Water-stable ammonium-terminated carbosilane ...

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www.rsc.org/dalton | Dalton Transactions

Water-stable ammonium-terminated carbosilane dendrimers as efficient antibacterial agents† Beatriz Rasines,a José Manuel Hernández-Ros,a Natividad de las Cuevas,b José Luis Copa-Patiño,c Juan Soliveri,c M. Angeles Muñoz-Fernández,b Rafael Gómeza and F. Javier de la Mataa

Downloaded by Universidad de Burgos on 17 February 2011 Published on 27 August 2009 on http://pubs.rsc.org | doi:10.1039/B909955G

Received 20th May 2009, Accepted 6th August 2009 First published as an Advance Article on the web 27th August 2009 DOI: 10.1039/b909955g A new family of amine- and ammonium-terminated carbosilane dendrimers of the type Gn -[Si(CH2 )3 N(Et)CH2 CH2 NMe2 ]x and Gn -{[Si(CH2 )3 N+ R(Et)CH2 CH2 N+ RMe2 ]x (X- )y } (where n = 1, 2 and 3; R = H, X = Cl; R = Me, X = I) respectively has been synthesized by hydrosilylation of N,N-dimethyl-N¢-allyl-N¢-ethyl-ethylenediamine, [(CH2 =CH–CH2 )(Et)N(CH2 )2 NMe2 ] with the corresponding hydride-terminated dendrimers and subsequent quaternization with HCl or MeI. Quaternized dendrimers are soluble and stable in water or other protic solvents for long time periods. The antibacterial properties of the quaternary ammonium functionalized dendrimers have been evaluated showing that they act as potent biocides in which the multivalency along with the biopermeability of the carbosilane dendritic skeleton play an important role in the antibactericidal activity of these compounds.

1.

Introduction

Monovalent molecules like traditional antibiotics have been widely used in treating and controlling bacterial infections, however new biocide drugs are needed due to the increasing cases of antibiotic resistance among human and animal bacteria.1 One approach for the search of new types of antibacterial agents consists of the use of polyvalent interactions to treat infections.2 Conventional polymeric systems have demonstrated high activity as biocides due to having a high local density of active groups in close vicinity with a clear relationship between structure and activity in some cases.3 In this context, dendrimers emerge as an alternative approach to traditional polymers. In addition to the presence of multiple sites of attachment and the versatility to modify their skeletons and surfaces, their major advantage is their uniform structure, ideally monodisperse, which permits a precise characterization of the system structure and thus a more accurate and systematic analysis of the biomedical responses.4 Surface charged dendritic systems are membrane disruptive, causing the formation of holes followed by the leakage of cytosolic protein and the entry of dendrimers into the cells normally via an endocytosis-independent mechanism.5 As a general rule, antibacterial dendrimers are cationic, though some anionic dendrimers a Departamento de Qu´ımica Inorgánica, Universidad de Alcalá, Campus Universitario. Edificio de Farmacia, E-28871 Alcalá de Henares (Spain). Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain. E-mail: [email protected], [email protected]; Fax: (+34) 91 885 4683; Tel: (+34) 91 8854654 b Laboratorio de Inmunobiolog´ıa Molecular, Hospital General Universitario Gregorio Maraño´ n, Madrid, Spain. Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain. E-mail: [email protected] c Departamento de Microbiolog´ıa y Parasitolog´ıa, Universidad de Alcalá, E-28871, Alcalá de Henares, Spain. E-mail: [email protected] † Electronic supplementary information (ESI) available: Selected data as NMR spectra and MALDI-TOF mass spectrometry of derivatives 1–13. See DOI: 10.1039/b909955g

8704 | Dalton Trans., 2009, 8704–8713

have been reported.6,7 As a working model described elsewhere,8,9 the mode of action is based on the adsorption of cationic systems onto the usually negatively charged bacterial cell surface, diffusion through the cell wall, binding to the cytoplasmic membrane with subsequent disruption of the membrane. The destabilization of lipid membranes involves the displacement of the membranestabilizing divalent cations, which are extremely important in the neutralization of the anionic groups of the phospholipid membrane. In the case of cationic dendrimers such displacement may occur through competition with the divalent cations such as calcium or in the case of anionic dendrimers by scavenging the cations. One may expect that the strength of the membrane disruption and hence the biocide efficiency of dendrimers may depend on two major factors: (i) the number of charged groups mainly located on the surface, because the polycationic nature may increase the binding rate during the initial adsorption step. In this sense, a higher activity may be expected when increasing the dendrimer generation; (ii) the biopermeability of the dendritic system which may depend on parameters like dendrimer size, because the permeability of biocides through the cell wall should be lower for polymeric systems in comparison to small molecules, or higher due to the presence of appropriate lipophilic groups or domains like the presence of long aliphatic chains at the dendritic surface. It has been reported that quaternary ammonium functionalized poly(propyleneimine) dendrimers (PPI) behave as very potent biocides against both Gram-positive and Gramnegative bacteria and the antibacterial properties depend on the dendrimer generation, the length of hydrophobic chains and the counteranion.8 Dendrimeric peptides have also been used in order to generate efficient bacteriocides. In this case, the use of peptides did not show any antimicrobial activity on their own, but become broadly active when conjugated to an asymmetric dendritic polylysine core.9 Another example is the use of aminoterminated polyamidoamine dendrimers (PAMAM) and their This journal is © The Royal Society of Chemistry 2009


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partially PEG-coated derivatives which possess attractive antimicrobial properties, particularly against Gram-negative bacteria, being surprisingly less toxic for Gram-positive bacteria.10 Finally, dendrimers based on poly(propyleneoxide) amines (Jeffamines) showed broad spectrum activity against both Gram-positive and Gram-negative bacteria being comparatively higher or equipotent to antibiotics and antifungal agents.7 We have recently reported the synthesis of cationic carbosilane dendrimers as a new type of systems useful for biomedical applications.11 Novel amine- and ammonium-terminated carbosilane dendrimers of the type Gn -[Si{CH2 O-(C6 H4 )-3-NMe2 }]x or Gn -[Si{CH2 O-(C6 H4 )-3-NMe3 + I- }]x have been reported elsewhere and synthesized up to the second generation.12 A study of the antimicrobial activity of these cationic dendrimers of the first and second generation against both Gram-positive and Gramnegative bacteria has been described showing that the new ammonium-terminated carbosilane dendrimers can be considered as multivalent biocides. However, these quaternized carbosilane dendrimers are not soluble in water, though they can be solubilised after addition of less than 1% of dimethyl sulfoxide. Here we describe the synthesis of a new family of water-soluble and -stable cationic carbosilane dendrimers up to the third generation that overcome the water insolubility problem and allow the rationalization of the role of the carbosilane skeleton in their behaviour as antibacterial agents. The synthetic approach shown here constitutes an important procedure for water-soluble and -stable cationic carbosilane dendrimers as very lipophilic systems, only prepared by two different groups using different synthetic approaches.13,14

2. 2.1.

Results and discussion Synthesis and characterization of dendrimers

We have designed new amine-terminated carbosilane dendrimers by the hydrosilylation reaction of Si–H terminated dendrimers with a new allyl amine. This allyl amine depicted in Scheme 1 can

be prepared in a two step reaction starting from N,N-dimethyl-N¢ethyl-ethylenediamine. The addition of n-BuLi to this amine leads to a lithium salt that is not isolated and is treated in situ with allyl bromide to give N,N-dimethyl-N¢-allyl-N¢-ethyl-ethylenediamine, [(CH2 =CHCH2 )(Et)N(CH2 )2 NMe2 ] (1) (see Scheme 1). The final product is initially isolated with lithium bromide as an adduct (see Experimental Section) as a white crystalline solid but can be purified by washing with a water solution of sodium carbonate and subsequent extraction with diethyl ether to give 1 in a 60% yield as a yellow oil. The 1 H NMR spectra of 1 and 1·LiBr are slightly different, with a general deshielding in the case of the adduct product (see Experimental section).

Scheme 1

Triethylsilane and Si–H terminated carbosilane dendrimers of the type Gn -(SiH)x (n = 1, 2 and 3; x = 4, 8 and 16)15 were used in the hydrosilylation of N,N-dimethyl-N¢-allyl-N¢ethyl-ethylenediamine. The reactions were performed in ampoules with J. Young valves, using toluene as solvent and the Karstedt catalyst during 12 h at 120 ◦ C, to afford the corresponding system [Et3 Si(CH2 )3 N(Et)CH2 CH2 NMe2 ] (2) and dendrimers Gn -[Si(CH2 )3 N(Et)CH2 CH2 NMe2 ]x (n = 1, x = 4 (3); n = 2, x = 8 (4); n = 3, x = 16 (5)) in high yield as colourless oils (see Scheme 2), in which n is the number of generation G, and x is the number of peripheral units. The hydrosilylation reaction occurred exclusively by b-addition and any by-products were detected in the 1 H NMR spectra of the crude products. All of these dendrimers are soluble in chlorinated solvents and aromatic and aliphatic hydrocarbons but insoluble in water.

Scheme 2

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Dalton Trans., 2009, 8704–8713 | 8705


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The NMR spectroscopic, MALDI-TOF MS and analytical data for compounds 2–5 are consistent with their proposed structures (Scheme 2 and see ESI† for structures). The 1 H NMR spectra of compounds 2–5 show, for the carbosilane skeleton, almost identical chemical shifts for analogous nuclei in different generations, although broader and less structured resonances are present with increasing generation. For the SiCH2 CH2 CH2 Si branches two broad multiplets are observed, one due to the middle methylene groups centered at d = 1.30 ppm, whilst the methylene groups bonded directly to silicon atoms are located around d = 0.60 ppm. With respect to the outer group, four methylene groups bonded to nitrogen atoms (Fig. 1) appear as two multiplets, one at about d = 2.50 ppm that we ascribed to the signals due to the protons of the methylene groups f and d overlapped, and the other at d = 2.40 ppm due to the protons of the methylene groups c and e that are also overlapped. The resonances at d = 1.40 ppm and d = 0.42 ppm are assigned to methylene groups b and a respectively. The methyl fragment of the ethyl group bonded to nitrogen appears as a triplet at d = 1.10 ppm, and the methyl protons of the dimethyl amine fragment gives a singlet at d = 2.30 ppm. This assignment has been performed on the basis of NOESY 1D, TOCSY 1D and gHMBC-{1 H-15 N} experiments. The 13 C NMR spectra for the methylene groups bonded to nitrogen show four resonances located at d = 57.9 (c), 57.7 (e), 51.6 (d) and 48.0 (f) ppm based on HMQC-{1 H-13 C} and HMBC-{1 H-13 C} experiments. The –NMe2 group gives a singlet at d = 45.9 ppm and the methyl fragment of the ethyl group bonded to nitrogen appears as a singlet at d = 11.7 ppm. The methylene groups of the SiCH2 CH2 CH2 Si branches gives signals in the range d = 21.5 to 17.7 ppm (the number of signals depends on the dendrimer generation). Finally, methyl groups bonded to silicon give signals in the expected zone, around 0 ppm, in both 1 H and 13 C NMR spectra. The 29 Si-NMR spectra show signals with the expected values although the signals corresponding to the most internal silicon atoms are generally not observed. This spectroscopic behaviour has been observed in other related carbosilane dendrimers.11b,16 Finally, 15 N-NMR spectra of complexes 2–5 show two signals around -356 and -337 ppm corresponding to the outer and inner nitrogen atoms of each branch respectively.17

Fig. 1 Labeled external branches of amine (A) or ammonium (B)-terminated carbosilane dendrimers.

MALDI-TOF mass spectra of the carbosilane dendrimers were obtained for dendrimers 3 and 4 using dithranol as the matrix in which the molecular peaks were identified. No molecular peak was detected for the third generation dendrimer 4 where the ionization becomes more difficult.16a,18 Finally, the hydrodynamic diameters have been determined for dendrimers 3–5 using DOSY experiments in chloroform as solvent at 25 ◦ C, and reveals a value 8706 | Dalton Trans., 2009, 8704–8713

of 1.28 nm for the first generation (3), 1.58 nm for the second generation (4) and 1.92 nm for the third generation (5), which confirm their nanoscopic dimension. The ammonium-terminated dendrimers were cleanly prepared by adding a little excess of HCl or MeI to parent derivatives 2–5 in diethyl ether (see Scheme 2). The addition of HCl leads to the quaternized systems {[Et3 Si(CH2 )3 N+ (H)(Et)CH2 CH2 N+ HMe2 ](Cl- )2 } (6) and Gn -{[Si(CH2 )3 N+ (H)(Et)CH2 CH2 N+ HMe2 ]x (Cl- )y } (n = 1, x = 4, y = 8 (7); n = 2, x = 8, y = 16 (8); n = 3, x = 16, y = 32 (9)) in quantitative yields. Addition of MeI over the amine functionalized dendrimers also produces the corresponding cationic ammonium-terminated derivatives of type {[Et3 Si(CH2 )3 N+ (Me)(Et)CH2 CH2 N+ Me3 ](I- )2 }(10) and Gn -{[Si(CH2 )3 N+ (Me)(Et)CH2 CH2 N+ Me3 ]x (I- )y } (n = 1, x = 4, y = 8 (11); n = 2, x = 8, y = 16 (12); n = 3, x = 16, y = 32 (13)) again in quantitative yields. All compounds 6–13 are white solids, soluble in water, alcohols (like methanol or ethanol), and dimethylsulfoxide. Both cationic non-dendrimer and dendrimer systems are stable in protic solvents and can be stored without decomposition for long time periods. The NMR spectroscopic and analytical data for compounds 6–13 are consistent with their proposed structures (Scheme 2 and Fig. 2). The 1 H NMR spectra were recorded in DMSO-d6 or D2 O at room temperature. In these solvents the line widths of these spectra tended to be broader than those of the derivatives soluble in common organic solvents. The 1 H and 13 C NMR spectra of the quaternized dendrimers exhibited identical resonance patterns to those observed in their neutral counterparts 3–5 for the carbosilane framework, although broader signals were seen with increasing generation (see Experimental section and ESI†). In general for the 1 H NMR spectra (see Fig. 1), the quaternization of the amine groups result in a deshielding of the chemical shifts of about 0.8–1.5 ppm of the geminal methylene (c, d, e and f ) and methyl (h and i) groups directly bound to the charged nitrogens, whereas small downfield shifts are detected around d = 0.3–0.4 ppm for the vicinal methylene and methyl groups (b and g). Beyond these positions, no displacement is observed on the chemical shift due to the positive charge on the nitrogen atoms. However, this effect is more evident in the quaternized systems by MeI than those prepared using HCl. Analogous behaviour is observed for the carbon atoms in the 13 C NMR spectra. For the methylene groups bonded to nitrogen in the derivatives quaternized by MeI (10–13), the four resonances are located at d = 63.3 (c), 56.6 (f ), 56.0 (e) and 51.9 (d) ppm. Respecting the methyl groups bound to nitrogen, these appear at d = 52.4 (h) and 47.1 (i) ppm. In the case of systems quaternized with HCl (6–9), the geminal groups attached to nitrogen are located at d = 54.2 (c), 49.5 (f ), 46.3 (e) and 44.9 (d) ppm for methylenes and d = 41.8 ppm for the methyl groups. Again, the 1 H and 13 C resonance assignments are based on TOCSY 1D, HMQC-{1 H13 C} and HMBC-{1 H-13 C} experiments. The 15 N-NMR spectra of derivatives 6–9 quaternized with HCl show two signals at about d = -340 and -325 ppm while for compounds 10–13 quaternized by MeI an expected deshielding is observed at d = -331 and -320 ppm, corresponding in both cases to the outer and inner nitrogen atoms, respectively. Both situations mean a downfield shifting with respect to the neutral counterparts, 2–5, as a consequence of the positive charge present on both nitrogen atoms.17 The presence of only these two signals This journal is © The Royal Society of Chemistry 2009


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Fig. 2

Molecular representation of ammonium-terminated carbosilane dendrimers 7–9 and 11–13.

again corroborates that all the nitrogen atoms present in these dendrimers are quaternized even at the higher generation. Attempts to prepare mono quaternized amine derivatives failed due to the difficulty to reproduce the synthetic procedure. The stoichiometric reaction of dendrimer 4 containing eight units with identical equivalents of MeI gave the corresponding outermost quaternized terminal amine derivative G2 -{[Si(CH2 )3 N(Et)CH2 CH2 N+ Me3 ]8 (I- )8 } (14) with small amounts of non-methylated branches on the basis of the HMBC{1 H-15 N} experiment. Dendrimer 14 shows two signals at about This journal is © The Royal Society of Chemistry 2009

d = -331 and -342 ppm corresponding to the outer and inner nitrogen atoms of each branch respectively, in addition to two weak signals located at d = -356 and -339 ppm corresponding to the non-methylated branches. In any case, signals attributed to the mono-methylated forms holding the innermost quaternized amine or double-methylated branches were observed, even when a defect of the quaternized agent was added. This feature strongly suggests that the quaternization process starts in the outermost amine and subsequently proceeds to the innermost when a slight excess of MeI is added. Dalton Trans., 2009, 8704–8713 | 8707

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Table 1 Bacteriostatic (MIC)a and bactericidal (MBC)b effects of monofunctional derivative 10 and dendrimers 8, 11–13c 10

Escherichia coli (-) Staphylococcus aureus (+) a

12

13

Penicillin G

8

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

>920 115

>920 230

3.65 0.46

3.65 1.82

1.70 0.85

1.70 1.70

1.65 0.82

1.65 0.82

42.49 42.49

84.98 84.98

766.0 0.09

766.0 0.19

MIC denotes minimal inhibitory concentration. b MBC denotes minimal bactericidal concentration. c All concentration data are measured as mM.

2.2.


11

Antibacterial activity of dendrimers

We have evaluated the antibacterial activity of dendrimers 11–13 containing iodide as counteranion as well as their monofunctional counterpart 10, on Gram+ and Gram- bacteria. In particular, we have measured the MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) of our dendrimers against Staphylococcus aureus (Gram+) and Escherichia coli (Gram-). The term MIC states the minimal concentration of an agent that can inhibit the growth of a microorganism (bacteriostatic effect) while MBC indicates the minimal concentration of an agent that can kill a microorganism (bactericidal effect). The value of MIC is always less than or equal to MBC for the same biocide and microorganism. Data for these activities are shown in Table 1. The results of the antibacterial tests clearly show a high activity for the carbosilane dendrimers both against Gram+ and Grambacteria in contrast to their monofunctional counterpart 10, since they are over two orders of magnitude more potent. This significant improvement of biocidal action of dendritic biocides may be due to the high number of functional groups located in a compact space and their polycationic structure. Moreover, the data reveal lower MIC and MBC values for Gram+ bacteria than for Gram- bacteria, in all the dendrimer generations used. This difference has been observed by other authors in lysine based-dendrimers9 and may arise from differences in the cell wall structure. In Gram+ bacteria, there is a membrane formed by a single bilayer while in Gram- bacteria it is composed of two bilayer membranes, making the latter bacteria more resistant to an external attack. Interestingly, in the case of Gram- bacteria both bacteriostatic and bactericidal effects are identical for each generation meaning that at such a concentration the dendritic system not only inhibits bacterial growth but also kills the bacteria. For Gram+ bacteria, this behaviour is only observed when using the higher generation. The antibacterial activity of carbosilane dendrimers depends on the increasing generation: 13 > 12 > 11 regarding the MBC values of both types of bacteria. However, for Gram+ bacteria, the MIC data suggests an opposite trend. Comparing the data with the antibiotic penicillin G potassium, a very effective antibiotic against Gram+ bacteria, the cationic dendrimers are two orders of magnitude more active against Gram- bacteria and slightly less active against Gram+ bacteria. Although penicillin is not an antibiotic for Gram- bacteria, the values found for the multivalent systems indicate their high potential as antibacterial agents. As mentioned above, the strength of the membrane disruption depends basically on two parameters: (i) the number of charged groups and (ii) the biopermeability processes (size and molecular weight or lipophilic groups or domains). Concerning the first one, 8708 | Dalton Trans., 2009, 8704–8713

when increasing the generation, the number of quaternary groups grows and thus the system should be more potent. However, regarding the second parameter, if we take the size and molecular weight of the dendritic system into account this trend should be the opposite. The dendrimers used here are small enough to be able to cross the cell wall of both types of bacteria since the theoretical molecular weights range from 2193 to 9713 and the size measured by DOSY on neutral systems is around 1.3– 2.0 nm of diameter. Another aspect of the biopermeability is the presence and length of hydrophobic groups on the quaternized nitrogen atoms. From several studies on quaternary ammonium salts, conventional cationic polymers or ammonium-terminated PPI dendrimers, a parabolic relationship has been observed in which a chain length of C10 –C16 tends to contribute the most effective lipophilic groups for biocide activity.3b,8 It is worth noting that the lipophilicity of the carbosilane dendrimers is different in comparison to that shown by other related dendritic systems where the lipophilic groups are attached on the surface.8 In the case of dendrimers 11–13, the ammonium groups are bound to a very lipophilic skeleton which may aid in the biopermeability processes. Such lipophilicity may increase on going from the first to the third generation. The combination of all these factors gives a complicated response but the fact that the antibacterial activity increases with the generation implies that the outer charged functional groups along with the lipophilicity of the dendritic skeleton play an important role in the biocide action. However, if the antibacterial activity is determined taking into account the concentration per branch, a slightly different scenario is found. Although, it is clear the existence of a dendritic effect in both types of bacteria, for Gram- the MBC values are of the same magnitude for the first (11) and second (12) generation and double for the third (13), while for Gram+ bacteria, the second and third generations are of the same magnitude and a half value is found for the first generation. These data suggest that the antibacterial activity is not only proportional to the multivalency (number of charges) or to the lipophilicity of the dendrimer but also increases synergistically up to a maximum centered on the first or second generation depending on the type of bacteria. From this fact, the final activity must be a compromise of different factors like size, molecular weight, lipophilicity and the number of charged groups. The counteranion nature could be important in the biocide efficiency of these molecules. Such an influence has been studied and the results are shown in Table 1. According to the MIC values, the second generation dendrimer 8 containing chloride as the counteranion is about 12 times and 50 times less effective against Gram+ and Gram- bacteria respectively than dendrimer 12 having iodide as the counteranion. The same behaviour has been observed in PPI dendrimers8b though different values are not expected since in both types of dendrimers the ions tend This journal is © The Royal Society of Chemistry 2009

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to dissociate freely in water. An explanation emerges from the fact that iodides form weaker anionic pairs with ammonium units than chloride anions, leading to more exposed cations and thus stronger electrostatic attraction to the negatively charged bacterial membranes. However, in this case the different nature of the ammonium units may also be responsible for such a different behaviour. Dendrimer 8 seems to be more hydrophilic than the corresponding counterpart 12 because of the presence of N–H bonds, decreasing the biopermeability and thus the antibacterial activity.


3.

Conclusions

A new family of amine- and ammonium-terminated carbosilane dendrimers has been synthesized by the hydrosilylation reaction of N,N-dimethyl-N¢-allyl-N¢-ethyl-ethylenediamine, [(CH2 =CH– CH2 )(Et)N(CH2 )2 NMe2 ] (1) with hydride-terminated dendrimers and subsequent quaternization with HCl or MeI. These dendrimers have been fully characterized using elemental analysis, NMR spectroscopy and mass spectrometry. Quaternized dendrimers are soluble and stable in water or other protic solvents for long time periods (NMR solutions of these dendrimers show the same spectra after several months). The antimicrobial studies show that the multivalency of dendrimers plays an important role in the antibactericidal activity of these compounds, since the dendrimer biocides are over two orders of magnitude more potent than their monofunctional counterpart. The activity increases on increasing the generation mainly for the MBC values although taking into account the concentration per branch a maximum of activity is centered on the first or second generation depending on the type of bacteria. The different behaviour observed in the MIC for Gramand Gram+ bacteria may be related to the different structure of the cell wall of these systems. In addition, the different nature of the peripheral ammonium salt in the dendrimers also contributes to modulate the antibacterial activity. A comparison of the activity of dendrimers 11–13 with the penicillin G antibiotic reveals the high potential of these systems as biocides.

4. 4.1.

Experimental General remarks

All manipulations of oxygen- or water-sensitive compounds were carried out under an atmosphere of argon using standard Schlenk techniques or an argon-filled glove box. Solvents were dried and freshly distilled under argon prior to use: hexane from sodiumpotassium, toluene from sodium, tetrahydrofuran and ethyl ether from sodium benzophenone ketyl, and methylene chloride over P4 O10 .19 Unless otherwise stated, reagents were obtained from commercial sources and used as received. Et3 SiH or the analogous hydride-terminated carbosilane dendrimers of different generations Gn -(SiH)x were prepared according to reported methods.15 1 H, 13 C, 15 N and 29 Si NMR spectra were recorded on Varian Unity VXR-300 and Varian 500 Plus Instruments. Chemical shifts (d, ppm) were measured relative to residual 1 H and 13 C resonances for chloroform-d1 , DMSO-d6 and water-d2 used as solvents. The 15 N and 29 Si chemical shifts were referenced to external CH3 NO2 and SiMe4 (0.00 ppm) respectively. The NMR signal assignments of the compounds have been performed by different This journal is © The Royal Society of Chemistry 2009

1D and 2D NMR experiments (COSY, TOCSY, ROESY {1 H-1 H}, HSQC, HMBC{1 H-13 C}, HMBC{1 H-15 N} or HMBC{1 H-29 Si} where appropriate). C, H and N analyses were carried out with a Perkin-Elmer 240 C microanalyzer. MALDI-TOF MS samples were prepared in a 1,8,9-trihydroxyanthracene (dithranol) matrix, and spectra were recorded on a Bruker Reflex II spectrometer equipped with a nitrogen laser emitting at 337 nm and operated in the reflection mode at an accelerating voltage in the range 23–25 kV. DOSY experiments. The diffusion coefficients were measured in chloroform at 25 ◦ C by using the Dbppste (DOSY Bipolar Pulse Simulated Echo) pulse sequence in aVarian NMRSystem 500 equipped with a high accuracy variable temperature unit (± 0.1 ◦ C), a Performa IV PFG amplifier, and a Z-PFG Triple Resonance 5-mm probe. Fine calibration of the PFG strength (DAC to G unit) was performed with an H2 O/HDO (2 Hz) sample as standard supplied by Varian (D = 19.04 ¥ 10-10 m2 s-1 at 25 ◦ C). The diffusion NMR data were acquired over 64 scans, with settings pw90, an acquisition time of 3 s, a relaxation delay of 2 s, in each one of the 15 steps of the gradient level array between 1 and 50 G cm-1 (50 ms of diffusion delay and 2 ms of total defocusing time). The experimental data (32 K ¥ 1 K) was treated with the “DOSY” software from VNMRJ2.1B. The hydrodynamic radii, RH , were calculated from the diffusion coefficients of a certain molecular species using the Stokes–Einstein equation RH = kB T/6pg D where kB is the Boltzmann constant, T the absolute temperature, and g the viscosity of the solution. 4.2. Synthesis of [(CH2 =CHCH2 )N(Et)(CH2 CH2 NMe2 )] (1) To a solution of the amine [H(Et)NCH2 CH2 NMe2 ] (7.5 g, 64.5 mmol) in Et2 O (250 mL) cooled at -70 ◦ C was added another solution of n-BuLi drop by drop (64.5 mmol, 2.5 M in Et2 O). The mixture was stirred for 1 h at room temperature, then cooled again at -70 ◦ C and 1 equivalent of CH2 =CHCH2 Br (7.81 g, 64.5 mmol) added and stirred for 12 h at room temperature affording a white crystalline solid of the adduct 1·LiBr. The off-salt ligand was obtained by treating the solid with a water solution of Na2 CO3 and the subsequent extraction with Et2 O (3 ¥ 50 mL). The organic phase was dried with MgSO4 , the solution filtered and the volatiles removed under vacuum to give 1 as a yellow oil (4.5 g, 45% yield). Data for 1. 1 H-NMR (CDCl3): d 5.83 (m, 1H, CH2 =CHCH2-), 5.10 (m, 2H, CH 2 =CHCH2 -), 3.08 (d, 2 H, CH2 =CHCH 2 -), 2.52 (m, 4H, -NCH 2 CH2 NMe2 and -NCH 2 CH3 ), 2.35 (m, 2H, -NCH2 CH 2 NMe2 ), 2.19 (s, 6H, -NMe2 ), 0.99 (t, 3H, -NCH2 CH 3 ). 13 C-NMR (CDCl3 ): d 135.8 (CH2 =CHCH2 -), 117.2 (CH2 =CHCH2 -), 57.5 (-NCH2 CH2 NMe2 ), 57.2 (CH2 =CHCH2 -), 51.0 (-NCH2 CH2 NMe2 ), 47.7 (-NCH2 CH3 ), 45.9 (-NCH2 CH2 NMe2 ), 11.6 (-NCH2 CH3 ). 15 N-NMR (CDCl3 ): d -355.8 (NMe2 ), -338.6 (NCH2 CH3 ). C9 H20 N2 : calcd. C, 69.23; H, 12.82; N, 17.95; found C, 69.61; H, 12.35; N, 17.15. APCI MS (M+ [H+ ]): calcd. 157; found 157. Data for 1·LiBr. 1 H-NMR (CDCl3 ): d 6.14 (m, 1H, CH2 =CHCH2 -), 5.18 (m, 2H, CH 2 =CHCH2 -), 3.28 (d, 2 H, CH2 =CHCH 2 -), 2.71 (q, 2H, -NCH 2 CH3 ), 2.52 (m, 2H, -NCH2 CH 2 NMe2 or -NCH 2 CH2 NMe2 ), 2.39 (m, 2H, NCH 2 CH2 NMe2 or -NCH2 CH 2 NMe2 ), 2.32 (s, 6H, -NMe2 ), 1.09 Dalton Trans., 2009, 8704–8713 | 8709

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(t, 3H, -NCH2 CH 3 ). BrC9 H20 LiN2 : calcd. C, 44.46; H, 8.29; N, 11.52; found C, 44.32; H, 7.70; N, 11.56.


4.3. (2)

Synthesis of [(Et3 SiCH2 CH2 CH2 )N(Et)(CH2 CH2 NMe2 )]

To 1 equivalent of diamine [(CH2 =CHCH2 )N(Et)(CH2 CH2 NMe2 )] (1) (0.284 g, 1.82 mmol), was added 1 equivalent of HSiEt3 (0.21 g, 1.82 mmol) in 3 mL of toluene, and then one drop of the Karsted catalyst was added. The reaction mixture was stirred for 12 h at 120 ◦ C and then evaporated to dryness to remove the solvent. Compound 2 was obtained as a brown oil (0.36 g, 73%). 1 H-NMR (CDCl3 ): d 2.51 (m, 4H, -NCH 2 CH3 and -NCH 2 CH2 NMe2 ), 2.36 (m, 4H, -NCH2 CH 2 NMe2 and -SiCH2 CH2 CH 2 N-), 2.21 (s, 6H, -NMe2 ), 1.40 (m, 2H, -SiCH2 CH 2 CH2 N-), 0.99 (t, 3H, -NCH2 CH 3 ), 0.89 (t, 9H, -SiCH2 CH 3 ), 0.47 (q, 6H, -SiCH 2 CH3 ), 0.41 (q, 2H, -SiCH 2 CH2 CH2 N-). 13 C-NMR (CDCl3 ): d = 58.1 (-SiCH2 CH2 CH2 N-), 57.7, (-NCH2 CH2 NMe2 ), 51.6 (-NCH2 CH2 NMe2 ), 47.9 (-NCH2 CH3 ), 45.9 (-NMe2 ), 21.3 (-SiCH2 CH2 CH2 N-), 11.6 (-NCH2 CH3 ), 8.9 (-SiCH2 CH 2 CH2 N-), 7.3(-SiCH2 CH3 ), 3.2 (-SiCH2 CH3 ). 15 N-NMR (CDCl3 ): d -355.7 (-NMe2 ), -338.1 (-NCH2 CH3 ). 29 Si-NMR (CDCl3 ): d 7.0 (-SiEt3 ). C15 H36 N2 Si: calcd. C, 66.17; H, 13.23; N, 10.29; found C, 65.41; H, 12.83; N, 9.57. 4.4. (3)

Synthesis of G1 -[Si(CH2 CH2 CH2 )N(Et)(CH2 CH2 NMe2 )]4

To 4.2 equivalents of diamine [(CH2 =CHCH2 )N(Et)(CH2 CH2 NMe2 )] (1) (1.19 g, 7.60 mmol), was added a toluene solution (3 mL) of 1 equivalent of dendrimer G1 -(SiH)4 (0.78 g, 1.81 mmol), then one drop of the Karsted catalyst was added. The reaction mixture was stirred for 12 h at 120 ◦ C and then evaporated to dryness to remove the solvent. Compound 3 was obtained as a brown oil (1.85 g, 97%). Further purification was accomplished by exclusion chromatography using toluene as eluent. 1 H-NMR (CDCl3 ): d 2.51 (m, 16H, -NCH 2 CH3 and -NCH 2 CH2 NMe2 ), 2.36 (m, 16H, -NCH2 CH 2 NMe2 and -SiCH2 CH2 CH 2 N-), 2.20 (s, 24H, NMe2 ), 1.39 (m, 8H, -SiCH2 CH 2 CH2 N-), 1.26 (m, 8H, -SiCH2 CH 2 CH2 Si-), 0.98 (t, 12H, -NCH2 CH 3 ), 0.52 (m, 16H, -SiCH 2 CH2 CH 2 Si-), 0.38 (m, 8H, -SiCH 2 CH2 CH2 N-), -0.08 (s, 24H, -SiMe2 ). 13 C-NMR (CDCl3 ): d = 57.9 (-SiCH2 CH2 CH2 N-), 57.7, (-NCH2 CH2 NMe2-), 51.6 (-NCH2 CH2 NMe2-), 48.0 (-NCH2 CH3 ), 45.9 (NMe2 ), 21.4 (-SiCH2 CH2 CH2 N-), 20.3 (-SiCH2 CH2 CH2 Si-), 18.5 (-SiCH2 CH2 CH2 Si-), 17.5 (-SiCH2 CH2 CH2 Si-), 13.0 (SiCH2 CH2 CH2 N-), 11.7 (-NCH2 CH3 ), -3.3 (-SiMe2 ). 15 N-NMR (CDCl3 ): d -356.0 (NMe2 ), -337.8 (NCH2 CH3 ). 29 Si-NMR (CDCl3 ): d 0.6 (G0 -Si), 1.95 (G1 -Si). Assignments are based on TOCSY 1D, HMQC-{1 H-13 C} and HMBC-{1 H-13 C}, HMBC{1 H-28 Si} and HMBC-{1 H-15 N} experiments. C56 H132 N8 Si5 : calcd. C, 63.56; H, 12.57; N, 10.59; found C, 63.11; H, 12.10; N, 10.07. MALDI-TOF MS (M+ [H+ ]): calcd. 1057.9; found 1058.0. 4.5. (4)

Synthesis of G2 -[Si(CH2 CH2 CH2 )N(Et)(CH2 CH2 NMe2 )]8

This dendrimer was prepared using a similar method to that described for 3 starting from 8.2 equivalents of diamine, 8710 | Dalton Trans., 2009, 8704–8713

[(CH2 =CHCH2 )N(Et)(CH2 CH2 NMe2 )] (1) (1.06 g, 6.75 mmol) and 1 equivalent of dendrimer G2 -(SiH)8 (0.97 g, 0.82 mmol) to obtain compound 4 as a yellow oil (1.61 g, 81%). 1 H-NMR (CDCl3 ): d = 2.51 (m, 32 H, -NCH 2 CH3 and -NCH 2 CH2 NMe2 ), 2.36 (m, 32 H, -NCH2 CH 2 NMe2 , -SiCH2 CH2 CH 2 N-), 2.21 (s, 48 H, -NMe2 ), 1.38 (m, 16 H, -SiCH2 CH 2 CH2 N-), 1.27 (m, 24 H, -SiCH2 CH 2 CH2 Si-), 0.98 (t, 24 H, -NCH2 CH 3 ), 0.52 (m, 48 H, -SiCH 2 CH2 CH 2 Si-), 0.38 (t, 16 H, -SiCH 2 CH2 CH2 N-), -0.08 (s, 48H, -SiMe2 ), -0.012 (s,12 H, -SiMe). 13 C-NMR (CDCl3 ): d 57.9 (-SiCH2 CH2 CH2 N-), 57.7 (-NCH2 CH2 NMe2 ), 51.7 (-NCH2 CH2 NMe2 ), 48.0 (-NCH2 CH3 ), 45.9 (NMe2 ), 21.5 (-SiCH2 CH2 CH2 N-), 20.1–17.7 (-SiCH2 CH2 CH2 Si- and -SiCH2 CH2 CH2 Si-), 13.0 (-SiCH2 CH2 CH2 N-), 11.7 (-NCH2 CH3 ), -3.3 (-SiMe2 ), -5.0 (-SiMe). 15 N-NMR (CDCl3 ): d = -356.0 (NMe2 ), -337.8 (NCH2 CH3 ). 29 Si-NMR (CDCl3 ): d (G0 –Si) is not observed, 1.0 (G1 –Si), 1.9 (G2 –Si). C128 H300 N16 Si13 (2429.0): calcd. C, 63.29; H, 12.45; N, 9.23; found C, 61.98; H, 11.93; N, 7.86. MALDI-TOF MS (M+ [H+ ]): calcd. 2429.0; found 2429.2. 4.6. Synthesis of G3 -[Si(CH2 CH2 CH2 )N(Et)(CH2 CH2 NMe2 )]16 (5) This dendrimer was prepared using a similar method to that described for 3, starting from 18 equivalents of diamine, [(CH2 =CHCH2 )N(Et)(CH2 CH2 NMe2 )] (1) (0.53 g, 3.36 mmol) and 1 equivalent of dendrimer G3 -(SiH)16 (0.50 g, 0.187 mmol) to obtain compound 5 as a very viscous brown oil (0.97 g, 99%). 1 H-NMR (CDCl3 ): d 2.53 (m, 64 H, -NCH 2 CH3 and -NCH 2 CH2 NMe2 ), 2.41 (m, 64 H, -NCH2 CH2 NMe2 and -SiCH2 CH2 CH 2 N-), 2.21 (s, 96 H, NMe2 ), 1.40 (m, 32 H, -SiCH2 CH 2 CH2 N-), 1.27 (m, 56 H, -SiCH2 CH 2 CH2 Si-), 0.98 (t, 48 H, -NCH2 CH 3 ), 0.52 (m, 112 H, -SiCH 2 CH2 CH 2 Si-), 0.38 (t, 32H, -SiCH 2 CH2 CH2 N-), -0.08 (s, 96H, -SiMe2 ), -0.012 (s, 36 H, -SiMe). 13 C-NMR (CDCl3 ): d 58.0 (-SiCH2 CH2 CH2 N-), 57.8 (-NCH2 CH2 NMe2 ), 51.7 (-NCH 2 CH2 NMe2 ), 48.0 (-NCH2 CH3 ), 46.0 (-NMe2 ), 21.5 (-SiCH2 CH2 CH2 N-), 20.2- 18.5 (-SiCH2 CH2 CH2 Si- and -SiCH2 CH2 CH2 Si-), 13.1 (-SiCH2 CH2 CH2 N-), 11.7 (-NCH2 CH3 ), -3.2 (-SiMe2 ), -4.9 (-SiMe). 15 N-NMR (CDCl3 ): d -356.0 (NMe2 ), -338.0 (-NCH2 CH3 ). 29 Si-NMR(CDCl3 ): d (G0 -Si) and (G1 -Si) are not observed, 0.9 (G2 –Si), 1.9 (G3 –Si). C272 H636 N32 Si29 (5170.7): calcd. C, 63.18; H, 12.40; N, 8.67; found C, 61.7; H, 11.62; N 7.81. 4.7. Synthesis of {[Et3 Si(CH2 CH2 CH2 )N+ H(Et)(CH2 CH2 N+ HMe2 )] 2Cl- } (6) To a THF solution (25 mL) of [Et3 Si(CH2 CH2 CH2 )N(Et)(CH2 CH2 NMe2 )] (2) (0.42 g, 1.5 mmol) was added an excess of HCl (1 M) in diethyl ether (7.0 mL, 6.89 mmol). The resulting solution was stirred for 48 h at room temperature and then evaporated under reduced pressure to give 6 as a white water soluble solid (0.51 g, 96%). 1 H-NMR (DMSO): d 11.30, 11.10 (s wide, 2H, N+ H, N+ HMe2 ), 3.47 (m, 4 H, -NH+ CH 2 CH 2 N+ HMe2 ), 3.10 (m, 2H, -N+ CH 2 CH3 ), 3.02 (m, 2H, -SiCH2 CH2 CH 2 N+-), 2.80 (s, 6H, N+ HMe2 ), 1.65 (m, 8H, -SiCH2 CH 2 CH2 N+ -), 1.22 (t, 3H, -NCH2 CH 3 ), 0.91(t, 9H, SiCH2 CH 3 ) 0.49 (m, 8H, -SiCH 2 CH3 and -SiCH 2 CH2 CH2 N+ -). 13 C-NMR (DMSO): d 54.2 (-SiCH2 CH2 CH2 N+ ), 49.0 (-N+ CH2 CH2 N+ Me3 ), 46.4 (-N+ CH2 CH3 ), 44.7 (-N+ CH2 CH2 N+ Me3 ), 41.8 (N+ HMe2 ), 17.3 This journal is © The Royal Society of Chemistry 2009

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(-SiCH2 CH2 CH2 N+ -), 7.8 (-N+ CH2 CH3 ), 7.3 (-SiCH2 CH2 CH2 N+ -), 6.8 (-SiCH2 CH3 ), 2.2 (-SiCH2 CH3 ). 15 N-NMR (DMSO): d -340.4 (N+ HMe2 ), -325.3 (N+ H). 29 Si-NMR (CDCl3 ): d 7.2 (-SiEt3 ).


4.8. Synthesis of G1 -{[Si(CH2 CH2 CH2 )N+ H(Et)(CH2 CH2 N+ HMe2 )]4 8Cl- } (7) To a THF solution (25 mL) of G1 -[Si(CH2 CH2 CH2 )N(CH2 CH3 )(CH2 CH2 NMe2 )]4 (3) (0.46 g, 0.043 mmol) was added a large excess of HCl (1 M) in diethyl ether (7.0 mL, 6.89 mmol). The resulting solution was stirred for 48 h at room temperature and then evaporated under reduced pressure to give 7 as a white water soluble solid (0.55 g, 95%). 1 H-NMR (DMSO): d 11.51, 11.35 (s wide, 8H, N+ H, N+ HMe2 ), 3.54 (m, 16 H, -NH+ CH 2 CH 2 N+ HMe2 ), 3.15 (m, 8H, -N+ CH 2 CH3 ), 3.02 (m, 8H, -SiCH2 CH2 CH 2 N+ -), 2.80 (s, 24H, N+ HMe2 ), 1.72 (m, 8H, -SiCH2 CH 2 CH2 N+ -), 1.24 (m, 20H, -NCH2 CH 3 and -SiCH2 CH 2 CH2 Si-), 0.56 (m, 16H, -SiCH 2 CH2 CH 2 Si-), 0.43 (m, 8H, -SiCH 2 CH2 CH2 N+ -), -0.03 (s, 24H, -SiMe2 ). 13 C-NMR (DMSO): d 54.0 (-SiCH2 CH2 CH2 N+ ), 49.0 (-N+ CH2 CH2 N+ HMe2 ), 46.3 (-N+ CH2 CH3 ), 44.8 (-N+ CH2 CH2 N+ HMe2 ), 41.7 (N+ HMe2), 18.9 (-SiCH2 CH2 CH2 Si-), 17.6 (-SiCH2 CH2 CH2 Si-), 17.1 (-SiCH2 CH2 CH2 Si-), 16.4 11.2 (-SiCH2 CH2 CH2 N+ -), 7.8 (-SiCH2 CH2 CH2 N+ -), (-N+ CH2 CH3 ), -3.9 (-SiMe2 ). 15 N-NMR (DMSO): d -340.3 (N+ HMe2 ), -325.1 (N+ H). 29 Si-NMR (DMSO): d 0.9 (G0 –Si), 2.5 (G1 –Si). C56 H140 Cl8 N8 Si5 (1349.8): calcd. C, 49.83; H, 10.45; N, 8.30; found C, 50.68; H, 11.30; N, 7.43. 4.9. Synthesis of G2 -{[Si(CH2 CH2 CH2 )N+ H(Et)(CH2 CH2 N+ HMe2 )]8 16Cl- } (8) This dendrimer was prepared using a similar method to that described for 7, starting from a THF (25 mL) solution of 4 (0.37 g, 0.015 mmol) and a large excess of HCl (1 M) in diethyl ether (3 mL, 3.05 mmol). This compound, 8, was isolated as a white water soluble solid (0.42 g, 91%). 1 H-NMR (DMSO): d N+ H and N+ HMe2 were not observed, 3.56 (m, 32H, -N+ CH 2 CH 2 N+ Me3 ), 3.17 (m, 16H, -N+ CH 2 CH3 ), 3.03 (m, 16H, -SiCH2 CH2 CH 2 N+ -), 2.81 (s, 48H, N+ HMe2 ), 1.69 (m, 16H, -SiCH2 CH 2 CH2 N+ -), 1.24 (m, 48H, -NCH2 CH 3 and -SiCH2 CH 2 CH2 Si-), 0.53 (m, 48H, -SiCH 2 CH2 CH 2 Si-), 0.44 (m, 16H, -SiCH 2 CH2 CH2 N+ -), -0.03 (s, 48H, -SiMe2 ), -0.10 (s, 12H, -SiMe). 13 C-NMR (DMSO): d 54.0 (-SiCH2 CH2 CH2 N+ -), 49.2 (-N+ CH2 CH2 N+ Me3 ), 46.3 (-N+ CH2 CH3 ), 44.8 (-N+ CH2 CH2 N+ Me3 ), 41.7 (N+ HMe2 ), 18.9 (-SiCH2 CH2 CH2 Si-), 17.6 (-SiCH2 CH2 CH2 Si-), 17.2 (-SiCH2 CH2 CH2 Si-), 16.4 (-SiCH2 CH2 CH2 N+-), 11.2 (-SiCH2 CH2CH2 N+ -), 7.8 (-N+ CH2 CH3 ), -3.9 (-SiMe2 ), -5.3 (-SiMe). 15 NNMR (DMSO): d -340.5 (N+ HMe2 ), -325.0 (N+ H). 29 Si-NMR (DMSO): d (G0 –Si) is not observed, 1.1 (G1 –Si), 2.3 (G2 –Si). C128 H316 Cl16 N16 Si13 (3012.3): calcd. C, 51.04; H 10.57, N 7.44; found C 51.29, H 10.79, N 7.02. 4.10. Synthesis of G3 -{[Si(CH2 CH2 CH2 )N+ H(Et)(CH2 CH2 N+ HMe2 )]16 32Cl- } (9) This dendrimer was prepared using a similar method to that described for 7, starting from a THF solution (25 mL) of 5 (0.32 g, 0.06 mmol) and a large excess of HCl (1 M) in diethyl This journal is © The Royal Society of Chemistry 2009

ether (2.5 mL, 2.45 mmol). Compound 9 was isolated as a white water soluble solid (0.39 g, 100%). 1 H-NMR (DMSO): d 11.5 (br, N+ H, N+ HMe2 ), 3.56 (m, 64H, -N+ CH 2 CH 2 N+ Me3 ), 3.17 (m, 32H, -N+ CH 2 CH3 ), 3.03 (m, 32H, -SiCH2 CH2 CH 2 N+ -), 2.82 (s, 96H, N+ HMe2 ), 1.69 (m, 32H, -SiCH2 CH 2 CH2 N+ -), 1.26 (m, 104H, -NCH2 CH 3 and -SiCH2 CH 2 CH2 Si-), 0.52 (m, 144H, -SiCH 2 CH2 CH 2 Si- and -SiCH 2 CH2 CH2 N+ -), -0.03 (s, 96H, -SiMe2 ), -0.10 (s, 36H, -SiMe). 13 C-NMR (DMSO): d 54.0 (-SiCH2 CH2 CH2 N+ -), 49.0 (-N+ CH2 CH2 N+ Me3 ), 46.3 (-N+ CH2 CH3 ), 44.6 (-N+ CH2 CH2 N+ Me3 ), 41.7 (N+ HMe2 ), 18.9 (-SiCH2 CH2 CH2 Si-), 17.7–17.1 (-SiCH2 CH2 CH2 Si-, -SiCH2 CH2 CH2 Si- and -SiCH2 CH2 CH2 N+ -), 11.2 (-SiCH2 CH2 CH2 N+ -), 7.8 (-N+ CH2 CH3 ), -3.9 (-SiMe2 ), -5.4 (-SiMe). 15 N-NMR (DMSO): d -340.5 (N+ HMe2 ), -325.0 (N+ H). 29 Si-NMR (DMSO): d (G0 –Si) and (G1 –Si) is not observed, 1.1 (G2 –Si), 2.3 (G3 -Si). C272 H664 Cl32 N32 Si29 (6333.4): calcd. C 51.58, H 10.57, N 7.08; found C 50.58, H 10.43, N 7.02. 4.11. Synthesis of {[Et3 Si(CH2 CH2 CH2 )N+ Me(Et)(CH2 CH2 N+ Me3 )] 2I- } (10) To a THF solution (25 mL) of Et3 Si(CH2 CH2 CH2 )N(Et)(CH2 CH2 NMe2 )] (2) (0.315 g, 1.15 mmol) was added an excess of MeI (0.5 mL, 2.31 mmol). The resulting solution was stirred for 48 h at room temperature and then evaporated under reduced pressure to give 10 as a white water soluble solid (0.505 g, 79%). 1 H-NMR (DMSO): d 3.88 (m, 2H, -N+ CH2 CH 2 N+ Me3 ), 3.80 (m, 2H, -N+ CH 2 CH2 N+ Me3 ) 3.38 (m, 2H, -N+ CH 2 CH3 ), 3.29 (m, 2H, -SiCH2 CH2 CH 2 N+ -), 3.19 (s, 9H, -N+ Me3 ), 3.05 (s, 3H, -N+ Me), 1.60 (m, 2H, -SiCH2 CH 2 CH2 N+-), 1.26 (t, 3H, -NCH2 CH 3 ), 0.91(t, 9H, -SiCH2 CH 3 ) 0.50 (q, 6H, -SiCH 2 CH3 ), 0.45 (m, 2H, -SiCH 2 CH2 CH2 N+ -). 13 C-NMR (DMSO): d 63.3 (-SiCH2 CH2 CH2 N+ ), 56.6 (-N+ CH2 CH3 ), 56.0 (-N+ CH2 CH2 N+ Me3 ), 52.4 (-N+ Me3 ), 52.0 (-N+ CH2 CH2 N+ Me3 ), 47.1 (-N+ Me), 15.8 (-SiCH2 CH2 CH2 N+ -), 7.3 (-N+ CH2 CH3 ), 6.8 (-SiCH2 CH3 ), 6.7 (-SiCH2 CH2 CH2 N+ -), 2.1 (-SiCH2 CH3 ). 15 NNMR (DMSO): d = -332.6 (N+ Me3 ), -320.5 (N+ Me). 29 Si-NMR (CDCl3 ): d 7.5 (-SiEt3 ). 4.12. Synthesis of G1 -{[(CH2 CH2 CH2 )N+ (Me)(Et)(CH2 CH2 NMe+ 3 )]4 8I- } (11) To a THF solution (25 mL) of G1 -[(CH2 CH2 CH2 )N(Et)(CH2 CH2 NMe2 )]4 (3) (1.04 g, 0.98 mmol) was added a slight excess of MeI (0.49 mL, 7.88 mmol). The resulting solution was stirred for 48 h at room temperature and then evaporated under reduced pressure to give 11 as a white water soluble solid (0.31 g, 94%). 1 H-NMR (DMSO): d = 3.93 (m, 8 H, -N+ CH2 CH 2 N+ Me3 ), 3.85 (m, 8H, -N+ CH 2 CH2 N+ Me3 ), 3.41 (m, 8H, -N+ CH 2 CH3 ), 3.33 (m, 8H, -SiCH2 CH2 CH 2 N+ -), 3.22 (s, 36H, -N+ Me3 ), 1.63 (m, 8H, -SiCH2 CH 2 CH2 Si-), 1.27 (m, 20H, -SiCH2 CH 2 CH2 Siand -N+ CH2 CH 3 ), 0.57 (16H, -SiCH 2 CH2 CH 2 Si-), 0.43, (m, 8H, -SiCH 2 CH2 CH2 N+ -), 0.005 (s, 24H, -SiMe2 ). 13 C-NMR (DMSO): d = 63.3 (-SiCH2 CH2 CH2 N+ -), 56.6 (-N+ CH2 CH3 ), 56.0 (-N+ CH2 CH2 N+ Me3 ), 52.5 (-N+ Me3 ), 52.0 (-N+ CH2 CH2 N+ Me3 ), 47.2 (-N+ Me), 18.9 (-SiCH2 CH2 CH2 N+ -), 17.6 (-SiCH2 CH2 CH2 Si-), 16.4 (-SiCH2 CH2 CH2 Si-), 16.0 10.6 (-SiCH2 CH2 CH2 N+ -), 7.4 (-SiCH2 CH2 CH2 Si-, + 15 (-N CH2 CH3 ), -3.8 (-SiMe2 ). N-NMR (DMSO): d = -332.1 Dalton Trans., 2009, 8704–8713 | 8711

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(N+ Me3 ), -320.2 (N+ Me). 29 Si-NMR (DMSO): d = 0.7 (G0 –Si), 2.6 (G1 –Si). C64 H156 I8 N8 Si5 (2193.7): calcd. C, 35.04; H, 7.17; N, 5.11; found. C, 36.74; H, 7.15; N, 5.61.


4.13. Synthesis of G2 -{[(CH2 CH2 CH2 )N+ (Me)(Et)(CH2 CH2 NMe+ 3 )]8 16I- } (12) This dendrimer was prepared using a similar method to that described for 11, starting from a THF solution (25 mL) of G2 [(CH2 CH2 CH2 )N(Et)(CH2 CH2 NMe2 )]8 (4) (1.18 g, 0.48 mmol) and MeI (1.2 mL, 7.75 mmol). Compound 12 was isolated as a white water soluble solid (2.11 g, 92%). 1 H-NMR (DMSO): d = 3.95 (m, 16H, -N+ CH2 CH 2 N+ Me3 ), 3.87 (m, 16H, -N+ CH 2 CH2 N+ Me3 ), 3.42 (m, 24H, -N+ CH 2 CH3 ), 3.31 (m, 16H, -SiCH2 CH2 CH 2 N+ -), 3.23 (s, 96H, -N+ Me3 and -N+ Me), 1.63 (m, 16H, -SiCH2 CH 2 CH2 N+ -), 1.27 (m, 56H, -SiCH2 CH 2 CH2 Siand -N+ CH2 CH 3 ), 0.55 (48H, -SiCH 2 CH2 CH 2 Si-), 0.46 (m, 16H, -SiCH 2 CH2 CH2 N+ -), 0.00 (s, 48H, -SiMe2 ), -0,10 (s, 12H, -SiMe). 13 C-NMR (DMSO): d = 63.3 (-SiCH2 CH2 CH2 N+ -), 56.6 (-N+ CH2 CH3 ), 56.0 (-N+ CH2 CH2 N+ Me3 ), 52.5 (-N+ Me3 ), 52.0 (-N+ CH2 CH2 N+ Me3 ), 47.2 (-N+ Me), 18.9 (-SiCH2 CH2 CH2 N+ -), 17.8–16.1 (-SiCH2 CH2 CH2 Si-), 10.6 (-SiCH2 CH2 CH2 N+ -), 7.5 (-N+ CH2 CH3 ), -3.8 (-SiMe2 ) -5.4 (-SiMe). 15 N-NMR (DMSO): d = -331.4 (N+ Me3 ), -320.0 (N+ Me). 29 Si-NMR (DMSO): d = (G0 –Si) is not observed, 1.1 (G1 –Si), 2.8 (G2 –Si). C144 H348 I16 N16 Si13 (4696.0): calcd. C, 36.80, H, 7.46; N, 4.77; found C, 38.26; H, 7.00; N, 4.77. 4.14. Synthesis of G3 -{[(CH2 CH2 CH2 )N+ (Me)(Et)(CH2 CH2 NMe+ 3 )]16 32I- } (13) This dendrimer was prepared using a similar method to that described for 11, starting from a THF solution (25 mL) of (5) (0.31 g, G3 -[(CH2 CH2 CH2 )N(Et)(CH2 CH2 NMe2 )]16 0.06 mmol) and MeI (0.15 mL, 2.37 mmol). Compound 13 was isolated as a white water soluble solid (0.58 g, 99%). 1 H-NMR (DMSO): d = 4.00 (m, 32H, -N+ CH2 CH 2 N+ Me3 ) 3.90 (m, 32H, -N+ CH 2 CH2 N+ Me3 ), 3.42 (m, 64H, -N+ CH 2 CH3 and -SiCH2 CH2 CH 2 N+ - overlapped), 3.25 (s, 144H, -N+ Me3 ), 3.17 (s, 48H, -N+ Me), 1.65 (m, 32H, -SiCH2 CH 2 CH2 Si), 1.29 (m, 104H, -SiCH2 CH 2 CH2 Si- and -N+ CH2 CH 3 ), 0.53 (m, 144H, -SiCH 2 CH2 CH 2 Si- and -SiCH 2 CH2 CH2 N+ overlapped), 0.00 (s, 96H, -SiMe2 ), -0.10 (s, 36H, -SiMe). 13 C-NMR (DMSO): d = 63.3 (-SiCH2 CH2 CH2 N+ -), 56.6 (-N+ CH2 CH3 ), 56.0 (-N+ CH2 CH2 N+ Me3 ), 52.5 (-N+ Me3 ), 52.1 (-N+ CH2 CH2 N+ Me3 ), 47.2 (-N+ Me), 18.9 (-SiCH2 CH2 CH2 N+ -), 17.7–16.1 (-SiCH2 CH2 CH2 Si-), 10.6 (-SiCH2 CH2 CH2 N+ -), 7.5 (-N+ CH2 CH3 ), -3.8 (-SiMe2 ) -5.3 (-SiMe). 15 N-NMR (DMSO): d -332.0 (N+ Me3 ), -320.0 (N+ Me). 29 Si-NMR (DMSO): d (G0 –Si) and (G1 –Si) is not observed, 1.2 (G2 –Si), 2.5 (G3 –Si). ppm. C304 H732 I32 N32 Si29 (9712.7): calcd. C, 37.59; H, 7.60; N, 4.61; found C, 38.26; H, 7.00; N, 4.77. 4.15.

Antimicrobial activity assay

The minimal inhibitory concentration (MIC) of the products was performed in 96-well tray microplates using the international standard methods ISO 20776-1 by microdilution trays preparation.20 The assay was carried out in duplicate microplates with three different wells for each concentration analysed in the microplate. 8712 | Dalton Trans., 2009, 8704–8713

The bacteria used in the analysis were Escherichia coli (Gram-) and Staphylococcus aureus (Gram+). Both strains were obtained ´ Espanola ˜ from the “Coleccion de cultivos tipo” (CECT). A stock solution of the products was obtained by dissolving 0.01024 g of the compound in 10 ml of distilled water. After that, distilled water was added to obtain the desired concentration. The microplates were incubated at 37◦ C using an ultramicroplate reader ELX808iu (Bio-Tek Instruments). The minimal bactericidal concentration (MBC) was calculated by inoculating 100 ml of the samples used to calculate the MIC in a PetriTM dish with Mueller–Hinton agar (peptone 17.5 g l-1 , starch 1.5 g l-1 , solids of meat infusion 2.0 g l-1 , pH = 7.4, Ref. 02-136, Scharlau). After 48 h of incubation at 37 ◦ C the presence of colonies was tested. The MBC was the minimal concentration where not growth was detected.

Acknowledgements This work has been supported by grants from and MNT-ERA ´ NET 2007 (ref. NAN2007-31135-E) and Fondo de Investigacion Sanitaria (ref. PI040993) to UA and FIS (ref. PI061479), Red RIS RD06-0006-0035, FIPSE (24632/07), MNT-ERA NET 2007 (ref. ´ Caja Navarra and Comunidad NAN2007-31198-E), Fundacion de Madrid (S-SAL-0159-2006) to MAMF.

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