Amine and ammonium functionalization of chloromethylsilane-ended dendrimers. Antimicrobial activity studies

Article abstract of DOI:10.1039/b809569h

Novel amine- and ammonium-terminated carbosilane dendrimers of type G _n-[Si{CH₂O-(C₆H₄)-3-NMe ₂}]_x or G_n-[Si{CH₂O-(C ₆H₄)-3-NMe₃⁺

Full text of DOI:10.1039/b809569h

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Amine and ammonium functionalization of chloromethylsilane-ended
dendrimers. Antimicrobial activity studies
Paula Ortega,a Jose Luis Copa-Patin˜o,c Ma Angeles Mun˜oz-Fernandez,b Juan Soliveri,c Rafael Gomez*a and
F. Javier de la Mata*a
Received 5th June 2008, Accepted 27th June 2008
First published as an Advance Article on the web 29th July 2008
DOI: 10.1039/b809569h
Novel amine- and ammonium-terminated carbosilane dendrimers of type
Gn-[Si{CH2O-(C6H4)-3-NMe2}]x or Gn-[Si{CH2O-(C6H4)-3-NMe3 I−}]x have been synthesized and
+
characterized up to second generation by phenolysis of (chloromethyl)silyl-terminated dendrimers with
3-dimethylamine phenol and subsequent quaternization with methyl iodide. Quaternized carbosilane
dendrimers are stable in protic solvents and can be solubilised in water after the addition of less than
1% of dimethyl sulfoxide. A study of the antimicrobial activity of these cationic dendrimers of first and
second generation against both Gram-positive and Gram-negative bacteria is also described. The
results obtained demonstrate that the new ammonium-terminated carbosilane dendrimers can be
considered as multivalent biocides.
peripheral amine groups and the subsequent transformation of
1. Introduction
these amine groups into ammonium units leading to polycationic
Several groups have reported high-yield divergent synthesis of
carbosilane dendrimers with amphiphilic properties.
hydrophobic carbosilane dendrimers by using repeating sequences
Cationic dendrimers with ammonium groups in the surface
have been described as effective antimicrobial agents.8 These
dendrimers have been shown to be more potent than their small
molecule counterparts. The use of these systems as biocides is
due to the fact that polyvalent interactions have been frequently
used by nature to either induce or combat infections9 and it has
been demonstrated that intermolecular as well as intramolecular
interactions are much more enhanced in polymeric systems.10
In this sense, dendrimers can offer a high local concentration
of functional groups and a compact structure with a narrow
polydispersity and a well-characterized molecular weight. If
these units present biologically active antimicrobial groups, one
might expect to see an increased potency associated with the high
local concentration. Nevertheless, the dendrimer size is important
since it has been shown that quaternary ammonium compounds
exert their antimicrobial action by disrupting and disintegrating
cell membranes.11 Thus bulkier dendrimers may not be able to pen-
etrate through the cell membrane barrier and may find difficulties
in reaching the target site for the anticipated antimicrobial action.
In this paper, we report the antimicrobial activity of our
ammonium terminated carbosilane dendrimers of first and sec-
ond generation against both Gram-positive and Gram-negative
bacteria. We think that the hydrophobic nature of the carbosilane
skeleton present in our dendrimers may induce some differences
(e.g. biopermeability) in the interaction of these systems with
membrane cells compared with other kinds of dendrimers de-
scribed in the literature and may well lead to differences in the
antimicrobial action. We have compared the activity of these
dendrimers with the activity of the corresponding monofunctional
counterpart and have established the range of concentrations
in which our dendrimers present bacteriostatic (refers to an
agent that halts the growth of bacteria and not necessarily
killing them) and bactericidal (refers to an antimicrobial agent
or condition that kills a bacterial cell) effects. For the same
of alternating hydrosilylations with substituted chlorosilanes and
alkenylations with Grignard reagents.1 There are many reports
about the employment of these dendrimers as frameworks upon
which to attach other functional groups.2 However, only a
few attempts to functionalize these dendrimers with peripheral
amphiphilic groups have been published.3
Seyferth et al.4 reported the synthesis of stable amphiphilic car-
bosilane dendrimers through the reaction of (chloromethyl)silyl-
terminated dendrimers with several mercapto-substituted am-
phiphilic compounds. The (chloromethyl)silyl dendrimers are
necessary for this purpose since most silicon–heteroatom bonds
(e.g., Si–N, Si–OR, Si–S) are moisture sensitive,5 which means
that simple nucleophilic reactions with chlorosilyl-terminated
dendrimers are not suitable.
More recently, Astruc et al. have reported the nucleophilic sub-
stitution by phenol derivatives of (chloromethyl)silyl-terminated
groups, helped by the previous exchange of the chloromethyl
termini for iodomethyls upon reaction with sodium iodide.6
Four years ago, van Leeuwen et al. reported the synthesis of
(chloromethyl)silyl-terminated dendrimers by hydrosilylation of
allylsilane-ended dendrimers with (chloromethyl)dimethyl silane.7
Here, we show the functionalization of these dendrimers with
aDepartamento de Qu´ımica Inorga´nica, Universidad de Alcala´, Campus
Universitario, E-28871 Alcala´ de Henares (Spain). Networking Research
Center on Bioengineering, biomaterials and nanomedicine (CIBER-BBN),
Alcala´ de Henares, Spain. E-mail: javier.delamata@uah.es; Fax: +34 91
885 4683; Tel: +34 91 885 4654
bLaboratorio de Inmunobiolog´ıa Molecular, Hospital General Universitario
Gregorio Maran˜o´n, Madrid (Spain). Networking Research Center on
Bioengineering, biomaterials and nanomedicine (CIBER-BBN), Spain.
E-mail: mmunoz@cbm.uam.es
cDepartamento de Microbiolog´ıa y Parasitolog´ıa, Universidad de Alcala´,
Campus Universitario, E-28871, Alcala´ de Henares, Spain. E-mail:
josel.copa@uah.es
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agent, the bacteriostatic concentration may be lower or equal to
the bactericidal concentration, but never higher. In other words,
higher concentrations are necessary to kill the bacteria.
Treatment of dendrimers 3 and 4 with methyliodide in acetoni-
trile during 48 hours causes the quaternization of the peripheral
aromatic amines to give the cationic dendrimers G1-[Si{CH2O-
(C6H4)-3-NMe3 I−}]4 (5) and G2-[Si{CH2O-(C6H4)-3-NMe3 I−}]8
(6) (Scheme 2). The use of acetonitrile is necessary since the
quaternization reaction failed in the presence of other organic
solvents such as diethyl ether or toluene.
+
+
2. Results and discussion
2.1 Synthesis and structural characterization of carbosilane
dendrimers
Dendrimers 5 and 6 are obtained as white solids in very
high yields and are soluble in acetonitrile, dimethylsulfoxide and
methanol. They can be solubilised in water adding small quantities
(less than 1%) of dimethylsulfoxide.
Preparative details and spectroscopic data for the new compounds
are given in the Experimental Section. Only selected data will
be presented for this discussion. Dendrimers containing (chloro-
methyl)dimethylsilane functionalities, Gn-[Si(Me2)CH2Cl]m (n =
1, m = 4 (1); n = 2, m = 8 (2), where “n” means the number of
generation (G) and “m” denotes the number of branches and hence
the number of peripheral groups) were conveniently obtained
through the hydrosilylation reaction of allyl-functionalized den-
drimers with (chloromethyl)dimethylsilane using Speier’s catalyst.
The reactions conditions are very similar to those described
previously by van Leeuwen7 for these complexes, but in our case,
reaction times are shorter, probably due to the different platinum
catalyst used in both cases (see Scheme 1).
Structural characterization of dendrimers 1–6 has been carried
out using elemental analysis, 1H, 13C and 29Si- RMN spectroscopy
1
and mass spectrometry. The H-NMR spectra of dendrimers 3
and 4 in CDCl3 show a complex group of signals at about 7.10–
6.28 ppm corresponding to the aromatic hydrogen atoms, a singlet
around 3.53 ppm for the methylene protons of the –SiCH2O–
fragment and a singlet at 2.90 ppm due to the methyl groups
bonded to nitrogen. The carbosilane framework is insignificantly
affected by the addition of the peripheral arylamines, thus the
general features of their NMR spectra are almost identical to
those described for their precursors.7
1
13C{ H}-NMR spectra show six resonances for the carbon
atoms of the aromatic ring due to their unequivalence. The Cipso
bonded to the –OCH2Si group appears at 162.7 ppm, while the Cipso
bonded to –NMe2 gives a resonance at higher field, 151.8 ppm.
The chemical shift of the carbon atom in –OCH2 and the methyl
groups of –NMe2 appear at 60.0 and 40.0 ppm respectively. Fig. 1
1
shows the 1H and 13C{ H}-NMR spectra of the second generation
dendrimer 4. The 29Si chemical shifts of these dendrimers have been
1
determined by a 2D { H–29Si}-HMBC experiment (see Fig. 2),
which shows connectivity of the silicon atoms with the protons
Scheme 1
The reaction of the first and second generation chloromethy-
lene-terminated dendrimers, 1–2, with 3-dimethylaminophenol,
3-(NMe2)-C6H4OH, in the presence of potassium carbonate and
potassium iodide, using dimethylformamide as solvent renders
high yields of first and second generation amine-terminated den-
drimers, G1-[Si{CH2O-(C6H4)-3-NMe2}]4 (3) and G2-[Si{CH2O-
(C6H4)-3-NMe2}]8 (4) as yellow oily products. The use of a polar
and aprotic solvent clearly helps the nucleophilic substitution
reaction due to the presence of naked anions in this kind of solvent,
which in this case increases the reactivity of the phenol derivative
(see Scheme 2).
Fig. 1 1H and 13C-NMR spectra of dendrimer 4.
Scheme 2
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1
Fig. 2 2D{ H–29Si}-HMBC spectrum of dendrimer 4.
of the alkyl chains. For example, the spectrum of dendrimer 4
shows for the signal of the SiMe2 fragment (SiIII) (−0.22 ppm),
two crossed peaks assigned to Hg and Hf and another peak due
to Hm. The signal due to SiMe (SiII) (1.05 ppm) gives two crossed
peaks corresponding to Hc and Hd, together with a peak produced
by Hl. The signal corresponding to SiI is overlapped by the peak
due to SiIII.
molecular peak of the cationic specie after losing an iodide anion.
Other important peaks at 1327.48 (corresponding to the loss of one
methyl group and two iodide anions) or at 1043.63 (corresponding
to the loss of three methyl groups and four iodide anions) are also
observed in the spectrum. It has not been possible to analyze
dendrimers of second generation 4 and 6 by these techniques since
their molecular weight is out of the range in the equipment used.
1
The H, 13C and 29Si NMR spectra of the quaternized den-
drimers, 5 and 6, were recorded in [D6]DMSO at room tempera-
ture. These spectra exhibit resonance patterns identical to those
observed in their neutral counterparts, 3 and 4, for the carbosilane
framework, although broader signals are observed with the
increasing generation. In general, in the 1H-NMR spectra, the pos-
itive charge of the ammonium groups results in the deshielding of
about 0.7 ppm, for the methyl group directly bonded to the charged
nitrogen atoms whereas small downfield shifts are found for the
rest of the protons of the peripheral unit. Analogous behaviour is
observed in the 13C NMR spectra for the carbon atoms.
In addition, the proposed structure for dendrimer 3 has been
confirmed by mass spectrometry using APCI (atmospheric pressure
chemical ionization) (see Fig. 3). The molecular peak [M + H]+
appears at 1030 uma (theoretical, 1028.66 uma). Dendrimer 5
was analysed by mass spectroscopy using ESI (electro spray).
The spectrum shows a peak to 1470.41 uma corresponding to the
2.2 Antimicrobial activity
The antibacterial activity of dendrimers 5 and 6 as well as of
+
their monofunctional counterpart, HO-(C6H4)-3-NMe3 I− (7),
has been tested on Staphylococcus aureus (Gram+) and Escherichia
coli (Gram−) and the MIC (minimum inhibitory concentration)
and the MBC (minimum bactericidal concentration) have been
measured. The MIC indicates the minimal concentration of an
agent that can inhibit the growth of a microorganism (bacterio-
static effect) while the MBC means the minimal concentration of
an agent that can kill a microorganism (bactericidal effect). For
the same agent and microorganism, the value of MIC is lower than
or equal to the MBC value (data shown in Table 1).
The results of the antibacterial tests in Table 1 show the
high activity of carbosilane dendrimers against both Gram+ and
Gram− bacteria. These data also show that the multivalency of
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Fig. 3 APCI spectrum of dendrimer 3.
Table 1 Bacteriostatic and bactericidal effects of compounds 5, 6 and 7
Products
Monofunctional
counterpart 7/mg l−1
Dendrimer 5/mg l−1
Dendrimer 6/mg l−1
Microorganism
MIC
MBC
MIC
MBC
MIC
MBC
Escherichia coli
Staphylococcus aureus
> 512
512
> 512
> 512
4
1
4
4
64
8
256
32
MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration.
dendrimers plays a major role in this antibactericidal activity since
the dendrimer biocides are over two orders of magnitude more
potent than their monofunctional counterpart. This significant
improvement of biocidal action of dendritic biocides can be
attributed to the high number of functional groups in a compact
space and their polycationic structure. These tests also show a
lower MIC for the Gram+ bacteria than for the Gram− bacteria,
mainly in the second generation dendrimers. This fact has been
observed by other authors12 in lysine-based dendrimers and is
probably due to the structure of the double layer membrane of
Gram− bacteria. This underlines the importance of the interaction
between dendrimers and membrane in the mode of action of
these compounds. In this sense, it has been proposed8d that
there are electrostatic interactions between the negatively charged
bacteria membrane and the dendrimer biocides that present a high
positive charge density. These dendrimers displace the divalent
surface ions such as calcium and magnesium and may destabilize
the membrane structure; this means that dendrimer biocides
behave as surface-active organic cations and therefore have a
destabilizing effect on phospholipid systems.13 Nevertheless, the
exact mechanism of their antimicrobial action is still unclear and
mostly attributed to their ability to increase cell permeability and
disrupt cell membranes.
is more potent than the second one, primarily in the case of
the Gram− bacteria. This result has also been shown by other
authors8d,12 and has been attributed to the size and molecular
weight of the dendrimer and its capacity to cross the membrane
of a bacteria. The higher decrease of the antimicrobial activity
observed for the second generation dendrimer against Gram−
bacteria may be due to the size of this dendrimer and its
difficulty to cross the first double-layer membrane present in
this type of bacteria. Nevertheless, there is always a balance
between the disadvantage of the smaller number of functionalites
in dendrimers of low generation and the disadvantage of a bulkier
size in the dendrimers of high generation. In addition, in our case,
we cannot rule out solubility problems in the dendrimer of second
generation since this dendrimer is less soluble in water.
3. Conclusions
A new family of amine- and ammonium-terminated carbosilane
dendrimers has been synthesized by phenolysis of chloro–carbon
bonds in chloromethyl–silicon derivatives and subsequent quater-
nization with MeI. These dendrimers have been fully characterized
using elemental analysis, NMR spectroscopy and mass spectrom-
etry. Quaternized dendrimers are 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
The generation dependence of dendrimer biocides was also
evaluated and we observed that the first generation dendrimer
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studies showed that the multivalency of dendrimers plays a major
role in the antibactericidal activity of these compounds, since
the dendrimer biocides are over two orders of magnitude more
potent than their monofunctional counterpart. Dendrimers 5 and
6 present antimicrobial activity against both Gram+ and Gram−
bacteria. This activity slightly decreases when the generation
dendrimer increases, mainly in the case of a Gram− bacteria,
which may well be due to the different structure of the cell wall in
these systems
analysis calcd. (%) for C64H148Cl8Si13: C 49.09, H 9.52; found C
48.56, H 9.43.
4.4 Synthesis of G1-[SiCH2O-(C6H4)-3-NMe2]4 (3)
Over a solution of 1 (0.27 g, 0.42 mmol) in DMF (30 ml),
3-dimethylaminophenol (0.24 g, 1.69 mmol), 1 g of potassium
carbonate and a small amount of KI were added. The reaction
mixture was stirred and heated to 80 ◦C during two days, then
evaporated to dryness and the residue extracted with pentane
(2 × 20 ml) to give a yellow solution. The solvent was removed
under vacuum leading to dendrimer 3 as a yellow oily product
(0.36 g, 85%). 1H-NMR (CDCl3): d 7.10 (4H, s, C6H4), 6.35
(12H, m, C6H4), 3.54 (8H, s, CH2O), 2.91 (24H, s, NMe2), 1.37
(8H, m, SiCH2CH2CH2Si), 0.68 (8H, m, SiCH2CH2CH2SiCH2O),
0.59 (8H, m, SiCH2CH2CH2Si), 0.08 (24H, s, SiMe2). 13C-NMR
(CDCl3): d 162.7 (CipsoOCH2), 151.8 (CipsoNMe2), 129.5, 105.4,
101.9 and 99.3 (C6H4), 60.0 (CH2O), 40.7 (NMe2), 18.6, 18.4
and 17.3 (SiCH2CH2CH2Si), −4.5 (SiMe2). 29Si-NMR (CDCl3):
d 0.5 (G0–Si), −0.2 (G1–Si). Elemental analysis calcd. (%) for
C56H96N4O4Si5: C 65.31, H 9.40, N 5.44; found C 64.87, H 9.06, N
5.62. APCI: [M + H+] = 1030 uma (calcd. = 1028.66 uma).
4. Experimental details
4.1 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: DMF from sodium
carbonate, pentane from sodium, diethyl ether from sodium ben-
zophenone ketyl and acetonitrile over calcium hydride.14 Unless
otherwise stated, reagents were obtained from commercial sources
=
and used as received, Gn-[SiMe2CH2CH CH2]m dendrimers were
prepared according to reported methods.2f
4.5 Synthesis of G2-[SiCH2O-(C6H4)-3-NMe2]8 (4)
1H, 13C and 29Si-NMR spectra were recorded on Varian Unity
VXR-300 and Varian 500 Plus instruments. Chemical shifts (d,
ppm) were measured relative to residual 1H and 13C resonances for
CDCl3 and [D6]DMSO used as solvents, and 29Si chemical shifts
were referenced to external SiMe4 (0.00 ppm). C, H and N analyses
were carried out with a Perkin-Elmer 240 C microanalyzer. APCI
samples were prepared in acetonitrile or methanol and spectra
were recorded on a Termoquest Finnigan Automass spectrometer.
This dendrimer was prepared using a similar method to that
described for 3, starting from G2-[SiCH2Cl]8 (0.09 g, 0.06 mmol),
3-dimethylaminophenol (0.07 g, 0.48 mmol), K2CO3 (1 g, 7.23
mmol) and a small amount of KI. Second generation den-
drimer 4, is obtained as a yellow-orange oily product (0.11 g,
1
82%). H-NMR (CDCl3): d 7.09 (8H, s, C6H4), 6.29 (24H, m,
C6H4), 3.53 (16H, s, CH2O), 2.90 (48H, s, NMe2), 1.37 (24H,
m, SiCH2CH2CH2Si), 0.69 (16H, m, SiCH2CH2CH2SiCH2O),
0.55 (32H, m, SiCH2CH2CH2Si), 0.08 (48H, s, SiMe2), −0.09
(12H, s, SiMe). 13C-NMR (CDCl3): d 162.7 (CipsoOCH2), 151.9
(CipsoNMe2), 129.5, 105.3, 101.9 and 99.3 (C6H4), 60.0 (CH2O),
40.7 (NMe2), 18.6, 18.5 and 17.4 (SiCH2CH2CH2Si, outer frag-
ment), 19.0–18.0 (rest of the CH2 signals), −4.5 (SiMe2), −5.0
(SiMe). 29Si-NMR (CDCl3): d 0.6 (G0–Si), 1.0 (G1–Si), −0.2 (G2–
Si). Elemental analysis calcd. (%) for C128H228N8O8Si13: C 64.80, H
9.69, N 4.72; found C 63.85, H 9.51, N 4.55.
4.2 Synthesis of G1-[SiCH2Cl]4 (1)
=
Over a toluene solution of tetraallylsilane, Si(CH2CH CH2)4
(0.97 g, 5.09 mmol), HSi(Me)2CH2Cl (22.3 mmol, 2.70 ml) and
two drops of the Speier catalyst were added. The reaction mixture
was stirred at 60 ◦C during 5 hours. Silane excess and solvent were
removed under vacuum and the residue extracted with hexane and
then filtered with celite and active carbon to eliminate the catalyst.
The resulting solution was evaporated under reduced pressure to
1
give 1 as a colorless oil (2.75 g, 87%). H-NMR (CDCl3): d 2.75
4.6 Synthesis of G1-[SiCH2O-(C6H4)-3-NMe3 I−]4 (5)
+
(8H, s, CH2Cl), 1.30 (8H, m, SiCH2CH2CH2Si), 0.50 (16H, m
SiCH2CH2CH2Si), 0.09 (24H, s, SiMe2). 13C-NMR (CDCl3): d
30.5 (CH2Cl), 18.6 (SiCH2CH2CH2Si), 18.4 (SiCH2CH2CH2Si),
17.3 (SiCH2CH2CH2Si), −4.4 (SiMe2). Elemental analysis calcd.
(%) for C24H56Cl4Si5: C 45.98, H 9.0; found C 45.88, H 8.86.
A solution of MeI in Et2O (2 M, 0.10 ml, 1.68 mmol) was added
to an acetonitrile (30 ml) solution of 3 (0.29 g, 0.28 mmol). The
resulting solution was stirred for 24 hours at room temperature
and then evaporated under reduced pressure to remove residual
MeI. The residue was washed with Et2O (2 × 5 ml) and dried
under vacuum to yield dendrimer 5 as a white solid compound
4.3 Synthesis of G2-[SiCH2Cl]8 (2)
1
(0.43 g, 94%). H-NMR ([D6]DMSO): d 7.48 (12H, m, C6H4),
+
This dendrimer was prepared using a similar method to that
7.15 (4H, s, C6H4), 3.71 (8H, s, CH2O), 3.57 (36H, s, NMe3 ), 1.39
=
described for 1, starting from G1-[SiCH2CH CH2]8 (1.03 g, 1.47
(8H, m, SiCH2CH2CH2Si), 0.70 (8H, m, SiCH2CH2CH2SiCH2O),
mmol), HSi(Me)2CH2Cl (12.34 mmol, 1.5 ml) and two drops of
the Speier catalyst to obtain compound 2 as a colorless oil (1.85 g,
80%). 1H-NMR (CDCl3): d 2.75 (16H, s, CH2Cl), 1.33 (24H,
m, SiCH2CH2CH2Si), 0.68 (16H, m SiCH2CH2CH2SiCH2Cl),
0.54 (32H, m SiCH2CH2CH2Si), 0.08 (48H, s, SiMe2), −0.08
(12H, s, SiMe). 13C-NMR (CDCl3): d 30.5 (CH2Cl), 18.6–17.6
(SiCH2CH2CH2SiCH2Cl), −4.5 (SiMe2), −5.0 (SiMe). Elemental
0.58 (8H, m, SiCH2CH2CH2Si), 0.08 (24H, s, SiMe2). 13C-NMR
+
([D6]DMSO): d 161.3 (CipsoOCH2), 147.7 (CipsoNMe3 ), 130.2,
+
114.8, 111.3 and 106.5 (C6H4), 60.5 (CH2O), 55.9 (NMe3 ),
17.4, 17.3 and 16.3 (SiCH2CH2CH2Si), −5.18 (SiMe2).29Si-NMR
(CDCl3): d 1.3 (G0–Si), 0.3 (G1–Si). Elemental analysis calcd. (%)
for C60H108I4N4O4Si5: C 45.11, H 6.81, N 3.51; found C 44.62, H
6.54, N 3.44. ESI: [M − I]+ = 1470.4 uma (calcd. = 1470.6 uma),
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[M − Me − 2I]+ = 1328.5 uma (calcd = 1328.7 uma), [M − 3Me −
4I]+ = 1044.6 uma (calcd 1044.8 uma).
Acknowledgements
We thank the Ministerio de Ciencia y Tecnolog´ıa (project
CTQ2005-00795/BQU), Fondo de Investigacio´n Sanitaria
(PI040993) and Fundacio´n Caja Navarra for financial support.
4.7 Synthesis of G2-[SiCH2O-(C6H4)-3-NMe3 I−]8 (6)
+
This dendrimer was prepared using a similar method to that de-
scribed for 5, starting from G2-[SiCH2O-(C6H4)-3-NMe2]8 (0.18 g,
0.75 mmol) and MeI (0.6 ml, 9.6 mmol). Second generation
dendrimer, 6, is obtained as a white solid compound (0.21 g,
80%). 1H-NMR ([D6]DMSO): d 7.49 (24H, m, C6H4), 7.26 (8H, s,
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Lorenz, J. Kressler, H. Frey and R. Mu¨lhaupt, Macromolecules, 1996,
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3 (a) K. Lorenz, R. Mu¨lhaupt, H. Frey, U. Rapp and F. J. Mayer-Posner,
Macromolecules, 1995, 28, 6657; (b) S. S. Sheiko, A. M. Muzafarov,
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+
C6H4), 3.71 (16H, s, CH2O), 3.58 (72H, s, NMe3 ), 1.36 (24H,
m, SiCH2CH2CH2Si), 0.69 (16H, m, SiCH2CH2CH2SiCH2O),
0.54 (32H, m, SiCH2CH2CH2Si), 0.08 (48H, s, SiMe2), −0.09
(12H, s, SiMe). 13C-NMR ([D6]DMSO): d 161.3 (CipsoOCH2), 147.7
+
(CipsoNMe3 ), 130.2, 114.8, 111.3 and 106.5 (C6H4), 60.5 (CH2O),
a´n and J. Losada, J. Chem. Soc., Chem.
+
55.9 (NMe3 ), 17.8–17.1 (SiCH2CH2CH2Si), −5.1 (SiMe2), −5.2
(SiMe).29Si-NMR (CDCl3): d −0.3 (G0–Si), 1.2 (G1–Si), 0.0 (G2-
Si). Elemental analysis calcd. (%) for C136H252I8N8O8Si13: C 46.57,
H 7.24, N 3.19; found C 45.23, H 6.98, N 2.99.
4.8 Synthesis of HO(C6H4)-3-NMe3 I− (7)
+
A solution of MeI in Et2O (2 M, 0.10 ml, 1.68 mmol) was added to
an acetonitrile (30 ml) solution of 3-dimethylaminophenol (0.21 g,
1.50 mmol). The resulting solution was stirred for 6 hours at
room temperature and then evaporated under reduced pressure
to remove residual MeI. The residue was washed with Et2O (2 ×
5 ml) and dried under vacuum to give 7 as a purple solid compound
(0.35 g, 84%). 1H-NMR ([D6]DMSO): d 7.40 (3H, m, C6H4),
6.96 (1H, d, C6H4), 3.53 (9H, s, NMe3 ). 13C-NMR ([D6]DMSO):
+
+
d 157.8 (CipsoOH), 147.8 (CipsoNMe3 ), 130.2, 116.0, 109.9 and
+
107.1 (C6H4), 55.7 (NMe3 ). Elemental analysis calcd. (%) for
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C9H14INO: C 38.73, H 5.06, N 5.02; found C 38.52, H 4.99, N 4.92.
4.9 Antimicrobial activity assay
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The minimal inhibitory concentration (MIC) of the products
was determined in 96-well tray microplates using the interna-
tional standard methods ISO 20776–1 by microdilution tray
preparation.15 The assay was carried out in duplicate microplates
with three different wells for each analysed concentration. The
bacteria used in the analysis were Escherichia coli (Gram−) and
Staphylococcus aureus (Gram+). Both strains were obtained from
the “Coleccio´n Espan˜ola de cultivos tipo” (CECT). A stock
solution of the products was prepared by dissolving 0.01024 g
of the compound with 60 ll of dimethyl sulfoxide (DMSO) and
10 ml of distilled water. After that, the desired concentration was
achieved by dilution with distilled water. The microplates were
incubated at 37 ◦C using an ultra microplate reader ELX808iu
(Bio-Tek Instruments). The minimal bactericidal concentration
(MBC) was calculated inoculating 100 ll of the samples used to
calculate the MIC in a Petri dish with Mueller–Hinton agar (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 no growth was detected.
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International Dendrimer Symposiun 5, P-036, Toulouse, France, 2007;
(b) J. Janiszewska and Z. Urbanczyk-Lipkowska, Acta Biochim. Pol.,
2006, 53, 77.
13 H. Hauser, K. Howell and M. C. Phillips, FEBS Lett., 1977, 80, 355.
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Laboratory Chemicals, Pergamon Press Ltd., Oxford, 3rd edn, 1988.
15 ANSI 2006. Reference methods for testing the “in vitro” activity of
antimicrobial agents against bacteria involved in infectous diseases ISO
20776-1.
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 3264–3269 | 3269
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