrimer interacts just with the charged surface of the lipid bilayer. This dendrimer “oscilated” on the surface during the whole 665 ns long simulation being unable to stably catch on inside the bilayer, even if there were several opportunities (Figure 4). Regarding POPG case, both dendrimers (1 and 11) were able to penetrate into the lipid bilayer and anchor there in a similar way as did dendrimer 1 in DPPG bilayer (Figures S8, 3 and 5). In Figure 5 we can notice that dendrimer 1 succeeded to move deeper in the lipid bilayer ca. after 30 ns while in case of dendrimer 11 it was ca. after 45 ns. Hence, the important difference in antibacterial activities of these two dendrimers is also reflected in their ability to penetrate into selected lipid bilayers, probably as consequence of their different hydrophobic/hydrophilic balance [19]. Taking into account these data and that bacterial membranes are combinations of different phospholipids and proteins, we can suppose that variations of membrane composition can have a remarkable impact in the interaction/permeability of these dendritic systems with them, and as consequence, in their bactericidal activities.

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Figure 4. Evolution of the minimal distance rmin of the dendrimer core atoms (top) and all dendrimer atoms (bottom) from the hydrophobic interior of the DPPG lipid bilayer, which is here composed of hydrocarbon (palmitoyl) tails. Blue color is used for dendrimer 1, green color is used for dendrimer 11.

Figure 5. Evolution of the minimal distance rmin of the dendrimer core atoms (top) and all dendrimer atoms (bottom) from the hydrophobic interior of the POPG lipid bilayer, which is here composed of hydrocarbon (palmitoyl-oleoyl) tails. Blue color is used for dendrimer 1, green color is used for dendrimer 11. Higher value of rmin for dendrimer 11 core atoms (comparing to dendrimer 1) for t > 50 ns – i.e. in situation 16

where dendrimer is already anchored in lipid bilayer - is caused by the fact, that core of dendrimer 11 model is composed just of single Si atom which has significantly bigger van der Waals radius than hydrogen, which is present in the core unit of the dendrimer 1 model. 4. Conclusions Low generation dendrimers and dendrons with –NH3+ and guanidine functions are effective microbicide agents against bacteria, both Gram- positive and Gram-negative, and also against multi-resistant S. aureus and amoebae. The activity values of these small systems were similar in all microorganisms and were below their cytotoxicity in eukaryote cells (HeLa and retinal MU-PH1). Thus, a sole compound could be potentially used as wide spectrum microbicide. The advantages of these systems, the simplicity of their synthesis, which would make them easy available, and their multivalency, which could be employed to anchor extra active groups, make them attractive for further exploration on their microbicide properties. On the other hand, molecular modeling and dynamics supports that an adequate hydrophobic/hydrophilic balance is very important to interact and penetrate membranes. Research in this way and further studies to gain a for better understanding of interactions with bacterial membranes are currently in progress. 5. Experimental Section 5.1. General Considerations. All reactions were carried out under inert atmosphere. Reagents and solvents were used as received without further purification. NMR spectra were recorded on a Varian Unity VXR-300 (300.13 (1H), 75.47 (13C) MHz) or on a Bruker AV400 (400.13 (1H), 100.60 (13C), 40.56 (15N), 79.49 (29Si) MHz). Chemical shifts (δ) are given in ppm. 1H and

13

C resonances were measured relative to internal

deuterated solvent peaks considering TMS = 0 ppm, whereas 17

15

N and

29

Si resonances

were measured relative to external MeNO and TMS, respectively. When necessary, assignment of resonances was done from HSQC, HMBC, COSY, TOCSY and NOESY NMR experiments. Elemental analyses were performed on a LECO CHNS-932. Mass Spectra were obtained from a Thermo Scientific TSQ Quantum LC-MS and an Agilent 6210. Thiol-ene reactions were carried out employing a HPK 125 W mercury lamp from Heraeus Noblelight with maximum energy at 365 nm, in normal glassware under an inert atmosphere. Compounds cysteamine hydrochloride (HS(CH2)2NH2·HCl), 2,2’dimethoxy-2-phenylacetophenone

(DMPA),

1H-pyrazole-1-carboxamidine

hydrochloride, diisopropylethylamine (DIPEA), were obtained from commercial sources. Compounds [GnO3(S-NH3)m]m+ (1-3) [39], [G1O3(S-GU)6]6+ (4) [43] and HOGnVm [46] were synthesized as published. 5.2. Synthesis of selected compounds. The synthesis of all compounds is described in Supporting Information and a selection is collected next. HOG2(S-NH3Cl)4 (7). This dendron was prepared from reaction of HOG2V4 (0.240 g, 0.52 mmol) and HS(CH2)2NH2·HCl (0.235 g, 2.10 mmol) in the presence of 2,2dimethoxy-2-phenylacetophenone (DMPA, 0.05 mmol, 2.5 % mol per vinyl group) in a THF/methanol solution (1:2 ratio, 6 mL). The reaction mixture was deoxygenated using an argon flow and then irradiated for 2 h with UV light at 365 nm. Next, DMPA was again added (2.5 % mol per vinyl group) and the reaction mixture irradiated for another 3 h.

1

H-NMR monitoring allowed checking the disappearance of vinyl groups.

Afterwards, the initial reaction mixture was concentrated by rotator evaporation and solved in the minimum amount of MeOH. Subsequently, the product was precipitated with Et2O under continuous stirring, eliminating the DMPA remains. After filtering the solution, the precipitate was again solved in water and dialyzed (MWCO = 500 Da) in

18

order to eliminate the excess of thiol and disulfide. The pure product was dried under vacuum to afford 7 as a white solid (0.282 g, 60 %). The final low yield is consequence of the last purification step. 1

H-NMR (D2O): δ -0.03 (s, 9 H, SiMe), 0.54 (m, 2 H, O(CH2)3CH2Si), 0.63 (m, 8 H,

CH2Si), 0.85 (m, 8 H, SiCH2CH2S), 1.25 (m, 6 H, SiCH2CH2CH2Si), 1.66 (m, 2 H, OCH2CH2), 2.50 (m, 8 H, SiCH2CH2S), 2.74 (m, 8 H, SCH2CH2NH3), 3.07 (m, 8 H, CH2NH3), 3.80 (m, 2 H, OCH2), 6.63 and 6.71 (m, 2 H, C6H4). 13C{1H}-NMR (D2O): δ -2.7 and -1.7 (SiMe), 16.3 (O(CH2)3CH2Si), 16.9 (SiCH2CH2S), 20.9-21.2 (SiCH2CH2CH2Si), 23.1 (OCH2CH2CH2), 29.6 (SCH2), 30.1 (CH2S), 36.0 (OCH2CH2), 41.4 (CH2NH3), 70.7 (OCH2), 119.4 and 119.9 (C6H4O2, C-H), 152.5 and 155.2 (C6H4O2, C-O). 15N-NMR (D2O):  -343.1 (-NH3+). 29Si-NMR (D2O):  1.8 (G1–SiMe), 2.6 (G2–SiMe). Anal. Calcd. C35H78Cl4N4O2S4Si3 (941.35 g/mol): C, 44.66; H, 8.35; N, 5.95; S, 13.63; Exp.: C, 43.98; H, 8.73; N, 5.17; S, 12.87. HOG2(S-GU-Cl)4 (GU+ = NHC(NH2)NH2+) (9). This dendron was obtained by reaction of 7 (0.145 g, 0.15 mmol) and 1H-pyrazole-1-carboxamidine hydrochloride (0.135 g, 0.92 mmol) in the presence of DIPEA (0.332 mL, 1.91 mmol) in EtOH (10 mL) at 55 ºC during 16 h under inert atmosphere. Afterwards, volatiles were removed under vacuum and the residue was solved in the minimum amount of MeOH and precipitated with acetone, yielding 9 as a yellow solid (0.316 g, 70 %). Also, purification can be done by dialysis (MWCO = 500 Da) in water, obtaining a similar yield. 1

H-NMR (D2O): δ -0.05 (s, 9 H, SiMe), 0.52 (m, 2 H, O(CH2)3CH2Si), 0.63 (m, 8 H,

CH2Si), 0.80 (m, 8 H, SiCH2CH2S), 1.30 (m, 6 H, CH2CH2CH2Si), 1.66 (m, 2 H, OCH2CH2), 2.52 (m, 8 H, SiCH2CH2S), 2.65 (m, 8 H, SCH2CH2N), 3.22 (m, 8 H, CH2N), 3.78 (m, 2 H, OCH2), 6.62 and 6.71 (m, 2 H, C6H4). 13C{1H}-NMR (D2O): δ -

19

2.5

and

-1.7

(SiMe),

16.8

(O(CH2)3CH2Si),

17.0

(SiCH2CH2S),

20.5-21.5

(SiCH2CH2CH2Si), 21.7 (OCH2CH2CH2), 29.4 (SCH2), 29.6 (CH2S), 38.1 (OCH2CH2), 42.7 (CH2N), 70.7 (OCH2), 118.7 and 119.8 (C6H4O2, C-H), 152.5 and 155.1 (C6H4O2, C-O), 159.7 (NHC(N). ESI: q = 2 (482.2504 [M-2H-4Cl]2+), q = 3 (321.8359 [M-H4Cl]3+). Anal. Calcd. C39H86Cl4N12O2S4Si3 (1109.51 g/mol): C, 42.22; H, 7.81; N, 15.15; S, 11.56; Exp.: C, 41.50; H, 7.11; N, 15.04; S, 12.01. 5.3. Antibacterial methodology Bacterial strains. Escherichia coli (CECT 515, Gram-negative), Staphylococcus aureus (CECT 240, Gram-positive) and Staphylococcus aureus with resistances to penicillin (CECT 4004, Gram-positive) were obtained from the Spanish Type Culture Collection (CECT). The S. aureus MRSA was obtained from the Hospital Príncipe de Asturias (Alcalá de Henares, Madrid, Spain) and was resistant to penicillin, amoxicillin, ampicillin, clavulanic acid, oxacillin, ciprofloxacin, levofloxacin, moxifloxacin, erythromycin, clindamycin, and azithromycin. MIC and MBC. The minimal inhibitory concentration (MIC) of the products was measured in 96-well tray microplates by microdilution tray preparations following the international standard methods ISO 20776-1.[48] Assays were run in duplicate microplates and three different wells for each concentration analyzed in the microplate. Solutions of the products were prepared in the range of 0.25 to 1024 ppm adding in each well 100 µL of one of these solutions, 100 µL of double concentration Mueller Hinton (Scharlau, ref. 02-136) and 5 µL of a bacteria suspension of 2 x 107 CFU/mL. Microplates were incubated at 37 ºC for 19 h using an ultra microplate reader ELX808iu (Bio-Tek Instruments), considering the MIC the minimal concentration for which no turbidity was observed. The minimal bactericidal concentration (MBC) was calculated by inoculating Petri dishes containing Mueller-Hinton agar with 3 μl of the samples

20

used for MIC assessment. Samples were tested as droplets on the plates. Microbial growth on plates was monitored after 24 h of incubation at 37 ºC. The MBC was determined as the minimal concentration at which no growth was detected. 5.4. Acanthamoeba strain: trophozoites and cysts Acanthamoeba polyphaga 2961 (a clinical isolate kindly supplied by Dr. E. Hadas, Poznan University of Medical Sciences, Poland) was grown in 25 cm2 flasks containing 5 ml of peptone–yeast extract–glucose medium supplemented with 2 % bactocasitone (PYG-B) [49] and incubated at 32 ºC. Cysts were obtained by culturing 3-4 days trophozoites in non-nutrient Neff’s Encystment Medium (NEM: 0.1 M KCl, 8 mM MgSO4·7H2O, 0.4 mM CaCl2·2H2O, 1 mM NaHCO3, 20 mM ammediol [2-amino-2-methyl-1,3-propanediol; Sigma] pH 8.8 at 32 ºC) as described previously [25]. 5.5. In vitro Assays Chlorhexidine digluconate (Sigma-Aldrich Ltd.) was used as positive control against Acanthamoeba trophozoites and cysts in concentrations ranging from 1 to 40 mg/L. Experiments for drug testing on trophozoites were performed in sterile 48-well microtiter plates (NUNCTM) and 96-well microtiter plates for cysts. Amoebae from logphase cultures were resuspended in PYG-B medium at a density of 4 x 105 trophozoites/ml and cysts were resuspended in NEM at a density of 10 5 cysts/ml. 200 µL of the calibrated trophozoite suspension or 100 µL of the cyst suspension were added to each well. Control wells containing trophozoites or cysts received 200 µL or 100 µL of distilled water instead of drug solutions, respectively. Plates were sealed with Parafilm® and incubated at 32 ºC for 24 h. Assays were performed in triplicate and were repeated at least twice. 5.6. Study of trophocidal properties

21

The viability of trophozoites treated for 24 hours was assessed by direct counting using the 0.2% Congo red exclusion assay [25]. Samples were placed in a Fuchs– Rosenthal manual counting chamber and trophozoites counted using optical microscopy (Carl Zeiss). Two parameters were defined: 1) the Minimum Trophozoite Amoebicidal Concentration (MTAC) was defined as the lowest concentration of test solution that produced a complete destruction of trophozoites [50]; 2) the half maximal inhibitory concentration (IC50) values, which means decreasing in 50 % of the population of trophozoites. Both parameters were calculated for the different compounds tested and chlorhexidine digluconate by linear regression analysis. 5.7. Study of cysticidal properties Cyst viability was studied by assessing excystment of samples treated for 24 h. Wells were washed twice with Phosphate Buffered Saline 1x (PBS) to eliminate residual drugs and 250 µL of fresh PYG-B medium were added to each well. Plates were observed microscopically after 4, 7, 10, 15 and 21 days of incubation in order to get the Minimum Cysticidal Concentration (MCC), defined as the lowest concentration of test solution at which cyst excystment and trophozoite growth are completely inhibited [50]. 5.8. Cytotoxicity assays on HeLa and MU-PH1 cells The cytotoxicity of the concentrations combined was determined using HeLa cells. Experiments were performed in 24-well plates (Greiner Bio-One) in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (SigmaAldrich Ltd.) and 1% antibiotic mix: 10.000 U penicillin, 10 mg streptomycin and 25 µg amphotericin B per mL (Sigma-Aldrich Ltd.). Cells were seeded at a density of 5 x 104 cells/well in 500 µl medium.

22

After incubating the plates for 3 days at 37 ºC in a 5% CO 2 atmosphere to form a confluent cell monolayer, the medium was replaced by 500 µl of fresh medium plus 500 µl of drug combinations mentioned above. Control wells received 500 µl of distilled water instead of drugs. After 24 h of incubation, the culture medium was discarded, wells were washed three times with PBS in order to eliminate any residual drug and 500 µl of fresh medium were added to each well. Cytotoxicity on HeLa cells was evaluated using the Microculture Tetrazolium Assay (MTT, Sigma-Aldrich Ltd.). Each well received 50 µl of MTT (5 mg/ml) and the medium was discarded after 4 h of incubation at 37 ºC. Subsequently, 500 µl of dimethyl sulfoxide (DMSO) were added to dissolve formazan crystals and the absorbance of the samples recorded in a microplate absorbance reader at 570-630 nm (BioTek Instruments Inc. Model: ELX 800). Assays were performed in triplicate for each combination and repeated at least twice. Assays were performed in triplicate for each dendrimer and repeated at least twice, and the IC50 values were calculated for each treatment (dendrimers, dendrons and chlorhexidine digluconate) by linear regression analysis with all results. Cytotoxicity values lower that 10% were considered non-cytotoxic. Values between 10 and 25% were considered low and values from 25% to 40% were considered moderate levels. Values higher than 40% were considered high cytotoxicity levels [51]. 5.9. Statistical analysis The statistical analysis was carried out using STATISTICA. As previously described, each experimental condition was done in triplicate; therefore, results are given as means±SD of values obtained from two independent experiments. The significance of the differences was determined using t test independent by groups. The statistical significance was defined as p < 0.05. The IC50 values were calculated with the

23

percent cell survival for each concentration at the aforementioned times by linear regression analysis using GraphPad Prism 5® with a 95 % confidence limit. 5.10. Computational details 3D computer models of dendrimer structures were created using dendrimer builder, as implemented in the Materials Studio software package from BIOVIA (formerly Accelrys) – see Figure 2. Models of solvated (35 Å water layer at each side, at one side later shortened to 20 Å using Materials Studio) dipalmitoylphosphatidylglycerol (DPPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) bilayers (200 and 160 lipid molecules at each side (leaflet) in DPPG and POPG case, respectively) were created using CHARMM-GUI Membrane Builder [52]. The RESP technique [53] was used for calculation of dendrimer atoms partial charges. For this charge parametrization the R.E.D.-IV tools [54] was used. The necessary QM calculations (QM structure minimisations, molecular electrostatic potential (MEP) calculations) were done using GAMESS [55,56]. The default, HF/6-31G*, level of theory was used for all charge-related QM calculations and the MEP potential was fitted on Connolly molecular surface. GAFF (Generalized Amber Force Field) [57] and LIPID 17 [58] force field were used for parameterization of dendrimers and lipids, respectively. Missing “Si containing“ force field parameters were fitted by minimizing the differences between QM and force field based relative energies of different configurations of properly chosen molecular fragment (i.e. ff parameters that most accurately ensures the following requirement were used: E iforce-field = Eiquantum + K for all configurations i - where Eiforce-field and Eiquantum is force field and QM based energy of molecular configuration i, K is constant). QM energies were calculated at MP2/HF/631G** level of theory using GAMESS and fitting was accomplished using paramfit routine from AMBER16 software [59]. Slightly adjusted van der Waals parameters for

24

Si atoms from MM3 force field were used in this study [60]. Models of Dendrimer 1 and 11 were complexed with the solvated (TIP3P water model used) lipid bilayers (see Figure S8) [61]. Proper number of Na+ ions was added to ensure neutrality of the whole simulated system. Such setup (neutrality) is here unrealistic as the intention of this preliminary simulation work was to study interactions of selected dendrimers with negatively charged bilayers itself i.e. without the stabilizing effect of ions which are moreover in reality rather divalent (e.g. Ca2+, Mg2+). On the other hand, neutrality is here prerequisite for usage of PME method. Monovalent counterions with the actual Van der Waals (Lennard-Jones) parameters (Joung and Cheatham) have been found to condense models of anionic bilayers to areas per lipid well below experimental values due to strong interactions with the negatively charged lipid head groups. So, we used the older Amber ff99 sodium parameters in this work, as a greater Lennard-Jones radius used for this sodium ions model most likely prevents them from engaging in strong interactions within the lipid–water interface region – i.e. condensing effect resulting from the unrealistic conditions (many monovalent counterions) is by this trick to a large extent eliminated [62]. First, the systems were minimized (50000 steps with 10 kcal/(mol Å2) restraint applied to lipids and dendrimer + 50000 steps without restraint), heated (250 ps NVT, dt = 0.5 fs) to 100 K, heated (50 ps NPT, P = 0.1 MPa, dt = 0.5 fs) from 100 K to 310.15 K (with 10 kcal/(mol Å2), restraint applied to lipids and dendrimer during both heating phases and finally equilibrated using 665 ns (DPPG) and 200 ns (POPG) long molecular dynamics simulations (NPT, T = 310.15 K, dt = 2 fs, P = 0.1 MPa, during the first 7 ns with 2 kcal/(mol Å2) restraint applied just to dendrimer core unit). Hydrogens were constrained with the SHAKE algorithm (except to first heating phase) to allow 2 fs time step [63] and Langevin thermostat with collision frequency 1 ps-1 was used for all MD runs [64]. The pressure relaxation time for weak-

25

coupling barostat was 1 ps, anisotropic pressure scaling was used. Particle mesh Ewald method (PME) was used to treat long range electrostatic interactions under periodic conditions with a direct space cutoff of 10 Å. The same cutoff was used for van der Waals interactions. The pmemd.cuda module from AMBER16 package was used for all simulation steps [65]. The cpptraj module from Amber16 was used for dendrimer-lipids distance analysis (see Figures 4 and 5) [66]. UCSF Chimera software was used for all visualizations and for preparation of all PDB files containing the initial configurations of simulated systems [67]. Acknowledgments This work was supported by grants from CTQ2017-86224-P (MINECO), Consortium NANODENDMED II-CM ref B2017/BMD-3703 (CAM) and UAH-Project CCG2016/BIO-023. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. I. H.-B. wishes to thank MINECO for a fellowship (grants nº FPU AP2010-1471). This work was co-financed by the Czech Science Foundation (project No. 15-05903S) and the Research Infrastructure NanoEnviCz, supported by the Ministry of Education, Youth and Sports of the Czech Republic under project No. LM2015073. Dr. Pedro de la Villa of Dpt. Of Biología de Sistemas (UAH) is acknowledge for MU-PH1 cells supply.

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30

For Graphical Abstract:

AMMONIUM AND GUANIDINE CARBOSILANE DENDRIMERS AND DENDRONS AS MICROBICIDES Irene Heredero-Bermejo,a José M. Hernández-Ros,a Leticia Sánchez-García,b Marek Maly, c Cristina Verdú-Expósito,a Juan Soliveri,a F. Javier de la Mata,b,d José L. CopaPatiño,a Jorge Pérez-Serrano,a Javier Sánchez-Nieves,b,d,* Rafael Gómezb,d,*

a

Department of Biomedicina y Biotecnología. Facultad de Farmacia, Universidad de

Alcalá, E-28805 Alcalá de Henares, Madrid, Spain. b

Department of Química Orgánica y Química Inorgánica, Universidad de Alcalá

(IRYCIS), Campus Universitario; Instituto de Investigación Química "Andrés M. del Río" (IQAR), Universidad de Alcalá; E-28805 Alcalá de Henares (Madrid) Spain. c

Faculty of Science, J. E. Purkinje University, Ceske mladeze 8, 400 96 Usti nad

Labem, Czech Republic. d

Networking Research Centre for Bioengineering, Biomaterials and Nanomedicine

(CIBER-BBN), Madrid, Spain. * Corresponding author: J. Sánchez-Nieves, [email protected]; Rafael Gómez, [email protected].

31

The microbicide (antibacterial and antiamoebae) activity of carbosilane dendrimers and dendrons with ammonium and guanidine moieties has been evaluated.

INSERT GRAPHICAL ABSTRACT

32

For Supporting Information AMMONIUM AND GUANIDINE CARBOSILANE DENDRIMERS AND DENDRONS AS MICROBICIDES

Irene Heredero-Bermejo,a José M. Hernández-Ros,a Leticia Sánchez-García,b Marek Maly, c Cristina Verdú-Expósito,a Juan Soliveri,a F. Javier de la Mata,b,d José L. CopaPatiño,a Jorge Pérez-Serrano,a Javier Sánchez-Nieves,b,d,* Rafael Gómezb,d,*

a

Department of Biomedicina y Biotecnología. Facultad de Farmacia, Universidad de

Alcalá, 28871 Alcalá de Henares, Madrid, Spain. b

Department of Química Orgánica y Química Inorgánica, Universidad de Alcalá

(IRYCIS), Campus Universitario; Instituto de Investigación Química "Andrés M. del Río" (IQAR), Universidad de Alcalá; E-28805 Alcalá de Henares (Madrid) Spain. c

Faculty of Science, J. E. Purkinje University, Ceske mladeze 8, 400 96 Usti nad

Labem, Czech Republic. d

Networking Research Centre for Bioengineering, Biomaterials and Nanomedicine

(CIBER-BBN), Madrid, Spain. * Corresponding author: J. Sánchez-Nieves, [email protected]; Rafael Gómez, [email protected].

33

S.1 Experimental S.1.1 General Considerations. All reactions were carried out under inert atmosphere. Reagents and solvents were used as received without further purification. NMR spectra were recorded on a Varian Unity VXR-300 (300.13 (1H), 75.47 (13C) MHz) or on a Bruker AV400 (400.13 (1H), 100.60 (13C), 40.56 (15N), 79.49 (29Si) MHz). Chemical shifts (δ) are given in ppm. 1H and

13

C resonances were measured

relative to internal deuterated solvent peaks considering TMS = 0 ppm, whereas 15N and 29

Si resonances were measured relative to external MeNO and TMS, respectively. When

necessary, assignment of resonances was done from HSQC, HMBC, COSY, TOCSY and NOESY NMR experiments. Elemental analyses were performed on a LECO CHNS-932. Mass Spectra were obtained from a Thermo Scientific TSQ Quantum LCMS and an Agilent 6210. Thiol-ene reactions were carried out employing a HPK 125 W mercury lamp from Heraeus Noblelight with maximum energy at 365 nm, in normal glassware

under

inert

atmosphere.

Compounds

cysteamine

hydrochloride

(HS(CH2)2NH2·HCl), 2,2’-dimethoxy-2-phenylacetophenone (DMPA), 1H-pyrazole-1carboxamidine hydrochloride, diisopropylethylamine (DIPEA), were obtained from commercial sources. Compounds [GnO3(S-NH3)m]m+ (1-3),[1] [G1O3(S-GU)6]6+ (4)[2] and HOGnVm[3] were synthesized as published. S.1.2. Synthesis of compounds G1O3(S-GU-Cl)6 (GU+ = NHC(NH2)NH2+) (4). A suspension of 1 (0.342 g, 0.270 mmol), 1Hpyrazole-1-carboxamidine hydrochloride (0.359 g, 2.450 mmol) and DIPEA (6.57 mmol) in EtOH was heated overnight at 55ºC in a teflon valved ampoule, under inert atmosphere. Next, volatiles are removed under vacuum. Afterward, the solution was washed with MeOH/acetone twice, rendering 4 as a pale yellow solid very hygroscopic (0.325, 83 %). Also, the wax can be solved in water, dialyzed (MWCO = 500), and finally lyophilized to give 4 with similar yield.

Data for 4: 1H-NMR (D2O): .δ -0.03 (s, 9 H, SiMe), 0.48 (m, 6 H, O(CH2)3CH2Si), 0.84 (m, 12 H, SiCH2CH2S), 1.25 (m, 6 H, O(CH2)2CH2), 1.31 (m, 6 H, OCH2CH2), 34

2.56 (m, 12 H, CH2S), 2.72 (m, 12 H, SCH2), 3.37 (m, 12 H, CH2NH), 3.74 (m, 12 H, OCH2), 5.93 (m, 3 H, C6H3);

13

C-NMR (D2O): .δ -2.4 (SiMe), 10.8 (O(CH2)3CH2Si),

17.2 (SiCH2CH2S), 18.3 (O(CH2)2CH2), 24.2 (CH2S), 27.3 (SCH2), 29.8 (OCH2CH2), 39.1 (CH2NH), 93.4 (CH, C6H3), 156.7 (NCN), 160.7 (Cipso, C6H3). 15N-NMR (D2O): .δ -303.5. Elemental analysis (C51H114Cl6N18O3S6Si3 , FW 1516,93): Teor (%): C, 40.38; H, 7.57; N, 16.62; S, 12.68. Obt (%):, 41.02; H, 7.11; N, 16.20; S, 12.29. ESI-MS (uma): [M-4H-6Cl]2+ = 649.33; [M-3H-6Cl]3+ = 433,22; [M-2H-6Cl]4+ = 325.17. G2O3(S-GU-Cl)12 (GU+ = NHC(NH2)NH2+) (5). Following the procedure described for first generation derivative, from 2 (0.250 g, 0.095 mmol), 1H-pyrazole-1carboxamidine hydrochloride (0.252 g, 1.72 mmol) and DIPEA (3.80 mmol), compound 5 was obtained as a pale yellow solid very hygroscopic (0.257, 87 %). Data for 5: 1H-NMR (D2O): .δ -0.11 (s, 9 H, SiMe), 0.02 (s, 18 H, SiMe), 0.53 (m, 30 H, CH2Si), 0.88 (m, 24 H, SiCH2CH2S), 1.30 (m, 24 H, CH2CH2CH2), 1.57 (m, 6 H, OCH2CH2), 2.60 (m, 24 H, CH2S), 2.73 (m, 24 H, SCH2), 3.37 (m, 24 H, CH2NH), 3.66 (m, 6 H, OCH2), 5.83 (m, 3 H, C6H3);

13

C-NMR (D2O): .δ -4.9 (SiMe), -4.4 (SiMe),

14.5 (CH2), 117.6 (CH2), 27.2 (SCH2), 30.3 (CH2), 41.2 (CH2NH), 67.1 (OCH2), 93.9 (CH, C6H3), 156.7 (NCN), C nuclei of O(CH2)4 chain and Cipso were not oserved; 15NNMR (D2O): .δ -303.5;

29

Si-NMR (D2O): δ 1.7, 2.2. Elemental analysis

(C105H246Cl12N36O3S12Si9, FW 3124,30): Teor (%): C, 40.36; H, 7.94; N, 16.14; S, 12.32. Obt (%):, 39.89; H, 7.31; N, 16.20; S, 12.67. ESI-MS (uma): [M-8 H-12 Cl]4+ = 672.86; [M-7 H-12 Cl]5+ = 538.29; [M-6 H-12 Cl]6+ = 448.72. G3O3(S-GU-Cl)24 (GU+ = NHC(NH2)NH2+) (6). Following the procedure described for first generation derivative, from 3 (0.200 g, 0.038 mmol), 1H-pyrazole-1carboxamidine hydrochloride (0.211 g, 1.44 mmol) and DIPEA (1.92 mmol), compound 6 was obtained as a pale yellow solid very hygroscopic (0.209, 88 %).

35

Data for 5: 1H-NMR (D2O): δ -0.08 (s, 27 H, SiMe), 0.05 (s, 36 H, SiMe), 0.53 (m, 72 H, CH2Si), 0.89 (m, 48 H, SiCH2CH2S), 1.32 (m, 48 H, CH2CH2CH2), 1.57 (m, 6 H, OCH2CH2), 2.63 (m, 48 H, CH2S), 2.75 (m, 48 H, SCH2), 3.40 (m, 48 H, CH2NH), 3.66 (m, 6 H, OCH2), 5.83 (m, 3 H, C6H3);

13

C-NMR (D2O): .δ -4.9 (SiMe), -4.5 (SiMe),

14.1 (O(CH2)3CH2Si), 14.6 (CH2), 18.8 (CH), 20.4 (O(CH2)2CH2), 27.4 (SCH2), 30.4 (CH2), 33.4 (OCH2CH2), 41.3 (CH2NH), 67.1 (OCH2), 93.9 (CH, C6H3), 156.8 (NCN), 160.6 (Cipso, C6H3); 15N-NMR (D2O): .δ -303.3; 29Si-NMR (D2O): δ 1.2, 2.2. Elemental analysis (C213H512Cl24N72O3S24Si21, FW 6341.05): Teor (%): C, 40.34; H, 8.14; N, 15.90; S, 12.14. Obt (%): C, 39.39; H, 7.90; N, 15.98; S, 12.54. HOG2(S-NH3Cl)4 (7). This dendron was prepared from reaction of HOG2V4 (0.480 g, 1.05 mmol) and HS(CH2)2NH2·HCl (0.470 g, 4.20 mmol) in the presence of 2,2dimethoxy-2-phenylacetophenone (DMPA, 0.10 mmol, 2.5 % mol per vinyl group) in a THF/methanol solution (1:2 ratio, 10 mL). The reaction mixture was deoxygenated using an argon flow and then irradiated for 2 h with UV light at 365 nm. Next, DMPA was again added (2.5 % mol per vinyl group) and the reaction mixture irradiated for another 3 h. 1H-NMR monitoring allowed checking the disappearance of vinyl groups. Afterwards, the initial reaction mixture was concentrated by rotator evaporation and solved in the minimum amount of MeOH. Subsequently, the product was precipitated with Et2O under continuous stirring, eliminating the DMPA remains. After filtering the solution, the precipitate was again solved in water and dialyzed (MWCO = 500 Da) in order to eliminate the excess of disulfide. The pure product was dried under vacuum to afford 7 as a white solid (0.564 g, 60 %). The low yield is consequence of the last purification step. 1

H-NMR (D2O): δ -0.03 (s, 9 H, SiMe), 0.54 (m, 2 H, O(CH2)3CH2Si), 0.63 (m, 8 H,

CH2Si), 0.85 (m, 8 H, SiCH2CH2S), 1.25 (m, 6 H, SiCH2CH2CH2Si), 1.66 (m, 2 H,

36

OCH2CH2), 2.50 (m, 8 H, SiCH2CH2S), 2.74 (m, 8 H, SCH2CH2NH3), 3.07 (m, 8 H, CH2NH3), 3.80 (m, 2 H, OCH2), 6.63 and 6.71 (m, 2 H, C6H4). 13C{1H}-NMR (D2O): δ -2.7 and -1.7 (SiMe), 16.3 (O(CH2)3CH2Si), 16.9 (SiCH2CH2S), 20.9-21.2 (SiCH2CH2CH2Si), 23.1 (OCH2CH2CH2), 29.6 (SCH2), 30.1 (CH2S), 36.0 (OCH2CH2), 41.4 (CH2NH3), 70.7 (OCH2), 119.4 and 119.9 (C6H4O2, C-H), 152.5 and 155.2 (C6H4O2, C-O). 15N-NMR (DMSO-d6):  -329.9 (-NMe3+). 29Si-NMR (DMSO-d6):  1.9 (G1–SiMe), 2.6 (G2–SiMe). ESI: q=1 (1347.31 [M-I]+), q=2 (610.22 [M-2I]2+), q=3 (364.51 [M-3I]3+), q=4 (241.66 [M-4I]4+). Anal. Calcd. C35H78Cl4N4O2S4Si3 (941.35 g/mol): C, 44.66; H, 8.35; N, 5.95; S, 13.63; Exp.: C, 43.98; H, 8.73; N, 5.17; S, 12.87. HOG3(S-NH3Cl)8 (8). This dendron was prepared from reaction of HOG3V8 (0.524 g, 0.56 mmol) and HS(CH2)2NH2·HCl (0.480 g, 4.50 mmol) in the presence of 2,2dimethoxy-2-phenylacetophenone (DMPA, 2 x 2.5 % mol per vinyl group) following the procedure described for 7. Compound 8 was obtained as a white solid (0.83 g, 80 %). 1

H-NMR (D2O): δ -0.11 (s, 12 H, SiMe), -0.07 (s, 9 H, SiMe), 0.52 (m, 18 H,

CH2Si), 0.85 (m, 16 H, SiCH2CH2S), 1.28 (m, 14 H, SiCH2CH2CH2Si), 1.68 (m, 2 H, OCH2CH2), 2.57 (m, 16 H, SiCH2CH2S), 2.79 (m, 16 H, SCH2CH2NH3), 3.12 (m, 16 H, CH2NH3), 3.80 (m, 2 H, OCH2), 6.67 and 6.74 (m, 2 H, C6H4). 13C{1H}-NMR (D2O): δ -2.5 and -1.6 (SiMe), 16.3 (O(CH2)3CH2Si), 17.0 (SiCH2CH2S), 20.9-23.0 (CH2CH2CH2Si), 29.6 (SCH2), 30.9 (CH2S), 37.0 (OCH2CH2), 41.4 (CH2NH3), 70.7 (OCH2), 11.8 and 119.6 (C6H4O2, C-H), 152.5 and 155.2 (C6H4O2, C-O). (D2O):  -342.9 (-NH3+).

29

15

N-NMR

Si-NMR (D2O):  1.3 (G2–SiMe), 2.3 (G3–SiMe). Anal.

Calcd. C67H158Cl8N8O2S8Si7 (1844.77 g/mol): C, 43.62; H, 8.63; N, 6.07; S, 13.91; Exp.: C, 44.15; H, 8.27; N, 5.69; S, 14.17.

37

HOG2(S-GU-Cl)4 (GU+ = NHC(NH2)NH2+) (9). This dendron was obtained by reaction of 7 (0.145 g, 0.15 mmol) and 1H-pyrazole-1-carboxamidine hydrochloride (0.135 g, 0.92 mmol) in the presence of DIPEA (0.33 mL, 1.91 mmol) in EtOH (10 mL) at 55 ºC during 16 h under inert atmosphere. Afterwards, volatiles were removed under vacuum and the residue was solved in the minimum amount of MeOH and precipitated with acetone, yielding 9 as a yellow solid (0.316 g, 70 %). Also, purification can be done by dialysis (MWCO = 500 Da) in water, obtaining a similar yield. 1

H-NMR (D2O): δ -0.05 (s, 9 H, SiMe), 0.52 (m, 2 H, O(CH2)3CH2Si), 0.63 (m, 8 H,

CH2Si), 0.80 (m, 8 H, SiCH2CH2S), 1.30 (m, 6 H, CH2CH2CH2Si), 1.66 (m, 2 H, OCH2CH2), 2.52 (m, 8 H, SiCH2CH2S), 2.65 (m, 8 H, SCH2CH2N), 3.22 (m, 8 H, CH2N), 3.78 (m, 2 H, OCH2), 6.62 and 6.71 (m, 2 H, C6H4). 13C{1H}-NMR (D2O): δ 2.5

and

-1.7

(SiMe),

16.8 (O(CH2)3CH2Si),

17.0

(SiCH2CH2S),

20.5-21.5

(SiCH2CH2CH2Si), 21.7 (OCH2CH2CH2), 29.4 (SCH2), 29.6 (CH2S), 38.1 (OCH2CH2), 42.7 (CH2N), 70.7 (OCH2), 118.7 and 119.8 (C6H4O2, C-H), 152.5 and 155.1 (C6H4O2, C-O), 159.7 (NHC(N)N).

15

N-NMR (DMSO-d6):  -330.0 (-NMe3+).

29

Si-NMR

(DMSO-d6):  2.0 (G1–SiMe), 1.1 (G2–SiMe), 2.5 (G3–SiMe). ESI: q = 2 (482.2504 [M2H-4Cl]2+), q = 3 (321.8359 [M-H-4Cl]3+). Anal. Calcd. C39H86Cl4N12O2S4Si3 (1109.51 g/mol): C, 42.22; H, 7.81; N, 15.15; S, 11.56; Exp.: C, 41.50; H, 7.11; N, 15.04; S, 12.01. HOG3(S-GU-Cl)8 (GU+ = NHC(NH2)NH2+) (10). This dendron was obtained by reaction of 8 (0.171 g, 0.093 mmol) and 1H-pyrazole-1-carboxamidine hydrochloride (0.163 g, 1.11 mmol) in the presence of DIPEA (0.40 mL, 2.24 mmol) in EtOH (10 mL) following the procedure described for 4. Compound 10 was obtained as a yellow solid (0.156 g, 83 %).

38

1

H-NMR (D2O): δ -0.10 (s, 12 H, SiMe), -0.05 (s, 9 H, SiMe), 0.53 (m, 18 H,

CH2Si), 0.86 (m, 16 H, SiCH2CH2S), 1.27 (m, 14 H, SiCH2CH2CH2Si), 1.70 (m, 2 H, OCH2CH2), 2.57 (m, 16 H, SiCH2CH2S), 2.65 (m, 8 H, SCH2CH2N), 3.33 (m, 8 H, CH2N), 3.81 (m, 2 H, OCH2), 6.64 and 6.73 (m, 2 H, C6H4). 13C{1H}-NMR (D2O): δ 2.4 and -1.7 (SiMe), 17.1 (SiCH2CH2S), 20.5-21.5 (SiCH2CH2CH2Si), 21.7 (OCH2CH2CH2), 29.9 (SCH2), 32.9 (CH2S), 38.1 (OCH2CH2), 43.7 (CH2N), 70.7 (OCH2), 118.7 and 119.8 (C6H4O2, C-H), 152.5 and 155.1 (C6H4O2, C-O), 159.4 (NHC(N)N).

15

N-NMR (D2O):  -303.2 (-CH2N).

29

Si-NMR (D2O):  1.1 (G2–SiMe),

2.2 (G3–SiMe). ESI-MS: [M-5 H-8 Cl]3+ = 630.66, [M-4 H-8 Cl]4+ = 473.25. Anal. Calcd. C75H174Cl8N24O2S8Si7 (2181.09 g/mol): C, 41.30; H, 8.04; N, 15.41; S, 11.76; Exp.: C, 40.76; H, 8.11; N, 15.04; S, 11.21. S.1.3. Antibacterial methodology Bacterial strains. Escherichia coli (CECT 515, Gram-negative), Staphylococcus aureus (CECT 240, Gram-positive) and Staphylococcus aureus with resistances to penicillin (CECT 4004, Gram-positive) were obtained from the Spanish Type Culture Collection (CECT). The S. aureus MRSA was obtained from the Hospital Principe de Asturias (Alcalá de Henares, Madrid, Spain) and was resistant to penicillin, amoxicillin, ampicillin, clavulanic acid, oxacillin, ciprofloxacin, levofloxacin, moxifloxacin, erythromycin, clindamycin, and azitromycin. MIC and MBC. The minimal inhibitory concentration (MIC) of the products was measured in 96-well tray microplates by microdilution tray preparations following the international standard methods ISO 20776-1.[4] Assays were run in duplicate microplates and three different wells for each concentration analyzed in the microplate. Solutions of the products were prepared in the range of 0.25 to 1024 ppm adding in each well 100 µL of one of these solutions, 100 µL of double concentration Mueller Hinton

39

(Scharlau, ref. 02-136) and 5 µL of a bacteria suspension of 2 x 107 CFU/mL. Microplates were incubated at 37 ºC for 19 h using an ultra microplate reader ELX808iu (Bio-Tek Instruments), considering the MIC the minimal concentration for which no turbidity was observed. The minimal bactericidal concentration (MBC) was calculated by inoculating Petri dishes containing Mueller-Hinton agar with 3 μl of the samples used for MIC assessment. Samples were tested as droplets on the plates. Microbial growth on plates was monitored after 24 h of incubation at 37 ºC. The MBC was determined as the minimal concentration at which no growth was detected. S.1.4. Acanthamoeba strain: trophozoites and cysts. Acanthamoeba polyphaga 2961 (a clinical isolate kindly supplied by Dr. E. Hadas, Poznan University of Medical Sciences, Poland) was grown in 25 cm2 flasks containing 5 ml of peptone–yeast extract–glucose medium supplemented with 2 % Bactocasitone (PYG-B),[5] and incubated at 32 ºC. Cysts were obtained by culturing 3-4 days trophozoites in non-nutrient Neff’s Encystment Medium (NEM: 0.1 M KCl, 8 mM MgSO4·7H2O, 0.4 mM CaCl2·2H2O, 1 mM NaHCO3, 20 mM ammediol [2-amino-2-methyl-1,3-propanediol; Sigma] pH 8.8 at 32 ºC) as described previously.[6] S.1.5. In vitro Assays. Chlorhexidine digluconate (Sigma-Aldrich Ltd.) was used as positive control against Acanthamoeba trophozoites and cysts in concentrations ranging from 1 to 40 mg/L. Experiments for drug testing on trophozoites were performed in sterile 48-well microtiter plates (NUNCTM) and 96-well microtiter plates for cysts. Amoebae from logphase cultures were resuspended in PYG-B medium at a density of 4 x 105 trophozoites/ml and cysts were resuspended in NEM at a density of 10 5 cysts/ml. 200 µL of the calibrated trophozoite suspension or 100 µL of the cyst suspension were

40

added to each well. Control wells containing trophozoites or cysts received 200 µL or 100 µL of distilled water instead of drug solutions, respectively. Plates were sealed with Parafilm® and incubated at 32 ºC for 24 h. Assays were performed in triplicate and were repeated at least twice. S.1.6. Study of trophocidal properties. The viability of trophozoites treated for 24 hours was assessed by direct counting using the 0.2% Congo red exclusion assay.[6] Samples were placed in a Fuchs–Rosenthal manual counting chamber and trophozoites counted using optical microscopy (Carl Zeiss). Two parameters were defined: 1) the Minimum Trophozoite Amoebicidal Concentration (MTAC) was defined as the lowest concentration of test solution that produced a complete destruction of trophozoites;[7] 2) the half maximal inhibitory concentration (IC50) values, which means decreasing in 50 % of the population of trophozoites. Both parameters were calculated for the different compounds tested and chlorhexidine digluconate by linear regression analysis. S.1.7. Study of cysticidal properties. Cyst viability was studied by assessing excystment of samples treated for 24 h. Wells were washed twice with Phosphate Buffered Saline 1x (PBS) to eliminate residual drugs and 250 µL of fresh PYG-B medium were added to each well. Plates were observed microscopically after 4, 7, 10, 15 and 21 days of incubation in order to get the Minimum Cysticidal Concentration (MCC), defined as the lowest concentration of test solution at which cyst excystment and trophozoite growth are completely inhibited. [7] S.1.8. Cytotoxicity assays on HeLa and MU-PH1 cells. The cytotoxicity of the concentrations combined was determined using HeLa cells. Experiments were performed in 24-well plates (Greiner Bio-One) in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (Sigma-Aldrich Ltd.) and 1% antibiotic mix: 10,000 U penicillin, 10 mg streptomycin and 25 µg amphotericin B per

41

mL (Sigma-Aldrich Ltd.). Cells were seeded at a density of 5 x 10 4 cells/well in 500 µl medium. After incubating the plates for 3 days at 37 ºC in a 5% CO 2 atmosphere to form a confluent cell monolayer, the medium was replaced by 500 µl of fresh medium plus 500 µl of drug combinations mentioned above. Control wells received 500 µl of distilled water instead of drugs. After 24 h of incubation, the culture medium was discarded, wells were washed three times with PBS in order to eliminate any residual drug and 500 µl of fresh medium were added to each well. Cytotoxicity on HeLa cells was evaluated using the Microculture Tetrazolium Assay (MTT, Sigma-Aldrich Ltd.). Each well received 50 µl of MTT (5 mg/ml) and the medium was discarded after 4 h of incubation at 37 ºC. Subsequently, 500 µl of dimethylsulfoxide (DMSO) were added to dissolve formazan crystals and the absorbance of the samples recorded in a microplate absorbance reader at 570-630 nm (BioTek Instruments Inc. Model: ELX 800). Assays were performed in triplicate for each combination and repeated at least twice. Assays were performed in triplicate for each dendrimer and repeated at least twice, and the IC50 values were calculated for each treatment (dendrimers, dendrons and chlorhexidine digluconate) by linear regression analysis with all results. Cytotoxicity values lower that 10% were considered non cytotoxic. Values between 10 and 25% were considered low and values from 25% to 40% were considered moderate levels. Values higher than 40% were considered high cytotoxicity levels.[8] S.1.9. Statistical analysis. The statistical analysis was carried out using STATISTICA. As previously described, each experimental condition was done in triplicate; therefore, results are given as means±SD of values obtained from two independent experiments. The significance of the differences was determined using t test independent by groups. The statistical significance was defined as p<0.05. The IC50

42

values were calculated with the percent cell survival for each concentration at the aforementioned times by linear regression analysis using GraphPad Prism 5® with a 95 % confidence limit. S.1.10. Computational details 3D computer models of dendrimer structures were created using dendrimer builder, as implemented in the Materials Studio software package from BIOVIA (formerly Accelrys) – see Figure 2. Models of solvated (35 Å water layer at each side, at one side later shortened to 20 Å using Materials Studio) dipalmitoylphosphatidylglycerol (DPPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) bilayers (200 and 160 lipid molecules at each side (leaflet) in DPPG and POPG case, respectively) were created using CHARMM-GUI Membrane Builder [9]. The RESP technique [10] was used for calculation of dendrimer atoms partial charges. For this charge parametrization the R.E.D.-IV tools [11] was used. The necessary QM calculations (QM structure minimisations, molecular electrostatic potential (MEP) calculations) were done using GAMESS [12,13]. The default, HF/6-31G*, level of theory was used for all charge-related QM calculations and the MEP potential was fitted on Connolly molecular surface. GAFF (Generalized Amber Force Field) [14] and LIPID 17 [15] force field were used for parameterization of dendrimers and lipids, respectively. Missing “Si containing“ force field parameters were fitted by minimizing the differences between QM and force field based relative energies of different configurations of properly chosen molecular fragment (i.e. ff parameters that most accurately ensures the following requirement were used: E iforce-field = Eiquantum + K for all configurations i - where Eiforce-field and Eiquantum is force field and QM based energy of molecular configuration i, K is constant). QM energies were calculated at MP2/HF/631G** level of theory using GAMESS and fitting was accomplished using paramfit

43

routine from AMBER16 software [16]. Slightly adjusted van der Waals parameters for Si atoms from MM3 force field were used in this study [17]. Models of Dendrimer 1 and 11 were complexed with the solvated (TIP3P water model used) lipid bilayers (see Figure S8) [18]. Proper number of Na+ ions was added to ensure neutrality of the whole simulated system. Such setup (neutrality) is here unrealistic as the intention of this preliminary simulation work was to study interactions of selected dendrimers with negatively charged bilayers itself i.e. without the stabilizing effect of ions which are moreover in reality rather divalent (e.g. Ca2+, Mg2+). On the other hand neutrality is here prerequisite for usage of PME method. Monovalent counterions with the actual Van der Waals (Lennard-Jones) parameters (Joung and Cheatham) have been found to condense models of anionic bilayers to areas per lipid well below experimental values due to strong interactions with the negatively charged lipid head groups. So we used the older Amber ff99 sodium parameters in this work, as a greater Lennard-Jones radius used for this sodium ions model most likely prevents them from engaging in strong interactions within the lipid–water interface region – i.e. condensing effect resulting from the unrealistic conditions (many monovalent counterions) is by this trick to a large extent eliminated [19]. First the systems were minimized (50000 steps with 10 kcal/(mol Å2) restraint applied to lipids and dendrimer + 50000 steps without restraint), heated (250 ps NVT, dt = 0.5 fs) to 100 K, heated (50 ps NPT, P = 0.1 MPa, dt = 0.5 fs) from 100 K to 310.15 K (with 10 kcal/(mol Å2) restraint applied to lipids and dendrimer during both heating phases and finally equilibrated using 665 ns (DPPG) and 200 ns (POPG) long molecular dynamics simulations (NPT, T = 310.15 K, dt = 2 fs, P = 0.1 MPa, during the first 7 ns with 2 kcal/(mol Å2) restraint applied just to dendrimer core unit). Hydrogens were constrained with the SHAKE algorithm (except to first heating phase) to allow 2 fs time step [20] and Langevin thermostat with collision frequency 1

44

ps-1 was used for all MD runs [21]. The pressure relaxation time for weak-coupling barostat was 1 ps, anisotropic pressure scaling was used. Particle mesh Ewald method (PME) was used to treat long range electrostatic interactions under periodic conditions with a direct space cutoff of 10 Å. The same cutoff was used for van der Waals interactions. The pmemd.cuda module from AMBER16 package was used for all simulation steps [22]. The cpptraj module from Amber16 was used for dendrimer-lipids distance analysis (see Figures 4 and 5) [23]. UCSF Chimera software was used for all visualizations and for preparation of all PDB files containing the initial configurations of simulated systems [24]. S.2 References [1]

[2]

[3]

[4] [5]

[6]

[7] [8]

[9]

E. Fuentes-Paniagua, J. M. Hernández-Ros, M. Sánchez-Milla, M. A. Camero, M. Maly, J. Pérez-Serrano, J. L. Copa-Patiño, J. Sánchez-Nieves, J. Soliveri, R. Gómez and F. J. de la Mata. Carbosilane cationic dendrimers synthesized by thiol–ene click chemistry and their use as antibacterial agents. RSC Adv., 4, (2014), 1256–1265. I. Heredero-Bermejo, J. Sánchez-Nieves, J. L. Copa-Patiño, J. Soliveri, R. Gómez, F. J. de la Mata and J. Pérez-Serrano. In vitro anti-Acanthamoeba polyphaga synergistic effect of chlorhexidine and cationic carbosilane dendrimers against both trophozoites and cysts forms. Int. J. Pharm., 509, (2016), 1-7. E. Fuentes-Paniagua, C. E. Pe a-Gon e , M. Ga n, R. G me , F. . de a Mata and . nc e -Nieves. Thiol-Ene Synthesis of Cationic Carbosilane Dendrons: a New Family of Synthons. Organometallics, 32, (2013), 1789−1796. Reference Methods for the testing the “in vitro” activity of antimicrobial agents against bacteria involved in infectious diseases. ISO 20776-1, (2006). I. Heredero-Bermejo, C. San Juan Martín, J. Soliveri, J. L. Copa-Patiño and J. PérezSerrano. Acanthamoeba castellanii: in vitro UAH-T17c3 trophozoite growth study in different culture media. Parasitol. Res., 110, (2012), 2563–2567. I. Heredero-Bermejo, J. L. Copa-Patiño, J. Soliveri, E. Fuentes-Paniagua, F. J. de la Mata, R. Gómez and J. Pérez-Serrano. Evaluation of the activity of carbosilane dendrimers on trophozoites and cysts of Acanthamoeba polyphaga. Parasitol. Res., 114, (2015), 473486. M. J. Elder, S. Kilvington and J. K. Dart. A clinicopathologic study of in vitro sensitivity testing and Acanthamoeba keratitis. Inv. Ophthalmol. Vis. Sci., 35, (1994), 1059-1064. J. Lorenzo-Morales, C. M. Martin-Navarro, A. Lopez-Arencibia, M. A. Santana-Morales, R. N. Afonso-Lehmann, S. K. Maciver, B. Valladares and E. Martinez-Carretero. Therapeutic potential of a combination of two gene-specific small interfering RNAs against clinical strains of Acanthamoeba. Antimicrob. Agents Chemother., 54, (2010), 5151–5155. E. L. Wu, X. Cheng, S. Jo, H. Rui, K. C. Song, E. M. Dávila-Contreras, Y. Qi, J. Lee, V. Monje-Galván, R. M. Venable, J. B. Klauda and W. Im. CHARMM-GUI Membrane Builder Toward Realistic Biological Membrane Simulations. J. Comput. Chem., 35, (2014), 1997-2004. 45

[10]

[11]

[12]

[13]

[14] [15]

[16]

[17] [18] [19]

[20]

[21] [22]

[23]

[24]

C. I. Bayly, P. Cieplak, W. Cornell and P. A. Kollman. A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. J. Phys. Chem., 97, (1993), 10269-10280. F.-Y. Dupradeau, A. Pigache, T. Zaffran, C. Savineau, R. Lelong, N. Grivel, D. Lelong, W. Rosanski and P. Cieplak. The R.E.D. Tools: Advances in RESP and ESP charge derivation and force field library building. Phys. Chem. Chem. Phys., 12, (2010), 7821-7839. M. W. Schmidt, K. K. Baldridge, J. A. Boatz, S. T. Elbert, M. S. Gordon, J. H. Jensen, S. Koseki, N. Matsunaga, K. A. Nguyen, S. Su, T. L. Windus, M. Dupuis and J. A. Montgomery. General Atomic and Molecular Electronic Structure System. J. Comput. Chem., 14, (1993), 1347-1363. M. S. Gordon and M. W. Schmidt. Advances in electronic structure theory: GAMESS a decade later. Advances in electronic structure theory: GAMESS a decade later in Theory and Applications of Computational Chemistry: the first forty years, (2005), 1167-1189. J. Wang, R. M. Wolf, J. W. Caldwell, P. A. Kollamn and D. A. Case. Development and testing of a general Amber force field J. Comput. Chem., 25, (2004), 1157–1174. I. R. Gould, A. A. Skjevik, C. J. Dickson, B. D. Madej and R. C. Walker. Lipid17: A Comprehensive AMBER Force Field for the Simulation of Zwitterionic and Anionic Lipids. In preparation, (2018). D. A. Case, D.S.Cerutti, T. E. C. III, T. A. Darden, R. E. Duke, T. J. Giese, H. Gohlke, A. W. Goetz, D. Greene, N. Homeyer, S. Izadi, A. Kovalenko, T. S. Lee, S. LeGrand, P. Li, C. Lin, J. Liu, T. Luchko, R. Luo, D. Mermelstein, K. M. Merz, G. Monard, H. Nguyen, I. Omelyan, A. Onufriev, F. Pan, R. Qi, D. R. Roe, A. Roitberg, C. Sagui, C. L. Simmerling, W. M. Botello-Smith, J. Swails, R. C. Walker, J. Wang, R. M. Wolf, X. Wu, L. Xiao, D. M. York and P. A. Kollman. AMBER 2017. (2017), University of California, San Francisco. J.-H. Lii, A. Norman and L. Allinger. The MM3 force field for amides, polypeptides and proteins. J. Comput. Chem., 12, (1991), 186-199. W. L. Jorgensen, J. Chandrasekhar, J. Madura and M. L. Klein. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys., 79, (1983), 926–935. B. D. M. Å.A. Skjevik, C.J. Dickson, C. Lin, K. Teigen, R.C. Walker, I.R. Gould. Simulation of lipid bilayer self-assembly using all-atom lipid force fields. Phys. Chem. Chem. Phys., 18, (2016), 10573–10584. J.-P. Ryckaert, G. Ciccotti and H. J. C. Berendsen. Numerical integration of the cartesian equations of motion of a system with constraints: Molecular dynamics of n-alkanes. J. Comput. Phys., 23, (1977), 327-341. X. Wu and B. R. Brooks. Self-guided Langevin dynamics simulation method. Chem. Phys. Lett., 381, (2003), 512-518. A. W. Götz, M. J. Williamson, D. Xu, D. Poole, S. L. Grand and R. C. Walker. Routine Microsecond Molecular Dynamics Simulations with AMBER on GPUs. 1. Generalized Born. J. Chem. Theory Comput., 8, (2012), 1542-1555. D. R. Roe and T. E. Cheatham. PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data. J. Chem. Theory Comput., 9, (2013), 3084-3095. E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch, D. M. Greenblatt, E. C. Meng and T. E. Ferrin. UCSF Chimera: a visualization system for exploratory research and analysis. J. Comput. Chem., 25, (2004), 1605-1612.

46

S.3 Schemes and Figures

Figure S1. Drawing of cationic guanidine carbosilane dendrimers GnO3(S-GU+)m (n = 1, m = 6 (4); n = 2, m = 12 (5); n = 3, m = 24 (6)). Chloride anions are omitted for clarity.

47

Figure S2. Drawing of ammonium and guanidine carbosilane dendrons HOGn(SNH3+)m (n = 2, m = 4 (7); n = 3, m = 8 (8)) and HOGn(S-GU+)m (n = 2, m = 4 (9); n = 3, m = 8 (10)). Chloride anions are omitted for clarity.

48

Figure S3. 1H-13C HSQC NMR spectrum of HOG2(S-NH3+)4 (7) in D2O.

Figure S4. 13C-NMR spectrum of of HOG2(S-NH3+)4 (7) in D2O.

49

Figure S5. 1H-NMR spectrum of HOG3(S-GU+)8 (10) in D2O.

Figure S6. 13C-NMR spectrum of HOG3(S-GU+)8 (10) in D2O.

50

Figure S7. Computer models of dendrimers 1 and 11, and DPPG and POPG molecules. Ball representation is used for atoms belonging to core units of dendrimer computer models and for atoms belonging to hydrocarbon (palmitoyl and oleoyl) tails. In dendrimer cases, hydrogens are omitted for better clarity except those belonging to core unit or terminal protonated amines. Colors: C – gray (In POPG case, black color is used to highlight “unsaturated” C-C double bond), H – white, O – red, P – orange, S – yellow, Si – beige.

Figure S8. Top (left) and side (right) view on initial configuration of the whole simulated molecular system: dendrimer 1/POPG, including water molecules and Na+ 51

ions. Sphere representation is used for atoms belonging to dendrimers and for Na+ ions, stick representation is used for lipids and wire representation for water molecules. Colors: C – gray (lipid carbons) or black (dendrimer carbons), H – white, O – red, P – orange, S – yellow, Si – beige, Na – purple.

52

S.4 Tables Table S1. Minimum inhibitory (MIC) and bactericidal (MBC) concentrations of dendrimers GnO3(S-NH3+)m (n = 1, m = 6 (1); n = 2, m = 12 (2); n = 3, m = 24 (3))[1] and GnO3(S-GU+)m (n = 1, m = 6 (4); n = 2, m = 12 (5); n = 3, m = 24 (6)). Data shown in µM of ammonium or guanidium groups for each compound. S. aureus

E. coli

MIC

MBC

MIC

MBC

[G1O3(S-NH3)6]6+ (1)

9

9

9

9

[G2O3(S-NH3)12]12+ (2)

18

18

37

37

[G3O3(S-NH3)24]24+ (3)

144

144

576

576

[G1O3(S-GU)6]6+ (4)

1

1

3

3

[G2O3(S-GU)12]12+ (5)

10

21

21

21

[G3O3(S-GU)24]24+ (6)

21

21

21

42

Table S2. Minimum inhibitory (MIC) and bactericidal (MBC) concentrations of dendrons HOGn(NH3+)m (n = 2, m = 4 (7); n = 3, m = 8 (8)) and HOGn(GU+)m (n = 2, m = 4 (9); n = 3, m = 8 (10)). Data shown in µM of ammonium or guanidium groups for each compound. S. aureus

E. coli

MIC

MBC

MIC

MBC

[HOG2(S-NH3)4]4+ (7)

2

2

2

4

[HOG3(S-NH3)8]8+ (8)

78

78

78

78

[HOG2(S-GU)4]4+ (9)

2

2

2

2

[HOG3(S-GU)8]8+ (10)

7

15

7

7

53

Table S3. Minimum inhibitory (MIC) and bactericidal (MBC) concentrations of dendrimers G1O3(S-NH3+)6 (1) and G1O3(S-GU+)6 (4) and dendrons HOG2(S-NH3+)4 (7) and HOGn(S-GU+)m (9) in multi resistant S. aureus (MRSA). Data shown in µM of peripheral groups for each compound. MRSA MIC

MBC

[G1O3(S-NH3)6]6+ (1)

12.6

12.6

[G1O3(S-GU)6]6+ (4)

1.3

1.3

[HOG2(S-NH3)4]4+ (7)

8.5

8.5

[HOG2(S-GU)4]4+ (10)

1.8

1.8

Table S4. IC50 of dendrimers GnO3(S-NH3+)m (n = 1, m = 6 (1); n = 2, m = 12 (2); n = 3, m = 24 (3))[6] and GnO3(S-GU+)m (n = 1, m = 6 (4); n = 2, m = 12 (5); n = 3, m = 24 (6)) in trophozoites A. polyphaga. Data shown in ppm (mg L-1). Exposure time (h) 4

24

48

72

[G1O3(S-NH3)6]6+ (1)

3.4 ± 0.2

2.4 ± 0.1

2.3 ± 0.1

2.1 ± 0.2

[G2O3(S-NH3)12]12+ (2)

31.8 ± 2.8

19.1 ± 0.6 19.4 ± 0.5 18.6 ± 0.4

[G3O3(S-NH3)24]24+ (3)

30.1 ± 0.3

27.5 ± 0.1 20.5 ± 0.6 30.6 ± 0.2

[G1O3(S-GU)6]6+ (4)

18.5 ± 0.6

7.6 ± 0.2

5.5 ± 0.2

4.1 ± 0.2

[G2O3(S-GU)12]12+ (5)

123.5 ± 2.9 73.9 ± 0.6 55.4 ± 1.1 44.4 ± 0.6

[G3O3(S-GU)24]24+ (6)

121.6 ± 2.7 63.9 ± 0.8 64.8 ± 0.3 62.8 ± 1.2

CLX

2.9 ± 0.1

1.7 ± 0.1

54

1.6 ± 0.1

1.2 ± 0.2

Table S5. IC50 of dendrons HOGn(S-NH3+)m (n = 2, m = 4 (7); n = 3, m = 8 (8)) and HOGn(S-GU+)m (n = 2, m = 4 (9); n = 3, m = 8 (10)) in trophozoites A. polyphaga. Data shown in ppm (mg L-1). Exposure time (h)

[HOG2(S-NH3)4]4+ (7)

4

24

48

72

4.2 ± 0.1

4.1 ± 0.2

4.4 ± 0.3

4.8 ± 0.3

[HOG3(S-NH3)8]8+ (8) 40.5 ± 0.7 39.8 ± 1.6 34.8 ± 0.3 32.8 ± 0.7 [HOG2(S-GU)4]4+ (9)

7.1 ± 0.1

4.6 ± 0.2

3.9 ± 0.1

4.1 ± 0.2

[HOG3(S-GU)8]8+ (10) 68.1 ± 1.4 43.5 ± 0.9 38.2 ± 0.3 24.7 ± 0.1 CLX

2.9 ± 0.1

1.7 ± 0.1

1.6 ± 0.1

1.2 ± 0.2

Tabla S6. IC50 of non-cytotoxic dendrimers and dendrons against HeLa, in MUPH1 cells after 24 h of treatment. Data shown in ppm (mg L -1). Acanthamoeba MU-PH1 cells

p-value

[G1O3(S-NH3)6]6+ (1)

2.4 ± 0.1

3.7 ± 0.6

0.034329

[G1O3(S-GU)6]6+ (4)

7.6 ± 0.2

14.3 ± 0.5

0.00007

[HOG2(S-NH3)4]4+ (7)

4.1 ± 0.2

7.2 ± 0.8

0.02087

[HOG2(S-GU)4]4+ (9)

4.6 ± 0.2

6.7 ± 0.5

0.03765

CLX

1.7 ± 0.1

3.3 ± 0.4

0.00186

55

Graphical abstract

120

Microbicide and Toxicity Activities

100

80

MIC S. aureus

IC50 Acanthamoeba

60 40 20 0

56

IC50 Hella

Highlights Carbosilane dendrimers and dendrons with ammonium or guanidine peripheral functions are easily obtained by thiol-ene addition and nucleophilic substitution. The low generation dendritic systems are the most active against bacteria and amoebae and the less cytotoxic. Molecular modelling and dynamics help to understand the activity of dendritic compounds by study of interactions between dendrimers and phospholipid bilayers.

57