water-stable ammonium-terminated carbosilane dendrimers as efficient antibacterial agents

PAPER www.rsc.org/dalton | Dalton Transactions

Water-stable ammonium-terminated carbosilane dendrimers as efficientantibacterial agents†

Beatriz Rasines,a Jose Manuel Hernandez-Ros,a Natividad de las Cuevas,b Jose Luis Copa-Patino,c

Juan Soliveri,c M. Angeles Munoz-Fernandez,b Rafael Gomeza and F. Javier de la Mataa

Received 20th May 2009, Accepted 6th August 2009First published as an Advance Article on the web 27th August 2009DOI: 10.1039/b909955g

A new family of amine- and ammonium-terminated carbosilane dendrimers of the typeGn-[Si(CH2)3N(Et)CH2CH2NMe2]x and Gn-{[Si(CH2)3N+R(Et)CH2CH2N+RMe2]x(X-)y} (where n = 1,2 and 3; R = H, X = Cl; R = Me, X = I) respectively has been synthesized by hydrosilylation ofN,N-dimethyl-N¢-allyl-N¢-ethyl-ethylenediamine, [(CH2=CH–CH2)(Et)N(CH2)2NMe2] with thecorresponding hydride-terminated dendrimers and subsequent quaternization with HCl orMeI. Quaternized dendrimers are soluble and stable in water or other protic solvents for long timeperiods. The antibacterial properties of the quaternary ammonium functionalized dendrimers havebeen evaluated showing that they act as potent biocides in which the multivalency along with thebiopermeability of the carbosilane dendritic skeleton play an important role in the antibactericidalactivity of these compounds.

1. Introduction

Monovalent molecules like traditional antibiotics have been widelyused in treating and controlling bacterial infections, however newbiocide drugs are needed due to the increasing cases of antibioticresistance among human and animal bacteria.1 One approach forthe search of new types of antibacterial agents consists of theuse of polyvalent interactions to treat infections.2 Conventionalpolymeric systems have demonstrated high activity as biocides dueto having a high local density of active groups in close vicinity witha clear relationship between structure and activity in some cases.3

In this context, dendrimers emerge as an alternative approachto traditional polymers. In addition to the presence of multiplesites of attachment and the versatility to modify their skeletonsand surfaces, their major advantage is their uniform structure,ideally monodisperse, which permits a precise characterization ofthe system structure and thus a more accurate and systematicanalysis of the biomedical responses.4

Surface charged dendritic systems are membrane disruptive,causing the formation of holes followed by the leakage of cytosolicprotein and the entry of dendrimers into the cells normally via anendocytosis-independent mechanism.5 As a general rule, antibac-terial dendrimers are cationic, though some anionic dendrimers

aDepartamento de Quımica Inorganica, Universidad de Alcala, Cam-pus Universitario. Edificio de Farmacia, E-28871 Alcala de Henares(Spain). Networking Research Center on Bioengineering, Biomaterialsand Nanomedicine (CIBER-BBN), Spain. E-mail: [email protected],[email protected]; Fax: (+34) 91 885 4683; Tel: (+34) 91 8854654bLaboratorio de Inmunobiologıa Molecular, Hospital General UniversitarioGregorio Maranon, Madrid, Spain. Networking Research Center on Bioengi-neering, Biomaterials and Nanomedicine (CIBER-BBN), Spain. E-mail:[email protected] de Microbiologıa y Parasitologıa, Universidad de Alcala,E-28871, Alcala de Henares, Spain. E-mail: [email protected]† Electronic supplementary information (ESI) available: Selected data asNMR spectra and MALDI-TOF mass spectrometry of derivatives 1–13.See DOI: 10.1039/b909955g

have been reported.6,7 As a working model described elsewhere,8,9

the mode of action is based on the adsorption of cationic systemsonto the usually negatively charged bacterial cell surface, diffusionthrough the cell wall, binding to the cytoplasmic membrane withsubsequent disruption of the membrane. The destabilization oflipid membranes involves the displacement of the membrane-stabilizing divalent cations, which are extremely important inthe neutralization of the anionic groups of the phospholipidmembrane. In the case of cationic dendrimers such displacementmay occur through competition with the divalent cations such ascalcium or in the case of anionic dendrimers by scavenging thecations.

One may expect that the strength of the membrane disruptionand hence the biocide efficiency of dendrimers may depend ontwo major factors: (i) the number of charged groups mainlylocated on the surface, because the polycationic nature mayincrease the binding rate during the initial adsorption step. Inthis sense, a higher activity may be expected when increasing thedendrimer generation; (ii) the biopermeability of the dendriticsystem which may depend on parameters like dendrimer size,because the permeability of biocides through the cell wall shouldbe lower for polymeric systems in comparison to small molecules,or higher due to the presence of appropriate lipophilic groupsor domains like the presence of long aliphatic chains at thedendritic surface. It has been reported that quaternary ammoniumfunctionalized poly(propyleneimine) dendrimers (PPI) behave asvery potent biocides against both Gram-positive and Gram-negative bacteria and the antibacterial properties depend onthe dendrimer generation, the length of hydrophobic chains andthe counteranion.8 Dendrimeric peptides have also been used inorder to generate efficient bacteriocides. In this case, the use ofpeptides did not show any antimicrobial activity on their own,but become broadly active when conjugated to an asymmetricdendritic polylysine core.9 Another example is the use of amino-terminated polyamidoamine dendrimers (PAMAM) and their

8704 | Dalton Trans., 2009, 8704–8713 This journal is © The Royal Society of Chemistry 2009

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http://dx.doi.org/10.1039/B909955G

partially PEG-coated derivatives which possess attractive antimi-crobial 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 andGram-negative bacteria being comparatively higher or equipotentto antibiotics and antifungal agents.7

We have recently reported the synthesis of cationic carbosilanedendrimers as a new type of systems useful for biomedicalapplications.11 Novel amine- and ammonium-terminated carbosi-lane dendrimers of the type Gn-[Si{CH2O-(C6H4)-3-NMe2}]x orGn-[Si{CH2O-(C6H4)-3-NMe3

+I-}]x have been reported elsewhereand synthesized up to the second generation.12 A study of theantimicrobial activity of these cationic dendrimers of the firstand second generation against both Gram-positive and Gram-negative bacteria has been described showing that the newammonium-terminated carbosilane dendrimers can be consideredas multivalent biocides. However, these quaternized carbosilanedendrimers are not soluble in water, though they can be solubilisedafter addition of less than 1% of dimethyl sulfoxide. Here wedescribe the synthesis of a new family of water-soluble and-stable cationic carbosilane dendrimers up to the third generationthat overcome the water insolubility problem and allow therationalization of the role of the carbosilane skeleton in theirbehaviour as antibacterial agents. The synthetic approach shownhere constitutes an important procedure for water-soluble and-stable cationic carbosilane dendrimers as very lipophilic systems,only prepared by two different groups using different syntheticapproaches.13,14

2. Results and discussion

2.1. Synthesis and characterization of dendrimers

We have designed new amine-terminated carbosilane dendrimersby the hydrosilylation reaction of Si–H terminated dendrimerswith 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 leadsto a lithium salt that is not isolated and is treated in situ with allylbromide to give N,N-dimethyl-N¢-allyl-N¢-ethyl-ethylenediamine,[(CH2=CHCH2)(Et)N(CH2)2NMe2] (1) (see Scheme 1). The finalproduct is initially isolated with lithium bromide as an adduct(see Experimental Section) as a white crystalline solid but can bepurified by washing with a water solution of sodium carbonateand subsequent extraction with diethyl ether to give 1 in a 60%yield as a yellow oil. The 1H NMR spectra of 1 and 1·LiBr areslightly different, with a general deshielding in the case of theadduct product (see Experimental section).

Scheme 1

Triethylsilane and Si–H terminated carbosilane dendrimersof 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 ampouleswith J. Young valves, using toluene as solvent and the Karstedtcatalyst during 12 h at 120 ◦C, to afford the correspondingsystem [Et3Si(CH2)3N(Et)CH2CH2NMe2] (2) and dendrimersGn-[Si(CH2)3N(Et)CH2CH2NMe2]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 ofperipheral units. The hydrosilylation reaction occurred exclusivelyby b-addition and any by-products were detected in the 1H NMRspectra of the crude products. All of these dendrimers are solublein chlorinated solvents and aromatic and aliphatic hydrocarbonsbut insoluble in water.

Scheme 2

This journal is © The Royal Society of Chemistry 2009 Dalton Trans., 2009, 8704–8713 | 8705

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The NMR spectroscopic, MALDI-TOF MS and analyticaldata for compounds 2–5 are consistent with their proposedstructures (Scheme 2 and see ESI† for structures). The 1H NMRspectra of compounds 2–5 show, for the carbosilane skeleton,almost identical chemical shifts for analogous nuclei in differentgenerations, although broader and less structured resonancesare present with increasing generation. For the SiCH2CH2CH2Sibranches two broad multiplets are observed, one due to themiddle methylene groups centered at d = 1.30 ppm, whilst themethylene groups bonded directly to silicon atoms are locatedaround d = 0.60 ppm. With respect to the outer group, fourmethylene groups bonded to nitrogen atoms (Fig. 1) appear astwo multiplets, one at about d = 2.50 ppm that we ascribed tothe signals due to the protons of the methylene groups f and doverlapped, and the other at d = 2.40 ppm due to the protonsof the methylene groups c and e that are also overlapped. Theresonances at d = 1.40 ppm and d = 0.42 ppm are assignedto methylene groups b and a respectively. The methyl fragmentof the ethyl group bonded to nitrogen appears as a triplet atd = 1.10 ppm, and the methyl protons of the dimethyl aminefragment gives a singlet at d = 2.30 ppm. This assignmenthas been performed on the basis of NOESY 1D, TOCSY 1Dand gHMBC-{1H-15N} experiments. The 13C NMR spectra forthe methylene groups bonded to nitrogen show four resonanceslocated at d = 57.9 (c), 57.7 (e), 51.6 (d) and 48.0 (f) ppm based onHMQC-{1H-13C} and HMBC-{1H-13C} experiments. The –NMe2

group gives a singlet at d = 45.9 ppm and the methyl fragmentof the ethyl group bonded to nitrogen appears as a singlet atd = 11.7 ppm. The methylene groups of the SiCH2CH2CH2Sibranches gives signals in the range d = 21.5 to 17.7 ppm (thenumber of signals depends on the dendrimer generation). Finally,methyl groups bonded to silicon give signals in the expected zone,around 0 ppm, in both 1H and 13C NMR spectra. The 29Si-NMRspectra show signals with the expected values although the signalscorresponding to the most internal silicon atoms are generallynot observed. This spectroscopic behaviour has been observedin other related carbosilane dendrimers.11b,16 Finally, 15N-NMRspectra of complexes 2–5 show two signals around -356 and-337 ppm corresponding to the outer and inner nitrogen atoms ofeach 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 wereobtained for dendrimers 3 and 4 using dithranol as the matrix inwhich the molecular peaks were identified. No molecular peakwas detected for the third generation dendrimer 4 where theionization becomes more difficult.16a,18 Finally, the hydrodynamicdiameters have been determined for dendrimers 3–5 using DOSYexperiments in chloroform as solvent at 25 ◦C, and reveals a value

of 1.28 nm for the first generation (3), 1.58 nm for the secondgeneration (4) and 1.92 nm for the third generation (5), whichconfirm their nanoscopic dimension.

The ammonium-terminated dendrimers were cleanly pre-pared by adding a little excess of HCl or MeI to parentderivatives 2–5 in diethyl ether (see Scheme 2). The addi-tion of HCl leads to the quaternized systems {[Et3Si(CH2)3-N+(H)(Et)CH2CH2N+HMe2](Cl-)2} (6) and Gn-{[Si(CH2)3N+(H)-(Et)CH2CH2N+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. Additionof MeI over the amine functionalized dendrimers also producesthe corresponding cationic ammonium-terminated derivativesof type {[Et3Si(CH2)3N+(Me)(Et)CH2CH2N+Me3](I-)2}(10) andGn-{[Si(CH2)3N+(Me)(Et)CH2CH2N+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 whitesolids, soluble in water, alcohols (like methanol or ethanol), anddimethylsulfoxide. Both cationic non-dendrimer and dendrimersystems are stable in protic solvents and can be stored withoutdecomposition for long time periods.

The NMR spectroscopic and analytical data for compounds6–13 are consistent with their proposed structures (Scheme 2 andFig. 2). The 1H NMR spectra were recorded in DMSO-d6 or D2Oat room temperature. In these solvents the line widths of thesespectra tended to be broader than those of the derivatives solublein common organic solvents. The 1H and 13C NMR spectra ofthe quaternized dendrimers exhibited identical resonance patternsto those observed in their neutral counterparts 3–5 for thecarbosilane framework, although broader signals were seen withincreasing generation (see Experimental section and ESI†).

In general for the 1H NMR spectra (see Fig. 1), the quaterniza-tion of the amine groups result in a deshielding of the chemicalshifts of about 0.8–1.5 ppm of the geminal methylene (c, d, eand f ) and methyl (h and i) groups directly bound to the chargednitrogens, whereas small downfield shifts are detected around d =0.3–0.4 ppm for the vicinal methylene and methyl groups (b andg). Beyond these positions, no displacement is observed on thechemical shift due to the positive charge on the nitrogen atoms.However, this effect is more evident in the quaternized systemsby MeI than those prepared using HCl. Analogous behaviouris observed for the carbon atoms in the 13C NMR spectra.For the methylene groups bonded to nitrogen in the derivativesquaternized by MeI (10–13), the four resonances are located atd = 63.3 (c), 56.6 (f ), 56.0 (e) and 51.9 (d) ppm. Respectingthe methyl groups bound to nitrogen, these appear at d = 52.4(h) and 47.1 (i) ppm. In the case of systems quaternized withHCl (6–9), the geminal groups attached to nitrogen are located atd = 54.2 (c), 49.5 (f ), 46.3 (e) and 44.9 (d) ppm for methylenesand d = 41.8 ppm for the methyl groups. Again, the 1H and 13Cresonance assignments are based on TOCSY 1D, HMQC-{1H-13C} and HMBC-{1H-13C} experiments.

The 15N-NMR spectra of derivatives 6–9 quaternized with HClshow two signals at about d = -340 and -325 ppm while forcompounds 10–13 quaternized by MeI an expected deshieldingis observed at d = -331 and -320 ppm, corresponding in bothcases to the outer and inner nitrogen atoms, respectively. Bothsituations mean a downfield shifting with respect to the neutralcounterparts, 2–5, as a consequence of the positive charge presenton both nitrogen atoms.17 The presence of only these two signals


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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 thesedendrimers are quaternized even at the higher generation.

Attempts to prepare mono quaternized amine derivatives faileddue to the difficulty to reproduce the synthetic procedure.The stoichiometric reaction of dendrimer 4 containingeight units with identical equivalents of MeI gave thecorresponding outermost quaternized terminal amine derivativeG2-{[Si(CH2)3N(Et)CH2CH2N+Me3]8(I-)8} (14) with smallamounts of non-methylated branches on the basis of the HMBC-{1H-15N} experiment. Dendrimer 14 shows two signals at about

d = -331 and -342 ppm corresponding to the outer and innernitrogen atoms of each branch respectively, in addition to twoweak signals located at d = -356 and -339 ppm corresponding tothe non-methylated branches. In any case, signals attributed tothe mono-methylated forms holding the innermost quaternizedamine or double-methylated branches were observed, even whena defect of the quaternized agent was added. This feature stronglysuggests that the quaternization process starts in the outermostamine and subsequently proceeds to the innermost when a slightexcess of MeI is added.


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

10 11 12 13 8 Penicillin G

MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC

Escherichia coli (-) >920 >920 3.65 3.65 1.70 1.70 1.65 1.65 42.49 84.98 766.0 766.0Staphylococcus aureus (+) 115 230 0.46 1.82 0.85 1.70 0.82 0.82 42.49 84.98 0.09 0.19

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

2.2. Antibacterial activity of dendrimers

We have evaluated the antibacterial activity of dendrimers 11–13containing iodide as counteranion as well as their monofunctionalcounterpart 10, on Gram+ and Gram- bacteria. In particular, wehave measured the MIC (minimum inhibitory concentration) andMBC (minimum bactericidal concentration) of our dendrimersagainst Staphylococcus aureus (Gram+) and Escherichia coli(Gram-). The term MIC states the minimal concentration of anagent that can inhibit the growth of a microorganism (bacterio-static effect) while MBC indicates the minimal concentration ofan agent that can kill a microorganism (bactericidal effect). Thevalue of MIC is always less than or equal to MBC for the samebiocide and microorganism. Data for these activities are shown inTable 1.

The results of the antibacterial tests clearly show a high activityfor the carbosilane dendrimers both against Gram+ and Gram-bacteria in contrast to their monofunctional counterpart 10,since they are over two orders of magnitude more potent. Thissignificant improvement of biocidal action of dendritic biocidesmay be due to the high number of functional groups locatedin a compact space and their polycationic structure. Moreover,the data reveal lower MIC and MBC values for Gram+ bacteriathan for Gram- bacteria, in all the dendrimer generations used.This difference has been observed by other authors in lysinebased-dendrimers9 and may arise from differences in the cell wallstructure. In Gram+ bacteria, there is a membrane formed bya single bilayer while in Gram- bacteria it is composed of twobilayer membranes, making the latter bacteria more resistant toan external attack. Interestingly, in the case of Gram- bacteriaboth bacteriostatic and bactericidal effects are identical for eachgeneration meaning that at such a concentration the dendriticsystem not only inhibits bacterial growth but also kills the bacteria.For Gram+ bacteria, this behaviour is only observed when usingthe higher generation. The antibacterial activity of carbosilanedendrimers depends on the increasing generation: 13 > 12 > 11regarding 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 cationicdendrimers are two orders of magnitude more active againstGram- 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 highpotential as antibacterial agents.

As mentioned above, the strength of the membrane disruptiondepends basically on two parameters: (i) the number of chargedgroups and (ii) the biopermeability processes (size and molecularweight or lipophilic groups or domains). Concerning the first one,

when increasing the generation, the number of quaternary groupsgrows and thus the system should be more potent. However,regarding the second parameter, if we take the size and molecularweight of the dendritic system into account this trend shouldbe the opposite. The dendrimers used here are small enough tobe able to cross the cell wall of both types of bacteria sincethe theoretical molecular weights range from 2193 to 9713 andthe size measured by DOSY on neutral systems is around 1.3–2.0 nm of diameter. Another aspect of the biopermeability is thepresence and length of hydrophobic groups on the quaternizednitrogen atoms. From several studies on quaternary ammoniumsalts, conventional cationic polymers or ammonium-terminatedPPI dendrimers, a parabolic relationship has been observed inwhich a chain length of C10–C16 tends to contribute the mosteffective lipophilic groups for biocide activity.3b,8 It is worth notingthat the lipophilicity of the carbosilane dendrimers is differentin comparison to that shown by other related dendritic systemswhere the lipophilic groups are attached on the surface.8 In thecase of dendrimers 11–13, the ammonium groups are bound toa very lipophilic skeleton which may aid in the biopermeabilityprocesses. Such lipophilicity may increase on going from the firstto the third generation. The combination of all these factors givesa complicated response but the fact that the antibacterial activityincreases with the generation implies that the outer chargedfunctional groups along with the lipophilicity of the dendriticskeleton play an important role in the biocide action. However,if the antibacterial activity is determined taking into account theconcentration per branch, a slightly different scenario is found.Although, it is clear the existence of a dendritic effect in bothtypes of bacteria, for Gram- the MBC values are of the samemagnitude for the first (11) and second (12) generation and doublefor the third (13), while for Gram+ bacteria, the second and thirdgenerations are of the same magnitude and a half value is foundfor the first generation. These data suggest that the antibacterialactivity is not only proportional to the multivalency (number ofcharges) or to the lipophilicity of the dendrimer but also increasessynergistically up to a maximum centered on the first or secondgeneration depending on the type of bacteria. From this fact, thefinal 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 biocideefficiency of these molecules. Such an influence has been studiedand the results are shown in Table 1. According to the MICvalues, the second generation dendrimer 8 containing chlorideas the counteranion is about 12 times and 50 times less effectiveagainst Gram+ and Gram- bacteria respectively than dendrimer12 having iodide as the counteranion. The same behaviour hasbeen observed in PPI dendrimers8b though different values arenot expected since in both types of dendrimers the ions tend


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to dissociate freely in water. An explanation emerges from thefact that iodides form weaker anionic pairs with ammonium unitsthan chloride anions, leading to more exposed cations and thusstronger electrostatic attraction to the negatively charged bacterialmembranes. However, in this case the different nature of theammonium units may also be responsible for such a differentbehaviour. Dendrimer 8 seems to be more hydrophilic than thecorresponding counterpart 12 because of the presence of N–Hbonds, decreasing the biopermeability and thus the antibacterialactivity.

3. Conclusions

A new family of amine- and ammonium-terminated carbosilanedendrimers has been synthesized by the hydrosilylation reactionof N,N-dimethyl-N¢-allyl-N¢-ethyl-ethylenediamine, [(CH2=CH–CH2)(Et)N(CH2)2NMe2] (1) with hydride-terminated dendrimersand subsequent quaternization with HCl or MeI. These den-drimers have been fully characterized using elemental analysis,NMR spectroscopy and mass spectrometry. Quaternized den-drimers are soluble and stable in water or other protic solvents forlong time periods (NMR solutions of these dendrimers show thesame spectra after several months). The antimicrobial studies showthat the multivalency of dendrimers plays an important role in theantibactericidal activity of these compounds, since the dendrimerbiocides are over two orders of magnitude more potent than theirmonofunctional counterpart. The activity increases on increasingthe generation mainly for the MBC values although taking intoaccount the concentration per branch a maximum of activity iscentered on the first or second generation depending on the type ofbacteria. The different behaviour observed in the MIC for Gram-and Gram+ bacteria may be related to the different structure of thecell wall of these systems. In addition, the different nature of theperipheral ammonium salt in the dendrimers also contributes tomodulate the antibacterial activity. A comparison of the activityof dendrimers 11–13 with the penicillin G antibiotic reveals thehigh potential of these systems as biocides.

4. Experimental

4.1. General remarks

All manipulations of oxygen- or water-sensitive compounds werecarried out under an atmosphere of argon using standard Schlenktechniques or an argon-filled glove box. Solvents were dried andfreshly distilled under argon prior to use: hexane from sodium-potassium, toluene from sodium, tetrahydrofuran and ethyl etherfrom sodium benzophenone ketyl, and methylene chloride overP4O10.19 Unless otherwise stated, reagents were obtained fromcommercial sources and used as received. Et3SiH or the analogoushydride-terminated carbosilane dendrimers of different genera-tions Gn-(SiH)x were prepared according to reported methods.15

1H, 13C, 15N and 29Si NMR spectra were recorded on VarianUnity VXR-300 and Varian 500 Plus Instruments. Chemical shifts(d , ppm) were measured relative to residual 1H and 13C resonancesfor chloroform-d1, DMSO-d6 and water-d2 used as solvents.The 15N and 29Si chemical shifts were referenced to externalCH3NO2 and SiMe4 (0.00 ppm) respectively. The NMR signalassignments of the compounds have been performed by different

1D and 2D NMR experiments (COSY, TOCSY, ROESY {1H-1H},HSQC, HMBC{1H-13C}, HMBC{1H-15N} or HMBC{1H-29Si}where appropriate). C, H and N analyses were carried out witha Perkin-Elmer 240 C microanalyzer. MALDI-TOF MS sampleswere prepared in a 1,8,9-trihydroxyanthracene (dithranol) matrix,and spectra were recorded on a Bruker Reflex II spectrometerequipped with a nitrogen laser emitting at 337 nm and operatedin the reflection mode at an accelerating voltage in the range23–25 kV.

DOSY experiments. The diffusion coefficients were measuredin chloroform at 25 ◦C by using the Dbppste (DOSY Bipolar PulseSimulated Echo) pulse sequence in aVarian NMRSystem 500equipped with a high accuracy variable temperature unit (± 0.1 ◦C),a Performa IV PFG amplifier, and a Z-PFG Triple Resonance5-mm probe. Fine calibration of the PFG strength (DAC to Gunit) was performed with an H2O/HDO (2 Hz) sample as standardsupplied by Varian (D = 19.04 ¥ 10-10 m2 s-1 at 25 ◦C). The diffusionNMR data were acquired over 64 scans, with settings pw90, anacquisition time of 3 s, a relaxation delay of 2 s, in each one ofthe 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). Theexperimental data (32 K ¥ 1 K) was treated with the “DOSY”software from VNMRJ2.1B. The hydrodynamic radii, RH, werecalculated from the diffusion coefficients of a certain molecularspecies using the Stokes–Einstein equation RH = kBT/6pg D wherekB is the Boltzmann constant, T the absolute temperature, and gthe viscosity of the solution.

4.2. Synthesis of [(CH2=CHCH2)N(Et)(CH2CH2NMe2)] (1)

To a solution of the amine [H(Et)NCH2CH2NMe2] (7.5 g,64.5 mmol) in Et2O (250 mL) cooled at -70 ◦C was added anothersolution of n-BuLi drop by drop (64.5 mmol, 2.5 M in Et2O). Themixture was stirred for 1 h at room temperature, then cooled againat -70 ◦C and 1 equivalent of CH2=CHCH2Br (7.81 g, 64.5 mmol)added and stirred for 12 h at room temperature affording a whitecrystalline solid of the adduct 1·LiBr. The off-salt ligand wasobtained by treating the solid with a water solution of Na2CO3

and the subsequent extraction with Et2O (3 ¥ 50 mL). The organicphase was dried with MgSO4, the solution filtered and the volatilesremoved under vacuum to give 1 as a yellow oil (4.5 g, 45% yield).

Data for 1. 1H-NMR (CDCl3): d 5.83 (m, 1H, CH2=CHCH2-),5.10 (m, 2H, CH2=CHCH2-), 3.08 (d, 2 H, CH2=CHCH2-),2.52 (m, 4H, -NCH2CH2NMe2 and -NCH2CH3), 2.35 (m,2H, -NCH2CH2NMe2), 2.19 (s, 6H, -NMe2), 0.99 (t, 3H,-NCH2CH3). 13C-NMR (CDCl3): d 135.8 (CH2=CHCH2-),117.2 (CH2=CHCH2-), 57.5 (-NCH2CH2NMe2), 57.2(CH2=CHCH2-), 51.0 (-NCH2CH2NMe2), 47.7 (-NCH2CH3),45.9 (-NCH2CH2NMe2), 11.6 (-NCH2CH3). 15N-NMR (CDCl3):d -355.8 (NMe2), -338.6 (NCH2CH3). C9H20N2: 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. 1H-NMR (CDCl3): d 6.14 (m, 1H,CH2=CHCH2-), 5.18 (m, 2H, CH2=CHCH2-), 3.28 (d, 2H, CH2=CHCH2-), 2.71 (q, 2H, -NCH2CH3), 2.52 (m,2H, -NCH2CH2NMe2 or -NCH2CH2NMe2), 2.39 (m, 2H, -NCH2CH2NMe2 or -NCH2CH2NMe2), 2.32 (s, 6H, -NMe2), 1.09


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(t, 3H, -NCH2CH3). BrC9H20LiN2: calcd. C, 44.46; H, 8.29; N,11.52; found C, 44.32; H, 7.70; N, 11.56.

4.3. Synthesis of [(Et3SiCH2CH2CH2)N(Et)(CH2CH2NMe2)](2)

To 1 equivalent of diamine [(CH2=CHCH2)N(Et)(CH2CH2-NMe2)] (1) (0.284 g, 1.82 mmol), was added 1 equivalentof HSiEt3 (0.21 g, 1.82 mmol) in 3 mL of toluene, andthen one drop of the Karsted catalyst was added. The re-action mixture was stirred for 12 h at 120 ◦C and thenevaporated to dryness to remove the solvent. Compound2 was obtained as a brown oil (0.36 g, 73%). 1H-NMR(CDCl3): d 2.51 (m, 4H, -NCH2CH3 and -NCH2CH2NMe2),2.36 (m, 4H, -NCH2CH2NMe2 and -SiCH2CH2CH2N-), 2.21(s, 6H, -NMe2), 1.40 (m, 2H, -SiCH2CH2CH2N-), 0.99(t, 3H, -NCH2CH3), 0.89 (t, 9H, -SiCH2CH3), 0.47 (q, 6H,-SiCH2CH3), 0.41 (q, 2H, -SiCH2CH2CH2N-). 13C-NMR(CDCl3): d = 58.1 (-SiCH2CH2CH2N-), 57.7, (-NCH2CH2NMe2),51.6 (-NCH2CH2NMe2), 47.9 (-NCH2CH3), 45.9 (-NMe2), 21.3(-SiCH2CH2CH2N-), 11.6 (-NCH2CH3), 8.9 (-SiCH2CH2CH2N-),7.3(-SiCH2CH3), 3.2 (-SiCH2CH3). 15N-NMR (CDCl3): d -355.7(-NMe2), -338.1 (-NCH2CH3). 29Si-NMR (CDCl3): d 7.0 (-SiEt3).C15H36N2Si: calcd. C, 66.17; H, 13.23; N, 10.29; found C, 65.41;H, 12.83; N, 9.57.

4.4. Synthesis of G1-[Si(CH2CH2CH2)N(Et)(CH2CH2NMe2)]4

(3)

To 4.2 equivalents of diamine [(CH2=CHCH2)N(Et)(CH2CH2-NMe2)] (1) (1.19 g, 7.60 mmol), was added a toluene solu-tion (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 andthen evaporated to dryness to remove the solvent. Compound3 was obtained as a brown oil (1.85 g, 97%). Further pu-rification was accomplished by exclusion chromatography us-ing toluene as eluent. 1H-NMR (CDCl3): d 2.51 (m, 16H,-NCH2CH3 and -NCH2CH2NMe2), 2.36 (m, 16H, -NCH2CH2

NMe2 and -SiCH2CH2CH2 N-), 2.20 (s, 24H, NMe2), 1.39(m, 8H, -SiCH2CH2CH2N-), 1.26 (m, 8H, -SiCH2CH2CH2Si-),0.98 (t, 12H, -NCH2CH3), 0.52 (m, 16H, -SiCH2CH2CH2Si-),0.38 (m, 8H, -SiCH2CH2CH2N-), -0.08 (s, 24H, -SiMe2).13C-NMR (CDCl3): d = 57.9 (-SiCH2CH2CH2N-), 57.7,(-NCH2CH2NMe2-), 51.6 (-NCH2CH2NMe2-), 48.0 (-NCH2CH3),45.9 (NMe2), 21.4 (-SiCH2CH2CH2N-), 20.3 (-SiCH2CH2CH2Si-),18.5 (-SiCH2CH2CH2Si-), 17.5 (-SiCH2CH2CH2Si-), 13.0 (-SiCH2CH2CH2N-), 11.7 (-NCH2CH3), -3.3 (-SiMe2). 15N-NMR(CDCl3): d -356.0 (NMe2), -337.8 (NCH2CH3). 29Si-NMR(CDCl3): d 0.6 (G0-Si), 1.95 (G1-Si). Assignments are based onTOCSY 1D, HMQC-{1H-13C} and HMBC-{1H-13C}, HMBC-{1H-28Si} and HMBC-{1H-15N} experiments. C56H132N8Si5: 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)

This dendrimer was prepared using a similar method to thatdescribed for 3 starting from 8.2 equivalents of diamine,

[(CH2=CHCH2)N(Et)(CH2CH2NMe2)] (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%). 1H-NMR(CDCl3): d = 2.51 (m, 32 H, -NCH2CH3 and -NCH2CH2NMe2),2.36 (m, 32 H, -NCH2CH2 NMe2, -SiCH2CH2CH2 N-), 2.21(s, 48 H, -NMe2), 1.38 (m, 16 H, -SiCH2CH2CH2N-), 1.27 (m,24 H, -SiCH2CH2CH2Si-), 0.98 (t, 24 H, -NCH2CH3), 0.52 (m,48 H, -SiCH2CH2CH2Si-), 0.38 (t, 16 H, -SiCH2CH2CH2N-),-0.08 (s, 48H, -SiMe2), -0.012 (s,12 H, -SiMe). 13C-NMR(CDCl3): d 57.9 (-SiCH2CH2CH2N-), 57.7 (-NCH2CH2NMe2),51.7 (-NCH2CH2NMe2), 48.0 (-NCH2CH3), 45.9 (NMe2), 21.5(-SiCH2CH2CH2N-), 20.1–17.7 (-SiCH2CH2CH2Si- and -SiCH2-CH2CH2Si-), 13.0 (-SiCH2CH2CH2N-), 11.7 (-NCH2CH3), -3.3(-SiMe2), -5.0 (-SiMe). 15N-NMR (CDCl3): d = -356.0 (NMe2),-337.8 (NCH2CH3). 29Si-NMR (CDCl3): d (G0–Si) is not ob-served, 1.0 (G1–Si), 1.9 (G2–Si). C128H300N16Si13 (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.


(5)

This dendrimer was prepared using a similar method to thatdescribed for 3, starting from 18 equivalents of diamine,[(CH2=CHCH2)N(Et)(CH2CH2NMe2)] (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%). 1H-NMR (CDCl3): d 2.53 (m, 64 H, -NCH2CH3 and-NCH2CH2NMe2), 2.41 (m, 64 H, -NCH2CH2NMe2 and-SiCH2CH2CH2N-), 2.21 (s, 96 H, NMe2), 1.40 (m, 32 H,-SiCH2CH2CH2N-), 1.27 (m, 56 H, -SiCH2CH2CH2Si-), 0.98 (t,48 H, -NCH2CH3), 0.52 (m, 112 H, -SiCH2CH2CH2Si-), 0.38 (t,32H, -SiCH2CH2CH2N-), -0.08 (s, 96H, -SiMe2), -0.012 (s, 36H, -SiMe). 13C-NMR (CDCl3): d 58.0 (-SiCH2CH2CH2N-),57.8 (-NCH2CH2NMe2), 51.7 (-NCH2CH2NMe2), 48.0(-NCH2CH3), 46.0 (-NMe2), 21.5 (-SiCH2CH2CH2N-),20.2- 18.5 (-SiCH2CH2CH2Si- and -SiCH2CH2CH2Si-), 13.1(-SiCH2CH2CH2N-), 11.7 (-NCH2CH3), -3.2 (-SiMe2), -4.9(-SiMe). 15N-NMR (CDCl3): d -356.0 (NMe2), -338.0(-NCH2CH3). 29Si-NMR(CDCl3): d (G0-Si) and (G1-Si) arenot observed, 0.9 (G2–Si), 1.9 (G3–Si). C272H636N32Si29 (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 {[Et3Si(CH2CH2CH2)N+H(Et)-(CH2CH2N+HMe2)] 2Cl-} (6)

To a THF solution (25 mL) of [Et3Si(CH2CH2CH2)-N(Et)(CH2CH2NMe2)] (2) (0.42 g, 1.5 mmol) was added an excessof HCl (1 M) in diethyl ether (7.0 mL, 6.89 mmol). The resultingsolution was stirred for 48 h at room temperature and thenevaporated under reduced pressure to give 6 as a white watersoluble solid (0.51 g, 96%). 1H-NMR (DMSO): d 11.30, 11.10 (swide, 2H, N+H, N+HMe2), 3.47 (m, 4 H, -NH+CH2CH2N+HMe2),3.10 (m, 2H, -N+CH2CH3), 3.02 (m, 2H, -SiCH2CH2CH2N+-),2.80 (s, 6H, N+HMe2), 1.65 (m, 8H, -SiCH2CH2CH2N+-),1.22 (t, 3H, -NCH2CH3), 0.91(t, 9H, SiCH2CH3) 0.49 (m,8H, -SiCH2CH3 and -SiCH2CH2CH2N+-). 13C-NMR (DMSO):d 54.2 (-SiCH2CH2CH2N+), 49.0 (-N+CH2CH2N+Me3), 46.4(-N+CH2CH3), 44.7 (-N+CH2CH2N+Me3), 41.8 (N+HMe2), 17.3


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(-SiCH2CH2CH2N+-), 7.8 (-N+CH2CH3), 7.3 (-SiCH2CH2CH2

N+-), 6.8 (-SiCH2CH3), 2.2 (-SiCH2CH3). 15N-NMR (DMSO):d -340.4 (N+HMe2), -325.3 (N+H). 29Si-NMR (CDCl3): d 7.2(-SiEt3).

4.8. Synthesis of G1-{[Si(CH2CH2CH2)N+H(Et)-(CH2CH2N+HMe2)]4 8Cl-} (7)

To a THF solution (25 mL) of G1-[Si(CH2CH2CH2)N(CH2-CH3)(CH2CH2NMe2)]4 (3) (0.46 g, 0.043 mmol) was addeda large excess of HCl (1 M) in diethyl ether (7.0 mL,6.89 mmol). The resulting solution was stirred for 48 hat room temperature and then evaporated under reducedpressure to give 7 as a white water soluble solid (0.55 g,95%). 1H-NMR (DMSO): d 11.51, 11.35 (s wide, 8H, N+H,N+HMe2), 3.54 (m, 16 H, -NH+CH2CH2N+HMe2), 3.15 (m,8H, -N+CH2CH3), 3.02 (m, 8H, -SiCH2CH2CH2N+-), 2.80(s, 24H, N+HMe2), 1.72 (m, 8H, -SiCH2CH2CH2N+-), 1.24(m, 20H, -NCH2CH3 and -SiCH2CH2CH2Si-), 0.56 (m, 16H,-SiCH2CH2CH2Si-), 0.43 (m, 8H, -SiCH2CH2CH2N+-), -0.03 (s,24H, -SiMe2). 13C-NMR (DMSO): d 54.0 (-SiCH2CH2CH2N+),49.0 (-N+CH2CH2N+HMe2), 46.3 (-N+CH2CH3), 44.8(-N+CH2CH2N+HMe2), 41.7 (N+HMe2), 18.9 (-SiCH2CH2CH2Si-),17.6 (-SiCH2CH2CH2Si-), 17.1 (-SiCH2CH2CH2Si-), 16.4(-SiCH2CH2CH2N+-), 11.2 (-SiCH2CH2CH2N+-), 7.8(-N+CH2CH3), -3.9 (-SiMe2). 15N-NMR (DMSO): d -340.3(N+HMe2), -325.1 (N+H). 29Si-NMR (DMSO): d 0.9 (G0–Si), 2.5(G1–Si). C56H140Cl8N8Si5 (1349.8): calcd. C, 49.83; H, 10.45; N,8.30; found C, 50.68; H, 11.30; N, 7.43.


This dendrimer was prepared using a similar method to thatdescribed 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 whitewater soluble solid (0.42 g, 91%). 1H-NMR (DMSO): d N+H andN+HMe2 were not observed, 3.56 (m, 32H, -N+CH2CH2N+Me3),3.17 (m, 16H, -N+CH2CH3), 3.03 (m, 16H, -SiCH2CH2CH2N+-),2.81 (s, 48H, N+HMe2), 1.69 (m, 16H, -SiCH2CH2CH2N+-), 1.24(m, 48H, -NCH2CH3 and -SiCH2CH2CH2Si-), 0.53 (m, 48H,-SiCH2CH2CH2Si-), 0.44 (m, 16H, -SiCH2CH2CH2 N+-), -0.03(s, 48H, -SiMe2), -0.10 (s, 12H, -SiMe). 13C-NMR (DMSO):d 54.0 (-SiCH2CH2CH2N+-), 49.2 (-N+CH2CH2N+Me3), 46.3(-N+CH2CH3), 44.8 (-N+CH2CH2N+Me3), 41.7 (N+HMe2),18.9 (-SiCH2CH2CH2Si-), 17.6 (-SiCH2CH2CH2Si-), 17.2(-SiCH2CH2CH2Si-), 16.4 (-SiCH2CH2CH2N+-), 11.2 (-SiCH2CH2-CH2N+-), 7.8 (-N+CH2CH3), -3.9 (-SiMe2), -5.3 (-SiMe). 15N-NMR (DMSO): d -340.5 (N+HMe2), -325.0 (N+H). 29Si-NMR(DMSO): d (G0–Si) is not observed, 1.1 (G1–Si), 2.3 (G2–Si).C128H316Cl16N16Si13 (3012.3): calcd. C, 51.04; H 10.57, N 7.44;found C 51.29, H 10.79, N 7.02.


This dendrimer was prepared using a similar method to thatdescribed 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

ether (2.5 mL, 2.45 mmol). Compound 9 was isolated as a whitewater soluble solid (0.39 g, 100%). 1H-NMR (DMSO): d 11.5(br, N+H, N+HMe2), 3.56 (m, 64H, -N+CH2CH2N+Me3), 3.17(m, 32H, -N+CH2CH3), 3.03 (m, 32H, -SiCH2CH2CH2N+-),2.82 (s, 96H, N+HMe2), 1.69 (m, 32H, -SiCH2CH2CH2N+-),1.26 (m, 104H, -NCH2CH3 and -SiCH2CH2CH2Si-), 0.52 (m,144H, -SiCH2CH2CH2Si- and -SiCH2CH2CH2 N+-), -0.03 (s,96H, -SiMe2), -0.10 (s, 36H, -SiMe). 13C-NMR (DMSO): d54.0 (-SiCH2CH2CH2 N+-), 49.0 (-N+CH2CH2N+Me3), 46.3(-N+CH2CH3), 44.6 (-N+CH2CH2N+Me3), 41.7 (N+HMe2),18.9 (-SiCH2CH2CH2Si-), 17.7–17.1 (-SiCH2CH2CH2Si-,-SiCH2CH2CH2Si- and -SiCH2CH2CH2N+-), 11.2 (-SiCH2-CH2CH2 N+-), 7.8 (-N+CH2CH3), -3.9 (-SiMe2), -5.4 (-SiMe).15N-NMR (DMSO): d -340.5 (N+HMe2), -325.0 (N+H).29Si-NMR (DMSO): d (G0–Si) and (G1–Si) is not observed, 1.1(G2–Si), 2.3 (G3-Si). C272H664Cl32N32Si29 (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 {[Et3Si(CH2CH2CH2)N+Me(Et)-(CH2CH2N+Me3)] 2I-} (10)

To a THF solution (25 mL) of Et3Si(CH2CH2CH2)-N(Et)(CH2CH2NMe2)] (2) (0.315 g, 1.15 mmol) was added anexcess of MeI (0.5 mL, 2.31 mmol). The resulting solutionwas stirred for 48 h at room temperature and then evaporatedunder reduced pressure to give 10 as a white water solublesolid (0.505 g, 79%). 1H-NMR (DMSO): d 3.88 (m, 2H,-N+CH2CH2N+Me3), 3.80 (m, 2H, -N+CH2CH2N+Me3) 3.38 (m,2H, -N+CH2CH3), 3.29 (m, 2H, -SiCH2CH2CH2N+-), 3.19 (s, 9H,-N+Me3), 3.05 (s, 3H, -N+Me), 1.60 (m, 2H, -SiCH2CH2CH2N+-),1.26 (t, 3H, -NCH2CH3), 0.91(t, 9H, -SiCH2CH3) 0.50 (q,6H, -SiCH2CH3), 0.45 (m, 2H, -SiCH2CH2CH2N+-). 13C-NMR(DMSO): d 63.3 (-SiCH2CH2CH2N+), 56.6 (-N+CH2CH3), 56.0(-N+CH2CH2N+Me3), 52.4 (-N+Me3), 52.0 (-N+CH2CH2N+Me3),47.1 (-N+Me), 15.8 (-SiCH2CH2CH2N+-), 7.3 (-N+CH2CH3), 6.8(-SiCH2CH3), 6.7 (-SiCH2CH2CH2N+-), 2.1 (-SiCH2CH3). 15N-NMR (DMSO): d = -332.6 (N+Me3), -320.5 (N+Me). 29Si-NMR(CDCl3): d 7.5 (-SiEt3).

4.12. Synthesis of G1-{[(CH2CH2CH2)N+(Me)(Et)-(CH2CH2NMe+

3)]4 8I-} (11)

To a THF solution (25 mL) of G1-[(CH2CH2CH2)-N(Et)(CH2CH2NMe2)]4 (3) (1.04 g, 0.98 mmol) was added a slightexcess of MeI (0.49 mL, 7.88 mmol). The resulting solution wasstirred for 48 h at room temperature and then evaporated underreduced pressure to give 11 as a white water soluble solid (0.31 g,94%). 1H-NMR (DMSO): d = 3.93 (m, 8 H, -N+CH2CH2N+Me3),3.85 (m, 8H, -N+CH2CH2N+Me3), 3.41 (m, 8H, -N+CH2CH3),3.33 (m, 8H, -SiCH2CH2CH2 N+-), 3.22 (s, 36H, -N+Me3), 1.63(m, 8H, -SiCH2CH2CH2Si-), 1.27 (m, 20H, -SiCH2CH2CH2Si-and -N+CH2CH3), 0.57 (16H, -SiCH2CH2CH2Si-), 0.43,(m, 8H, -SiCH2CH2CH2N+-), 0.005 (s, 24H, -SiMe2).13C-NMR (DMSO): d = 63.3 (-SiCH2CH2CH2N+-), 56.6(-N+CH2CH3), 56.0 (-N+CH2CH2N+Me3), 52.5 (-N+Me3), 52.0(-N+CH2CH2N+Me3), 47.2 (-N+Me), 18.9 (-SiCH2CH2CH2N+-),17.6 (-SiCH2CH2CH2Si-), 16.4 (-SiCH2CH2CH2Si-), 16.0(-SiCH2CH2CH2Si-, 10.6 (-SiCH2CH2CH2N+-), 7.4(-N+CH2CH3), -3.8 (-SiMe2). 15N-NMR (DMSO): d = -332.1


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(N+Me3), -320.2 (N+Me). 29Si-NMR (DMSO): d = 0.7 (G0–Si),2.6 (G1–Si). C64H156I8N8Si5 (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-{[(CH2CH2CH2)N+(Me)-(Et)(CH2CH2NMe+

3)]8 16I-} (12)

This dendrimer was prepared using a similar method to thatdescribed for 11, starting from a THF solution (25 mL) of G2-[(CH2CH2CH2)N(Et)(CH2CH2NMe2)]8 (4) (1.18 g, 0.48 mmol)and MeI (1.2 mL, 7.75 mmol). Compound 12 was isolated asa white water soluble solid (2.11 g, 92%). 1H-NMR (DMSO):d = 3.95 (m, 16H, -N+CH2CH2 N+Me3), 3.87 (m, 16H,-N+CH2CH2N+Me3), 3.42 (m, 24H, -N+CH2CH3), 3.31 (m, 16H,-SiCH2CH2CH2N+-), 3.23 (s, 96H, -N+Me3 and -N+Me), 1.63(m, 16H, -SiCH2CH2CH2N+-), 1.27 (m, 56H, -SiCH2CH2CH2Si-and -N+CH2CH3), 0.55 (48H, -SiCH2CH2CH2Si-), 0.46 (m,16H, -SiCH2CH2CH2N+-), 0.00 (s, 48H, -SiMe2), -0,10 (s, 12H,-SiMe). 13C-NMR (DMSO): d = 63.3 (-SiCH2CH2CH2N+-), 56.6(-N+CH2CH3), 56.0 (-N+CH2CH2N+Me3), 52.5 (-N+Me3), 52.0(-N+CH2CH2N+Me3), 47.2 (-N+Me), 18.9 (-SiCH2CH2CH2N+-),17.8–16.1 (-SiCH2CH2CH2Si-), 10.6 (-SiCH2CH2CH2N+-), 7.5(-N+CH2CH3), -3.8 (-SiMe2) -5.4 (-SiMe). 15N-NMR (DMSO):d = -331.4 (N+Me3), -320.0 (N+Me). 29Si-NMR (DMSO): d =(G0–Si) is not observed, 1.1 (G1–Si), 2.8 (G2–Si). C144H348I16N16Si13

(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-{[(CH2CH2CH2)N+(Me)(Et)-(CH2CH2NMe+

3)]16 32I-} (13)

This dendrimer was prepared using a similar method to thatdescribed for 11, starting from a THF solution (25 mL) ofG3-[(CH2CH2CH2)N(Et)(CH2CH2NMe2)]16 (5) (0.31 g,0.06 mmol) and MeI (0.15 mL, 2.37 mmol). Compound 13was isolated as a white water soluble solid (0.58 g, 99%).1H-NMR (DMSO): d = 4.00 (m, 32H, -N+CH2CH2N+Me3)3.90 (m, 32H, -N+CH2CH2N+Me3), 3.42 (m, 64H, -N+CH2CH3

and -SiCH2CH2CH2N+- overlapped), 3.25 (s, 144H, -N+Me3),3.17 (s, 48H, -N+Me), 1.65 (m, 32H, -SiCH2CH2CH2Si-), 1.29 (m, 104H, -SiCH2CH2CH2Si- and -N+CH2CH3),0.53 (m, 144H, -SiCH2CH2CH2Si- and -SiCH2CH2CH2N+-overlapped), 0.00 (s, 96H, -SiMe2), -0.10 (s, 36H, -SiMe).13C-NMR (DMSO): d = 63.3 (-SiCH2CH2CH2N+-), 56.6(-N+CH2CH3), 56.0 (-N+CH2CH2N+Me3), 52.5 (-N+Me3), 52.1(-N+CH2CH2N+Me3), 47.2 (-N+Me), 18.9 (-SiCH2CH2CH2N+-),17.7–16.1 (-SiCH2CH2CH2Si-), 10.6 (-SiCH2CH2CH2N+-), 7.5(-N+CH2CH3), -3.8 (-SiMe2) -5.3 (-SiMe). 15N-NMR (DMSO): d-332.0 (N+Me3), -320.0 (N+Me). 29Si-NMR (DMSO): d (G0–Si)and (G1–Si) is not observed, 1.2 (G2–Si), 2.5 (G3–Si). ppm.C304H732I32N32Si29 (9712.7): calcd. C, 37.59; H, 7.60; N, 4.61; foundC, 38.26; H, 7.00; N, 4.77.

4.15. Antimicrobial activity assay

The minimal inhibitory concentration (MIC) of the products wasperformed in 96-well tray microplates using the international stan-dard methods ISO 20776-1 by microdilution trays preparation.20

The assay was carried out in duplicate microplates with threedifferent wells for each concentration analysed in the microplate.

The bacteria used in the analysis were Escherichia coli (Gram-)and Staphylococcus aureus (Gram+). Both strains were obtainedfrom the “Coleccion Espanola de cultivos tipo” (CECT). A stocksolution of the products was obtained by dissolving 0.01024 g ofthe compound in 10 ml of distilled water. After that, distilled waterwas added to obtain the desired concentration. The microplateswere 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 usedto 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 ◦Cthe presence of colonies was tested. The MBC was the minimalconcentration where not growth was detected.

Acknowledgements

This work has been supported by grants from and MNT-ERANET 2007 (ref. NAN2007-31135-E) and Fondo de InvestigacionSanitaria (ref. PI040993) to UA and FIS (ref. PI061479), Red RISRD06-0006-0035, FIPSE (24632/07), MNT-ERA NET 2007 (ref.NAN2007-31198-E), Fundacion Caja Navarra and Comunidadde Madrid (S-SAL-0159-2006) to MAMF.

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