Design and synthesis of dendrimers with facile surface group functionalization, and an evaluation of their bactericidal efficacy

Article abstract of DOI:10.3390/molecules22060868

We report a versatile divergent methodology to construct dendrimers from a tetrafunctional core, utilizing the robust copper(I) catalyzed alkyne-azide cycloaddition (CuAAC, "click") reaction for both dendrimer synthesis and post-synthesis functionalization. Dendrimers of generations 1-3 with 8-32 protected or free OH and acetylene surface groups, were synthesized using building blocks that included acetylene- or azide-terminated molecules with carboxylic acid or diol end groups, respectively. The acetylene surface groups were subsequently used to covalently link cationic amino groups. A preliminary evaluation indicated that the generation one dendrimer with terminal NH₃⁺ groups was the most effective bactericide, and it was more potent than several previously studied dendrimers. Our results suggest that size, functional end groups and hydrophilicity are important parameters to consider in designing efficient antimicrobial dendrimers.

Full text of DOI:10.3390/molecules22060868

molecules
Article
Design and Synthesis of Dendrimers with Facile
Surface Group Functionalization, and an Evaluation
of Their Bactericidal Efficacy
Elizabeth Ladd 1,2,3, Amir Sheikhi 1,2,3, Na Li 1,3, Theo G.M. van de Ven 1,2,3,* and
Ashok Kakkar 1,3,
*
1
Department of Chemistry, McGill University, 801 Sherbrooke St. West, Montreal, QC H3A 0B8, Canada;
elizabeth.ladd@mail.mcgill.ca (E.L.); amir.sheikhi@mail.mcgill.ca (A.S.); Blinan04st@gmail.com (N.L.)
Pulp and Paper Research Centre, McGill University, 3420 rue University, Montreal, QC H3A 2A7, Canada
Centre for Self-Assembled Chemical Structures, McGill University, 801 Sherbrooke St. West, Montreal,
QC H3A 0B8, Canada
2
3
*
Correspondence: theo.vandeven@mcgill.ca (T.G.M.v.d.V.); ashok.kakkar@mcgill.ca (A.K.);
Tel.: +1-514-398-6912 (A.K.)
Academic Editor: Derek J. McPhee
Received: 5 May 2017; Accepted: 18 May 2017; Published: 24 May 2017
Abstract: We report a versatile divergent methodology to construct dendrimers from a tetrafunctional
core, utilizing the robust copper(I) catalyzed alkyne-azide cycloaddition (CuAAC, “click”) reaction
for both dendrimer synthesis and post-synthesis functionalization. Dendrimers of generations 1–3
with 8–32 protected or free OH and acetylene surface groups, were synthesized using building blocks
that included acetylene- or azide-terminated molecules with carboxylic acid or diol end groups,
respectively. The acetylene surface groups were subsequently used to covalently link cationic amino
+
groups. A preliminary evaluation indicated that the generation one dendrimer with terminal NH3
groups was the most effective bactericide, and it was more potent than several previously studied
dendrimers. Our results suggest that size, functional end groups and hydrophilicity are important
parameters to consider in designing efficient antimicrobial dendrimers.
Keywords: dendrimers; hyperbranched macromolecules; synthesis; click chemistry; bactericide
1. Introduction
Dendrimers are well-defined hyperbranched macromolecules in which structural regularity arises
from the controlled layer-by-layer build-up, with each additional layer leading to an exponential
increase in the number of surface groups [1–7]. The overall structure and properties of dendrimers
get contributions from their core, backbone, and the surface groups at the periphery [8]. The number
of reactive ends at the core molecule influences the density of end units at each generation that
primarily interact with the outside environment [9]. For example, a third generation dendrimer with a
tetrafunctional core has 32 surface groups, while a corresponding dendrimer with a trifunctional core
has 24. Flexibility of both the core and backbone also has a significant effect on the conformation of the
structure [10
core and backbones correspond to more compact, globular dendrimers, as the arms can fold back on
themselves [12 13]. The synthetic versatility and our ability to tailor their surfaces are some of the
,11]. A rigid backbone of the dendrimer leads to an open structure, while more flexible
,
features, which make these monodisperse macromolecules attractive for a wide variety of applications.
One of the topical areas of interest in which dendrimers are becoming increasingly important is
their ability to act as bactericides [14]. Multivalent surfaces of these hyperbranched macromolecules
offer opportunities to tailor the efficacy of antimicrobial agents using a single scaffold [15–19].
Molecules 2017, 22, 868; doi:10.3390/molecules22060868
www.mdpi.com/journal/molecules

Molecules 2017, 22, 868
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Cationic amino groups are well known to exert bactericidal effect by disrupting the integrity of bacterial
cell membranes, eventually leading to cell lysis and death [18 20]. It has been shown that amine- and
,
ammonium-terminated carbosilane dendrimers act as bactericides against Gram-positive and -negative
bacteria, with almost two orders of magnitude less minimum inhibitory concentration (MIC) and
minimum bactericidal concentration (MBC), than their monofunctional counterparts. However, these
dendrimers can only be solubilized in water when a small amount of DMSO (1%) is added [21].
In another study, polyamidoamine (PAMAM) dendrimers were functionalized with nitric oxide (NO)
releasing quaternary ammonium groups, which showed enhanced bactericidal activity and anti-biofilm
formation properties as compared to non-NO releasing counterparts [22,23]. Hyaluronic acid/PAMAM
multilayered dendrimers have also been used as antibacterial materials [24]. These studies suggest
that by carefully designing dendrimers, one could achieve high efficacy through tailoring of desired
functions [25].
We have developed a versatile synthetic methodology to dendrimers utilizing highly efficient
alkyne-azide “click” chemistry [26–28], which leads to surface terminated OH or acetylene groups.
The synthesis of dendrimers required the design of multifunctional building blocks including the one
bearing a diol at one end and an azide at the other. The azide can be clicked to the peripheral alkynes
of the growing dendrimer, regenerating the original alcohol functionality. The sequence can then be
repeated to construct subsequent generations in a divergent manner. These dendrimers provide a facile
method to introduce a variety of desired peripheral moieties, and we demonstrate the simplicity of this
approach by functionalizing the surfaces of these dendrimers with cationic amino groups. We have
evaluated the potential of these water-soluble cationic dendrimers for bactericidal activity, and we
demonstrate that a balance of size, the terminal end groups and hydrophilicity plays an important
role in their efficacy. Dendrimers functionalized with cationic amino groups were found to be highly
+
potent antimicrobial agents, and the generation 1 dendrimer with surface NH3 groups was the most
efficient bactericide among such macromolecules.
2. Results and Discussion
2.1. Synthesis
The synthesis of dendrimers was carried out using a careful design of building blocks with
compatible functionalities. Since we intended to employ click chemistry both in the synthesis and
functionalization of our dendrimers, we selected two building units, one with an acetylene moiety
and another with an azide. The azide functionalized 2,2-bis(hydroxymethyl)propanoic acid (bis-MPA,
Scheme 1) was prepared from bis-MPA (1) and bromoethanol (3). The diol terminal of the bis-MPA unit
was first protected with an acetonide group, to yield
2, which could be purified easily by neutralizing
with p-toluenesulfonic acid, followed by filtration.
O
O
O
OH
OH
O
O
O
HO
O
HO
PTSA, MgSO4
acetone, RT
(85%)
2
1
O
DCC, DMAP
N3
DCM, RT
O
H2O
NaN3
OH
4 (82%)
O
5
OH
(60%)
N3
70C
Br
3
Dowex
Cationic
Resin
OH
OH
O
N3
O
(95%)
6
Scheme 1. Synthesis of building blocks.

Molecules 2017, 22, 868
3 of 18
Bromoethanol (
3
) was reacted with sodium azide to yield
4
, which was then coupled with
2
using
N,N0-dicyclohexylcarbodiimide (DCC)/4-dimethylaminopyridine (DMAP), to yield
5
, a bifunctional
molecule with a protected diol at one end and an azide at the other. It was deprotected to yield
with azide and diol terminals. Propargyl bromide was coupled to the pentaerythritol core, yielding the
tetra-acetylene terminated G0 dendrimer (Scheme 2). The azide functionalized bis-MPA was then
clicked to G0 using CuAAC reaction to yield G1 dendrimer with eight terminal hydroxide groups.
It can also be prepared by first reacting core with protected version of the azide functionalized
, followed by deprotection. This method gave better yields
6,
7
6
8
7
bis-MPA
5
using CuAAC reaction to give
9
than the first route.
O
O
O
O
O
O
O
O
N
N
N
O
OH
OH
O
N
N
N
N
Br
(5)
O
O
O
HO
OH
KOH, DMF
O
N
N
C
uSO4
O
O
O
O
N
N
N
Na ascorbate
THF, H2O
(84%)
O
7
O
O
O
(83%)
9
O
O
Cu
SO4
Na ascorbate
THF, H2O
(6)
O
50%
O
O
O
O
O
O
HO
O
O
O
HO
OH
N
N
O
O
O
O
N
N
N
N
HO
O
O
O
OH
O
N
N
N
O
N
N
N
O
O
N
N
O
DCC, DMAP
DCM, pyridine
O
N
N
N
O
O
O
O
O
O
N
N
N
N
O
N
O
N
HO
O
N
OH
HO
O
O
O
O
O
O
O
(79%)
8
O
O
OH
10 (70%)
(5)
O
Na ascorbate
Cu
O
O
SO4
THF, H2O
O
O
O
N
N
N
N
O
N
N
O
N
N
N
N
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
N
O
N
O
O
O
O
N
O
N
N
O
N
N
N
N
N
N
N
O
N
N
O
O
N
O
O
O
O
O
O
N
N
O
O
O
O
O
O
O
O
O
O
O
N
O
N
O
N
N
N
O
N
O
N
N
N
O
O
O
O
O
O
11 (57%)
Scheme 2. Synthesis of G0–G2 dendrimers.

Molecules 2017, 22, 868
4 of 18
Several attempts to covalently link propargyl bromide to G1 dendrimer
to that used to prepare G0
termini, we considered a variation on this structure. An esterification with 4-pentynoic acid was
carried out to prepare G1 (compound 10 with eight terminal acetylene groups). Compound was then
8
using a similar procedure
7
failed. Since the important component was that the structure has acetylene
5
clicked onto the dendrimer to yield protected G2 dendrimer (p-G2, 11), with sixteen terminal protected
hydroxides, which was purified by dialysis. The deprotection using bismuth trichloride was employed
to give the desired hydroxide terminated G2 dendrimer. A simple filtration to remove the remaining
salts was the only purification necessary to yield G2 (12, Scheme 3). Another esterification with
4-pentynoic acid was then carried out to generate 13, with sixteen terminal acetylenes. The purification
of 13 was quite simple, employing only washing and filtration steps. A click reaction was then used to
attach an additional layer of 5 onto 13, generating pG3 14, with protected hydroxide groups, which was
purified by precipitation. The compound was then deprotected to yield G3 (15, Scheme 4) with thirty
two terminal hydroxyls, which were used to introduce terminal acetylene groups through esterification
(16, Scheme 4).
OH
O
OH
O
O
OH
O
HO
O
O
N
N
O
N
N
N
N
O
O
O
N
O
O
O
O
O
N
HO
O
N
63%
O
N
O
N
N
N
O
O
O
HO
N
O
O
N
N
O
O
O
N
N
O
OH
OH
O
N
O
O
N
O
O
N
N
O
O
O
O
O
O
O
O
O
O
O
BiCl3
O
N
O
O
O
O
N
N
N
N
OH
11
O
O
O
N
O
O
N
N
N
N
N
O
O
N
O
N
12 (89%)
N
HO
HO
N
EDC, DMAP
DCM, pyridine
O
O
O
O
O
O
O
N
O
O
N
N
N
N
O
O
N
N
O
O
O
N
O
N
O
N
N
N
O
O
N
O
O
OH
N
O
O
O
O
N
O
O
N
O
O
O
N
OH
O
N
O
O
O
N
N
O
N
O
N
N
O
N
O
O
O
O
N
O
O
N
O
O
O
O
O
O
OH
O
O
O
HO
O
O
O
O
O
N
O
N
N
N
O
HO
O
N
O
O
O
O
O
N
O
N
N
O
O
O
N
HO
N
N
O
O
N
O
O
N N
O
O
N
N
O
N
N
O
O
N
O
O
O
N
O
O
O
O
N
O
O
O
O
N
O
O
O
O
N
O
O
N
O
O
O
N
N
O
N
O
N
N
O
13 (63%)
O
O
O
O
N
N
O
N
N
N
O
N
N
N
O
O
O
O
O
N
N
O
N
O
O
O
O
O
O
(5)
O
O
O
N
O
N
O
N
CuS
Na ascorbate
THF, H2O
O4
O
O
O
N
O
O
N
O
O
N
O
N
O
O
N
N
O
N
N
N
N
N
N
O
O
N
O
O
N
N
N
N
N
O
O
O
O
O
O
O
O
O
N
O
N
O
O
O
O
O
O
O
O
O
O
O
O
N
N
O
O
O
O
N
N
N
N
O
N
N
O
O
O
N
N
O
O
N
N
O
N
O
N
N
O
O
O
O
O
O
O
O
N
N
O
N
O
N
O
O
N
O
N
N
N
N N
N
O
O
N
O
O
O
O
O
O
O
O
O
N
O
O
O
N
N
O
O
O
14 (30%)
O
O
O
Scheme 3. Synthesis of G2-G3 dendrimers.
Dendrimers with positively charged amine groups on the surface were synthesized from
the acetylene terminated dendrimers, by reacting them with protected amino-azides. The latter
were synthesized from the commercially available amino alcohol. The amine was first protected
with a t-butoxycarbonyl (BOC) group to yield 17 (Scheme 5), which was purified by washing in
sequence with a basic followed by an acidic solution, brine and finally water. The alcohol group
of 17 was then converted to a mesylate upon reaction with methane sulfonyl chloride to yield 18
which was subsequently azidified to afford 19. The amino-azide with a longer arm was prepared
from commercially available amino-bromide by reacting it with sodium azide to yield 20, which was
,

Molecules 2017, 22, 868
5 of 18
purified by a basic work-up, followed by extraction (Scheme 5). The amine moiety of 20 was then
protected with tBOC to yield 21.
OH
O
14
O
OH
OH
OH
O
N
N
N
O
OH
OH
O
OH
OH
O
O
O
HO
O
O
N N
O
OH
N
N
N
N
N
N
O
O
N
O
O
N
O
O
O
O
N
O
O
N
O
N
N
O
N
O
N
N
OH
O
OH
HO
O
N
N
O
N
N
OH
O
N
N
N
N
OH
O
O
O
O
O
N
N
O
N
O
O
O
O
O
O
EDC, DMAP
DCM, pyridine
O
O
O
N
O
N
O
N
O
O
O
N
O
O
N
O
O
O
O
N
O
N
O
O
N
N
HO
N
N
N
N
O
O
O
N
N
OH
N
O
N
N
N
O
N
N
O
O
HO
OH
O
O
O
O
O
O
O
O
O
O
O
N
O
O
N
O
O
O
N
O
O
O
N
N
O
O
O
O
O
O
O
O
O
O
O
O
O
N
N
O
O
O
O
O
N
N
N
O
N
O
O
O
O
N
O
N
O
HO
HO
O
N N
N
N
O
O
N
N
O
O
N
OH
N
O
N
N
N
HO
O
N
O
O
N
N
N
N
O
O
O
O
O
N
O
O
N
O
O
O
O
O
O
O
O
O
O
O
O
N
N
N
O
N
O
N
O
O
O
O
N
N
N
O
O
N
O
O
N
N
N
O
N
N
N
N
O
O
O
HO
HO
N
O
N
N
O
N N
HO
N
N
O
N
O
OH
O
O
N
O
N
O
O
O
O
O
N
N
O
O
O
O
O
O
O
O
N
HO
O
HO
O
HO
HO
O
N
O
O
N
O
O
N
O
N
O
N
HO
O
O
O
O
N
HO
O
O
O
O
N
O
O
N
O
N
O
O
N
N
N
O
N
N
N
N
N
O
O
15 (71%)
N
N
O
O
N
O
N
N
N
N
O
O
O
O
O
O
O
O
O
O
O
N
O
N
O
O
O
O
O
O
O
O
O
O
O
O
O
N
N
N
O
O
O
O
N
N
N
O
N
N
O
O
N
N
O
O
O
O
N
N
O
N
O
N
N
O
O
O
O
O
O
O
O
O
O
O
O
N
N
O
N
O
N
O
O
N
O
N
N
N
N
O
O
N
N N
O
O
O
O
O
O
O
O
O
O
O
O
N
N
O
O
O
O
O
O
N
O
O
O
O
O
16 (38%)
O
O
O
Scheme 4. Synthesis of G3 dendrimer with terminal OH and acetylene groups.
H
BOC2O
H
H
MsCl
DCM
NaN3
N
N
O
O
O
NH2
N
O
O
HO
HO
N3
NEt3
DCM
S
DMF / H2O
17
18
O
O
19
O
O
H
Br
NaN3
BO
C2O
N3
N3
NH2
HN
O
H2O, reflux
MeOH, RT
Br
NH2
20
21
O
Scheme 5. Synthesis of protected amine-azides.
The protected amine-azides 19 and 21 were clicked on to the acetylene terminated dendrimers
, 10 and 13, and the surface amine groups on the resulting dendrimers were deprotected using
7
trifluoroacetic acid (TFA), to yield the desired cationic amino terminated dendrimers (Figure 1:
+
+
+
G0-NH3 22, G1-NH3 23, and G2-NH3 24). We used amine 21 that was one carbon longer for
G1-2 dendrimers to reduce any steric hinderance on the surface for the larger dendrimers.
2.2. Bactericidal Activity of Dendrimers
We explored the bactericidal efficacy of the cationic amine terminated dendrimers, and one of the
hydroxide terminated water soluble dendrimer ((G1-OH), Figure 1) for comparision purposes, as it
is known that hydroxide terminated molecules may also show bactericidal effect [20]. Bactericidal
efficacy was assessed using two parameters: minimum inhibitory concentration (MIC), and minimum
bactericidal concentration (MBC). MIC is the lowest concentration of the antimicrobial (in this study,

Molecules 2017, 22, 868
6 of 18
the dendrimer) that inhibits the growth of a microorganism after overnight incubation. It gives a
measure of the inhibition of the growth of bacteria as a function of concentration. MBC is the lowest
concentration of the dendrimer that will prevent the growth of bacteria, and MBC is always larger
than MIC. Aqueous solutions of dendrimers with a range of different concentrations were prepared,
and then added to aliquots of bacteria solution. Escherichia coli (E. coli) with standard Gram-negative
ATCC 11229TM strain were chosen for bactericidal activity experiments in this study, as these are the
most widely studied microorganisms, and have a much faster growth rate which simplifies the time
scale of the experiments. The initial optical density (OD) of the solutions was measured, and they
were subsequently incubated overnight. After 18–24 h had elapsed, the OD was measured again.
The relative absorbance of solutions as a function of bactericide concentration for G2-NH3+ is shown
in Figure 2. The minimum inhibitory concentration (MIC) was determined as the lowest concentration
point where the curve drops sharply to zero.
O
F
F
O
NH3
O
F
F
F
O
F
N
N
H3
N
N
N
N
N
N
N
O
O
O
N
O
F
F
O
O
O
O
O
F
F
O
O
O
H3
N
F
F
O
N
N
F
F
O
N
O
O
F
NH3
O
O
O
N
N
N
O
N
N
O
N
N
NH3
N
N
F
N
N N
O
N
F
F
N
O
N
N
O
O
NH3
O
O
N
F
F
O
N
N
O
H3N
O
O
O
N
N
O
O
F
F
F
O
O
O
NH3
N
N
O
F
O
F
F
H3
N
O
O
O
O
N
OH
O
N
N
N
O
F
N
N
N
N
H3N
N
O
N
N
NH3
N
N
O
O
O
F
F
O
F
F
F
+
+
O
NH3
22, G0-NH3
F F
F
23, G1-NH3
O
F
F
F
O
N
NH3
F
O
F
F
O
O
O
F
F
F
N
N
O
F
F
NH3
O
F
NH3
F
O
NH3
F
O
F
F
O
N
N
O
N
N
N
N
N
N
NH3
O
O
N
F
F
O
N
F
O
O
O
O
N
O
O
N
O
O
O
F
F
N
N
O
N
N
N
N
NH3
O
O
F
N
O
N
N
N
O
H3N
N
N
N
N
O
O
O
O
N
O
N
O
O
O
O
O
O
O
O
N
O
N
N
O
N
O
O
F
F
F
O
O
O
N
O
N
N
O
O
O
NH3
N
O
N
N
N
N
N
N
O
N
N
O
N
O
N
N
N
F
F
O
N
O
O
O
H3
N
O
F
O
O
N
O
N
O
O
O
O
F
F
O
F
O
O
O
O
O
O
N
N
N
O
O
O
O
O
NH3
N
N
N
O
N
N
O
N
N
N
O
O
N
N
O
N
N
H3
N
O
O
O
O
O
O
O
F
N
O
N
O
F
N
F
F
N
O
O
O
N
F
NH3
F
N
N
N
N
N
O
O
N
N
F
H3N
F
F
H3N
O
N
N
O
O
H3N
F
O
F
F
N
O
F
F
F
O
H3N
O
O
+
24, G2-NH3
F
F
F
Figure 1. Dendrimers with positively charged surface groups.

Molecules 2017, 22, 868
7 of 18
140
120
100
80
60
40
20
0
0
10
20
30
40
50
60
70
Dendrimer concentration (mg/L)
Figure 2. Relative absorbance versus dendrimer concentration curve for the determination of MIC
+
for G2-NH3
.
The bactericidal efficacies of four different dendrimers, three of them functionalized with cationic
+
amino groups (G0-NH3+, G1-NH3 & G2-NH3+), and the fourth with hydroxide surface groups
(G1-OH) are shown in Table 1. The most efficient bactericide was the generation one cationic amine
+
+
terminated dendrimer, G1-NH3 (MIC = 0.9 mg/L, 8 NH3 groups, Table 1), and the least effective
+
+
was the smallest structure, G0-NH3 (MIC = 32–64 mg/L, 4 NH3 groups, Table 1). Dendrimer G1-OH
was found to have no bactericidal action at any of the concentrations tested (MIC > 64 mg/L, 8 OH
groups, Table 1), which clearly suggests the importance of cationic surface groups for bactericidal
activity. Since G1-NH3+ was more effective in its bactericidal activity than both the lower (G0-NH3
4 NH3 groups, MIC = 32–64 mg/L,) and higher (G2-NH3 16 NH3 groups, MIC = 1–16 mg/L)
generation dendrimers with the same backbone and type of end groups, it indicated that G1-NH3
+
+
+
+
+
provides the best balance between the number of bactericidal end groups and biopermeability.
These results may also suggest that the generation 2 dendrimer is past the tipping point where
the gain in bactericidal activity obtained from additional charged end groups, is outweighed by the
+
+
loss of biopermeability. The MBC for G1-NH3 (4–8 mg/L, 8 NH3 groups, Table 1) followed the
expected pattern of MBC > MIC for a given structure.
Table 1. MIC and MBC values of dendrimers for E. coli (ATCC 11229TM strain).
Type of End
Groups
No. of End
Groups
MW
(g/mol)
MIC
(mg/L)
MIC
(µM)
MBC
(mg/L)
MBC
(µM)
Structure
G1-OH
-OH
8
4
8
1101.1
1088.5
3454.9
8075.0
>64
32–64
>58
29–59
>64
>64
4–8
-
>58
>59
1.1–2.3
-
+
+
−
−
−
G0-NH3+
-NH3+ TFA
G1-NH3+
G2-NH3
-NH3+ TFA
-NH3 TFA
0.9 ± 0.1 0.26
±
0.03
16
1–16 0.12–2
To better evaluate the bactericidal efficacy of our dendrimers, a comparison with dendrimers
for which bactericidal activity has been reported earlier was carried out, and the data is presented
in Table 2. The data corresponds only to dendrimers that function by the membrane disruptive
mechanism (as opposed to anti-adhesive), where an MIC was measured [14]. Our best performing
dendrimer, G1-NH3+ (8 NH3+ groups), is a more effective bactericide than any of the dendrimers (4–32
end groups) presented in Table 2. Comparable both in size and number of charged end groups to
+
+
the generation 2 carbosilane dendrimer (MIC = 64 mg/L, 8 NMe3 groups, Table 2) [21], G1-NH3
+
(MIC = 0.9 mg/L, 8 NH3 groups, Table 1) is more effective by almost two orders of magnitude.

Molecules 2017, 22, 868
8 of 18
Equally comparable in size and number of charged end groups are the generation one carbosilane
+
+
+
dendrimers (MIC = 4 mg/L, 4 NR3 groups), and G0-NH3 (MIC = 32–64 mg/L, 4 NR3 groups,
Table 1). Interestingly, however, the trend is reversed in this case, with the generation one carbosilane
dendrimer showed about an order of magnitude more bactericidal effect than G0-NH3+. Since both
sets of structures present the same number of charged amino groups, approximately the same
molecular weight, and were tested against the same bacteria, the only major difference between
the two dendrimer backbones seems to be in their hydrophilicity. It is known that structures which
present more hydrophobic units can permeate the bacterial structure more easily to arrive at their site
of action [14]. These results may suggest that for dendrimers with hydrophilic backbones, such as
the ones presented here, the peak bactericidal efficiency is reached at a higher generation than for
dendrimers with hydrophobic backbones, such as carbosilane.
Table 2. Comparative MIC values for dendrimers.
Generation &
Backbone
No. End
Groups
Type of End
Groups
MW
(g/mol)
MIC
(mg/L)
MIC
(µM)
Type of Bacteria
Pseudomonas
aeruginosa (PA)
G3 PAMAM
31
-NH2
6859
6.3
0.92
G5 PAMAM
G1 Carbosilane
G2 Carbosilane
110
4
-NH2
26 373
1470.4
3493.8
12.5
4
0.47
2.7
PA
Escherichia coli (E coli)
E coli
+
+
−
−
-N(Me)3
-N(Me)3
I
I
8
64
18.3
The bactericidal efficacy of the neutral amine terminated generation three PAMAM dendrimer
(MIC = 6.3 mg/L, 31 NH2 groups Table 2) and generation five (12.5 mg/L, 110 NH2 groups), both fall
+
+
into the wide range determined for our G2-NH3 (MIC = 1–16 mg/L, 16 NH3 groups, Table 1)
dendrimer. The molecular mass of G2-NH3+ is comparable to that of the generation three PAMAM
dendrimer. It is difficult to draw definite conclusions about these results due to the fact that the
structures have different backbones, number of end groups, type of end groups, and were tested
against different bacteria. Interestingly, the neutral structures that were previously tested (generations
three and five PAMAM dendrimers) both performed better than the only neutral structure tested in
this study, G1-OH (MIC > 64 mg/L, 8 OH groups, Table 1).
Our results, as well as those for the dendrimers previously evaluated [29,30], indicate that higher
generation number may not be a necessary and important factor for bactericidal action. Although a
detailed evaluation is necessary in order to determine structure-property relationships, it should
be noted that the largest structure, the generation 5 PAMAM dendrimer with 110 peripheral amino
groups, is the least effective bactericide. This suggests that a balance between the size of the dendrimer
and the number of end groups is a key factor.
3. Materials and Methods
3.1. Generaal Information
The reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and
Fisher Scientific (Hampton, NH, USA), and used as received. NMR spectral acquisitions were
carried out on Mercury instruments (Varian, Palo Alto, CA, USA) and operated using VNMRJ 2.2D
(Chempack 5) and VNMRJ 2.3A (Chempack 5) software using a 5 mm Smart Probe. The chemical
1
shifts in ppm are reported relative to tetramethylsilane (TMS) as an internal standard for H,
and 13C. Mass spectra analyses (HRMS, ESI) were performed and analysed on an Exactive Plus
Orbitrap-API (Thermo Scientific, Waltham, MA, USA) high resolution mass spectrometer and on
MALDI Autoflex III-TOF (Bruker, Billerica, MA, USA). Procedures for the MIC and MBC determination
were followed as described in [31]. The Compounds 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid

Molecules 2017, 22, 868
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(2) [32], azidoethanol [33], and propargylated pentaerythritol [34], were prepared by adopting and
modifying the procedures reported earlier.
3.2. Building Blocks
2,2,5-Trimethyl-1,3-dioxane-5-carboxylic acid (
2): p-Toluenesulfonic acid (0.5975 g, 0.003141 mol) was
added to a stirred solution of 2,2-bis(hydroxymethyl)propanoic acid (1, 8.4265 g, 0.06282 mol) in
acetone (34 mL) in a 250 mL round bottom flask, under nitrogen. 2,2-Dimethoxypropane (9.8141 g,
0.09423 mol) and magnesium sulfate (0.7562 g, 0.006282 mol) were then added, and the reaction
mixture was left to stir for 48 h. A solution of ammonia in dioxane (6.27 mL, 0.5 M) was added to
neutralize the acid. The crude mixture was filtered, and the solvent evaporated to yield the product as a
1
white powder (9.30 g, 0.0534 mol, 85% yield). H-NMR (400 MHz, CDCl3):
δ
= 1.20 (s, 3H, -CO-C-C
2-C-CO-), 4.18 (d, 2H, -O-C 2-C-CO-)
H3), 22.03 (-O-C- H3), 25.07 (-O-C- H3),
41.71 (-CO-C-), 65.81 (-O-CH2-C-), 98.28 (-O-C-(CH3)2), 180.15 (-CO-) ppm.
H3),
1.40 (s, 3H, -O-C-C
H
3), 1.43 (s, 3H, -O-C-C
H
3), 3.67 (t, 2H, -O-C
= 18.42 (-CO-C-
H
H
ppm. 13C{1H}-NMR (300 MHz, CDCl3):
δ
C
C
C
Azidoethanol (
4
): A mixture of bromoethanol (
3
, 2.3742 g, 0.01900 mol) and sodium azide (2.4700 g,
◦
0.03800 mol) in water (4 mL) was left stirring overnight at 70 C. The reaction mixture was then
extracted with DCM, the organic layer was isolated and dried with magnesium sulfate. The solvent was
1
then evaporated to yield the product as yellow oil (1.36 g, 0.01561 mol, 82% yield). H-NMR (400 MHz,
CDCl3):
CDCl3): δ = 53.6 (N3-CH2-CH2-), 61.6 (N3-CH2-CH2-) ppm.
δ
= 3.46 (t, 2H, N3-CH2-CH2-), 3.79 (q, 2H, N3-CH2-CH2-) ppm. 13C{1H}-NMR (300 MHz,
2-Azidoethyl 2,2,5-trimethyl-1,3-dioxane-5-carboxylate ( ): A solution of azidoethanol (
5
4, 0.74 g,
0.008498 mol), 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid ( , 2.2204 g, 0.01275 mol) and DMAP
2
(0.5191 g, 0.004249 mol) in anhydrous DCM (10 mL) was stirred for 5 min under nitrogen.
DCC (2.1041 g, 0.01020 mol) was then added to the reaction mixture, and stirred under nitrogen,
at room temperature, overnight. The precipitate was filtered off, and the solvent was evaporated to
yield a residue that was purified by column chromatography (1:7 ethyl acetate–hexanes) to yield the
1
product as a white solid (1.25 g, 0.005139 mol, 60% yield). H-NMR (400 MHz, CDCl3):
δ = 1.21 (s, 3H,
-CO-C-CH3), 1.39 (s, 3H, -O-C-CH3), 1.44 (s, 3H, -O-C-CH3), 3.49 (t, 2H, N3-CH2-CH2-), 3.68 (d, 2H,
-O-C 2-C-CO-), 4.21 (d, 2H, -O-C 2-C-CO-), 4.33 (t, 2H, N3-CH2-C
2-) ppm. 13C{1H}-NMR (300 MHz,
CDCl3): = 18.5 (-CO-C- H3), 22.3 (-O-C- H3), 24.9 (-O-C- H3), 42.0 (-C-CH3), 49.8 (N3- H2-CH2-),
63.6 (N3-CH2-CH2-), 65.9 (-O-CH2-C-), 98.1 (-C-CH3), 174.0 (-CO-) ppm.
H
H
H
δ
C
C
C
C
2-Azidoethyl 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate ( ): A spoonful of Dowex cationic resin
6
was added to a solution of 5 (0.301 g, 0.001728 mol) in methanol (6 mL). The mixture was left stirring
overnight at room temperature. The supernatant was decanted off and the resin was rinsed several
times with methanol. The supernatant was then combined with rinsing and the solvent was evaporated
1
to yield the product as colorless oil (0.238 g, 0.001171 mol, 95% yield). H-NMR (400 MHz, CDCl3):
δ
= 1.09 (s, 3H, -CO-C-CH3), 2.47 (s, 2H, -O
3.80 (dd, 2H, -C-CH2-OH), 4.28 (t, 2H, N3-CH2-CH2-) ppm. 13C{1H}-NMR (300 MHz, CDCl3):
(CH3-C-), 49.3 (CH3-C), 49.8 (N3-CH2-CH2-), 63.5 (N3-CH2-CH2-), 68.2 (CH2-OH), 175.5 (-CO-) ppm.
H
), 3.41 (m, 2H, N3-CH2-CH2-), 3.50 (dd, 2H, -C-CH2-OH),
δ
= 17.0
Generation 0 ( ): Anhydrous DMF (25 mL) was added by syringe to a round bottom flask containing
7
pentaerythritol (2 g, 0.01469 mol) and KOH (12.5 g, 0.2228 mol), with stirring, under nitrogen.
The mixture was stirred for 5 min, under nitrogen, at 0 ◦C. A solution of propargyl bromide (20 g,
0.1681 mol) in toluene (80%) was then added dropwise to the reaction mixture over 30 min. The reaction
was heated at 40 ◦C overnight, under nitrogen. Water (100 mL) was added to the mixture, which was
then extracted with diethyl ether (3
×
50 mL). The organic layers were isolated, combined, and washed
50 mL). The organic layer was isolated and dried with
with water (3 50 mL) and brine (3
×
×
sodium sulfate. The solvent was removed to yield an orange oil which was purified by column
chromatography (2:8 ethyl acetate–hexanes) to give the product as an orange-brown solid (1.7468 g,

Molecules 2017, 22, 868
10 of 18
0.006058 mol, 84% yield). 1H-NMR (400 MHz, CDCl3):
δ
= 2.40 (t, 4H, C
H
-C-CH2-), 3.53 (s, 8H,
-C-CH2-O-), 4.12 (d, 8H, CH-C-CH2-) ppm. 13C{1H}-NMR (300 MHz, CDCl3):
δ
= 44.7 (-C-CH2-O-),
58.7 (CH-C-
[M + Na+].
CH2), 69.0 (-C-CH2-O-), 74.0 (CH-C-CH2), 80.0 (CH-C-CH2) ppm. ESI-MS: m/z = 311.1
Protected Generation 1 (pG1
(0.5 mL) was added to a round bottom flask containing a stirred solution of
and ( ) (0.2683 g, 0.001103 mol) in tetrahydrofuran (THF) (0.5 mL). Sodium ascorbate (0.0317 g,
,
9
): A solution of CuSO4
·
5H2O (0.0191 g, 0.00007669 mol) in water
7
(0.0768 g, 0.0002663 mol)
5
0.0001598 mol) was added, and the mixture was allowed to react overnight. The product was
purified by column chromatography (100% methanol) to yield a yellow oil (0.2778 g, 0.0002202 mol,
1
83% yield). H-NMR (400 MHz, CD3OD):
δ = 1.00 (s, 3H, -CO-C-CH3), 1.22 (s, 3H, -O-C-(CH3)2),
1.39 (s, 3H, -O-C-(CH3)2), 3.44 (s, 2H, -C-CH2-O-CH2), 3.63 (dd, 2H, -CO-C-CH2-O-), 4.07 (dd,
2H, -CO-C-CH2-O-), 4.51 (s, 2H, -O-CH2-C-N-), 4.54 (t, 2H, -N-CH2-CH2-O-), 4.72 (s, 2H,
-N-CH2-C
20.52 (-O-C-(
62.63 (-N-CH2-
97.97 (-O- -(CH3)2), 124.07 (-C-
m/z = 1267.3 [M + Li+].
H
2-O-), 8.04 (s, 1H, -C-C
H3)2), 24.93 (-O-C-(
H2-O-), 64.06 (-CH2-O-
H-N-), 145.00 (-
H
C
-N-) ppm. 13C{1H}-NMR (300 MHz, CD3OD):
H3)2), 41.78 (-C- -CH3), 45.11 (- -CH2-O-), 48.90 (-N-
H2-C-N-), 65.47 (-C- H2-O-C-), 68.65 (-C- H2-O-CH2-C-N-),
-N-), 173.80 (- -O-CH2-) ppm. MALDI-MS:
δ
= 17.19 (-C-
CH3),
C
C
C
C
H2-CH2-O-),
C
C
C
C
C
C
C
C
Generation G1 ). A solution of CuSO4
added to a round bottom flask containing a stirred solution of
1
(
,
8
·
5H2O (0.0191 g, 0.00007669 mol) in water (0.5 mL) was
(0.0768 g, 0.0002663 mol) and
7
6
(0.2597 g, 0.001278 mol) in THF (0.5 mL). Sodium ascorbate (0.0317 g, 0.0001598 mol) was then added
and the mixture was allowed to react overnight. The product was purified by column chromatography
(100% methanol) to yield an orange oil (0.1479 g, 0.0001343 mol, 50% yield).
G1
0.0077 mol) in methanol (485 mL). The mixture was then left stirring overnight. The resin was
then filtered off and the solvent evaporated to yield as a viscous orange oil (6.68 g, 0.0061 mol,
79% yield). 1H-NMR (400 MHz, CD3OD):
= 1.08 (s, 3H, -CO-C-CH3), 3.45 (s, 2H, -C-CH2-O-),
3.61 (q, 4H, -C-CH2-OH), 4.51 (t, 4H, -O-CH2-C-N-), 4.52 (s, 2H, -N-CH2-CH2-O-), 4.70 (t, 2H,
-N-CH2-CH2-O-), 8.04 (s, 1H, -C-C = 17.40
-N-) ppm. 13C{1H}-NMR (300 MHz, CD3OD):
H2-O-),
H-N-), 146.35 (-O-CH2- -N-),
(8) from pG1 (9): Dowex Cationic Resin (6.72 g) was added to a solution of pG1 (9, 9.69 g,
8
δ
H
δ
(-C-
C
H3), 46.53 (
C
-CH2-O-), 50.42 (-
C
-CH3), 51.78 (-N-CH2-CH2-O-), 63.86 (-N-CH2-
C
65.40 (-O-
C
H2-C-N-), 65.85 (-C-
C
H2-OH-), 70.09 (C-
C
H2-O-), 125.77 (-C-
C
C
176.06 (-C-CO-O-) ppm. MALDI-MS: m/z = 1107.6 [M + Li+].
G1-ester-acetylne (10). DMAP (2.97 g, 0.0243 mol) was added to a stirred mixture of G1 (8, 6.68 g,
0.0061 mol) in anhydrous DCM (267 mL) in a 500 mL round bottom flask under nitrogen. DCC (15.02 g,
0.0728 mol), pyridine (133 mL) and 4-pentynoic acid (7.14 g, 0.0728 mol) were added, and the reaction
mixture was then left stirring, under nitrogen, overnight. The crude mixture was filtered and the
solvent evaporated. The product was purified by column chromatography (ethyl acetate) to yield the
1
product as a yellow oil (7.39 g, 0.0042 mol, 70% yield). H-NMR (400 MHz, CD3OD):
δ
= 1.19 (s, 3H,
-C-CH3), 2.26 (t, 2H, -C-C
4.22 (q, 4H, -C-(CH2-O-C-)2), 4.54 (s, 2H, -C-CH2-O-CH2-C), 4.55 (t, 2H, -CH-N-CH2-CH2-),
4.73 (t, 2H, -C-CH-N-CH2-), 8.01 (s, 1H, -N-C = 15.10
-) ppm. 13C{1H}-NMR (300 MHz, CD3OD):
(- H2-C-CH), 18.20 (-C- H3), 34.31 (- H2-CH2-C-CH), 46.57 (- -CH2-O-CH2-C-N-), 47.70 (- -CH3),
H2-O-), 65.59 (-C-CH2-O- H2-C-N-), 66.61 (-C- H2-O-C-),
H), 83.54 (- -CH), 125.49 (-C- H-N-), 146.51 (- -CH-N-),
H), 2.46–2.53 (m, 8H, -O-C-CH2-CH2-C-), 3.45 (s, 2H, -C-CH2-O-CH2-),
H
δ
C
C
C
C
C
50.22 (-N-
C
H2-CH2-O-), 64.51 (-N-CH2-
C
C
C
C
C
70.19 (-C-
C
H2-O-CH2-C-N-), 70.55 (-C-
C
C
172.84 (-O-C-CH2-), 173.72 (O-C-C-) ppm. MALDI-MS: m/z = 1747.5 [M + Li+].
Protected Generation
(10 mL) was added to a round bottom flask containing a stirred solution of 10 (1.70 g, 0.0009766 mol)
and (2.2807 g, 0.009375 mol) in THF (10 mL). Sodium ascorbate (0.2322 g, 0.001172 mol) was
added and the mixture was allowed to react overnight. The product mixture was evaporated,
2
(
pG2
,
11). A solution of CuSO4
·
5H2O (0.1463 g, 0.000586 mol) in water
5

Molecules 2017, 22, 868
11 of 18
dissolved in a minimum of methanol and run through a silica plug to remove the copper salts.
The product was then purified by dialysis (MWCO = 1000 Da, methanol) to yield a brown solid
1
(2.05 g, 0.000556 mol, 57% yield). H-NMR (400 MHz, CD3OD):
δ
= 1.02 (s, 6H, CH3-C-O-CH2-C-CH3),
1.10 (s, 3H, C-C-O-CH2-C-C
H
3), 1.28 (s, 6H, C
H
3-C-O-CH2-C-CH3), 1.39 (s, 6H, C 3-C-O-CH2-C-CH3),
H
2.68 (t, 4H, -O-C-CH2-CH2-), 2.94 (t, 4H, t, 4H, -O-C-CH2-CH2-), 3.43 (s, 2H, -C-CH2-O-CH2-C-N-),
3.64 (dd, 4H, -C-CH2-O-C-CH3), 4.07 (dd, 4H, -C-CH2-O-C-CH3), 4.10 (s, 4H, -C-CH2-O-C-CH2),
4.50 (m, 8H, -N-CH2-CH2-O-C-C-CH2-O-C-CH3, -O-CH2-C-CH-N-CH2-CH2-), 4.68 (m, 6H,
-N-CH2-CH2-O-C-C-CH2-O-C-CH3, -O-CH2-C-CH-N-CH2-CH2-), 7.85 (s, 1H, -O-CH2-C-C
H-N-),
8.02 (s, 2H, -C-CH2-CH2-C-C = 18.10, 18.68, 21.85,
H
-N-) ppm. 13C{1H}-NMR (300 MHz, CD3OD):
δ
26.49, 34.24, 43.24, 46.57, 47.67, 50.03, 50.17, 50.27, 64.15, 64.48, 65.59, 66.51, 67.04, 70.22, 99.44, 124.20,
125.51, 146.45, 147.69, 173.37, 173.68, 175.31 ppm. MALDI-MS: m/z = 3710.1[M + Na+].
Generation
2 (G2, 12): BiCl3 (0.0009 g, 0.00000285 mol) and a drop of water were added to a
stirred solution of pG2
(
11, 0.02◦5 g, 0.00000678 mol) in acetonitrile (1.1 mL). The reaction mixture
was left stirring for 60 h at 45 C. The product mixture was evaporated, dissolved in methanol,
and filtered to remove the bismuth salts. The solvent was evaporated to yield a brown oil
1
(0.020 g, 0.00000604 mol, 89% yield). H-NMR (400 MHz, CD3OD):
δ = 1.10 (s, 9H, -C-CH3),
2.69 (t, 4H, -C-CH2-CH2-C-N-), 2.95 (t, 4H, -C-CH2-CH2-C-N-), 3.43 (s, 2H, -C-CH2-O-CH2-C-N-),
3.60 (q, 8H, -C-CH2-OH), 4.10 (s, 4H, -C-CH2-O-C-), 4.48 (m, 8H, -N-CH2-CH2-O-C-C-CH2-OH,
-O-CH2-C-CH-N-CH2-CH2-O-), 4.66 (m, 4H, -N-CH2-CH2-O-C-C-CH2-OH), 4.71 (s, 2H,
-O-CH2-C-CH-N-CH2-CH2-O-, -O-CH2-C-CH-N-CH2-CH2-O-), 7.86 (s, 2H, -CH2-CH2-C-C
H-N-),
8.03 (s, 1H, -O-CH2-C-C = ppm 17.41, 18.08, 34.18,
H
-N-) ppm. 13C{1H}-NMR (300 MHz, CD3OD):
δ
46.53, 47.65, 49.99, 50.17, 50.34, 51.79, 63.91, 64.45, 65.51, 65.86, 66.51, 70.13, 124.35, 125.61, 146.37,
147.58, 173.47, 173.70, 176.08. MALDI-MS: m/z = 3388.5[M + K+].
G2-ester-acetylene (13). DMAP (0.1586 g, 0.001298 mol) was added to a stirred mixture of 12 (0.2732 g,
0.00008113 mol) in anhydrous DCM (6.6 mL), under nitrogen. 1-ethyl-(3-dimethylaminopropyl)-
carbodiimide hydrochloride (EDC) (0.3732 g, 0.001947 mol), pyridine (3.3 mL) and 4-pentynoic acid
(0.1911 g, 0.001947 mol) were added to the flask. The reaction mixture was left stirring, under nitrogen,
overnight. The crude product mixture was then washed with methanol, dissolved in chloroform and
1
filtered to yield the product as brown oil (0.27 g, 0.0000581 mol, 63% yield). H-NMR (400 MHz, CDCl3):
δ
= 1.12 (s, 3H, -N-C-CH2-CH2-C-O-CH2-C-CH3), 1.20 (s, 6H, -CH-C-CH2-CH2-C-O-CH2-C-CH3),
1.98 (s, 4H, -C-C ), 2.50 (m, 16H, -C-C 2-C 2-C-CH), 2.70 (t, 4H, -O-C-C 2-CH2-C-N-), 2.97 (t, 4H,
H
H
H
H
-O-C-CH2-CH2-C-N-), 3.42 (s, 2H, -C-CH2-O-CH2-), 4.10 (m, 4H, -C-CH2-O-C-CH2-CH2-C-N-),
4.21 (m, 8H, -C-CH2-O-C-CH2-CH2-C-CH), 4.51 (m, 8H, -CH2-CH2-C-N-N-N-CH2-CH2-O-,
-C-CH2-O-C
-C-CH2-O-CH2-C-CH-N-CH2-CH2-), 7.48 (s, 2H, -O-C-CH2-CH2-C-C
-CH2-O-CH2-C-C = ppm 14.26, 17.58, 17.68,
-N-) ppm. 13C{1H}-NMR (300 MHz, CDCl3):
H
2-C-CH-N-C
H
2-CH2-), 4.59 (m, 4H, -C-CH2-O-CH2-C-CH-N-CH2-C
H2-), 4.65 (m, 2H,
H
-N-), 7.78 (s, 1H,
H
δ
20.76, 33.11, 33.15, 43.19, 45.17, 46.27, 46.29, 48.67, 63.10, 64.75, 65.10, 65.25, 69.02, 69.34, 77.20, 82.26,
121.87, 123.31, 145.29, 146.22, 171.09, 171.98, 172.09, 172.14. MALDI-MS: m/z = 4669.1 [M + Na+].
pG3
(
14). A solution of CuSO4
·
5H2O (0.0081 g, 0.000586 mol) in water (2 mL) was added to a round
(0.1268 g, 0.0005212 mol)
bottom flask containing a stirred solution of 13 (0.1262 g, 0.00002715 mol) and
5
in THF (2 mL). Sodium ascorbate (0.0129 g, 0.00006515 mol) was added and the mixture was allowed
to react overnight. The product mixture was evaporated, dissolved in a minimum of methanol and
run through a silica plug to remove the copper salts. The product was precipitated out of chloroform
1
with hexanes, and dried to yield a yellow oil (0.067 g, 0.00000785 mol, 30% yield). H-NMR (400 MHz,
CDCl3):
δ = 1.04 (s, 12H, CH3-C-O-CH2-C-CH3), 1.11 (s, 3H, C-C-O-CH2-C-CH3), 1.12 (s, 6H,
C-C-O-CH2-C-CH3), 1.33 (s, 12H, CH3-C-O-CH2-C-CH3), 1.41 (s, 12H, CH3-C-O-CH2-C-CH3),
2.69 (t, 12H, -O-C-CH2-CH2-), 2.97 (t, 12H, -O-C-CH2-CH2-), 3.40 (s, 2H, C-CH2-O-CH2-),
3.62 (dd, 8H, -C-CH2-O-C-CH3), 4.10 (s, 12H, -C-CH2-O-C-CH2), 4.12 (dd, 8H, -C-CH2-O-C-CH3),
4.40–4.75 (m, 30H, -CH2-CH2-C-N-N-N-CH2-CH2-O-, -C-CH2-O-CH2-C-CH-N-CH2-CH2-,

Molecules 2017, 22, 868
12 of 18
-N-CH2-CH2-O-C-C-CH2-O-C-CH3), 7.56 (s, 2H, -C
H
-N-CH2-CH2-O-C-C-CH2-O-C-CH2-), 7.61 (s, 4H,
-C
H
-N-CH2-CH2-O-C-C-CH2-O-C-CH3), 7.88 (s, 1H, -CH2-O-CH2-C-C
= ppm 17.48, 18.12, 20.71, 20.86, 26.22, 33.05, 41.98, 46.19, 48.68, 48.99, 62.82, 62.94,
H
-N-) ppm. 13C{1H}-NMR
(300 MHz, CDCl3):
δ
65.04, 65.85, 68.41, 77.20, 98.05, 122.22, 146.71, 171.86, 171.98, 172.05, 173.70. MALDI-MS: m/z = 8580.6
[M + K+].
G3
(
15). Dowex Cationic Resin (0.0629 g) was added to a solution of pG3
(
14,◦0.1531 g, 0.0000179 mol)
in methanol (8.5 mL). The mixture was left stirring for three days at 45 C. The resin was then
filtered off and the solvent evaporated to yield the product as a yellow oil (0.0983 g, 0.0000124 mol,
71% yield). 1H-NMR (400 MHz, CD3OD):
δ = 1.09 (s, 21H, -C-CH3), 2.69 (m, 12H, -O-C-CH2-CH2-C-N-),
2.95 (m, 12H, -O-C-CH2-CH2-C-N-), 3.43 (s, 2H, -C-CH2-O-CH2-C-), 3.60 (q, 16H, -C-CH2-OH),
4.10 (m, 12H, -C-CH2-O-C-), 4.48 (m, 16H, -C-CH2-O- CH2-C-N-, -N-CH2-CH2-O-), 4.66 (m, 14H,
-N-) ppm. 13C{1H}-NMR (300 MHz, DMSO-d6):
-N-CH2-CH2-O-), 7.82–8.07 (m, 7H, -C-CH δ = ppm
16.65, 16.85, 16.89, 20.38, 32.69, 45.77, 45.80, 48.05, 48.34, 48.53, 50.24, 51.25, 62.24, 62.94, 63.06,
63.68, 63.84, 64.82, 122.31, 122.44, 123.98, 144.08, 145.22, 145.26, 171.50, 171.83, 174.20. MALDI-MS:
m/z = 7894.2 [M + H+].
G3-ester-acetylene (16). DMAP (0.0266 g, 0.0002181 mol) was added to a stirred mixture of
G3 (0.0713 g, 0.000006814 mol) in anhydrous DCM (3.3 mL), under nitrogen. EDC (0.0627 g,
0.0003271 mol), pyridine (1.7 mL) and 4-pentynoic acid (0.0796 g, 0.0003271 mol) were added and
the reaction mixture was left stirring, under nitrogen, overnight. The crude product mixture was
dissolved in DCM and filtered to remove the salts. The product was then precipitated out of DCM
with hexanes. Ether and hexanes washes were then performed to obtain a light brown oil (38%
1
yield). H-NMR (400 MHz, CDCl3):
δ
= 1.07–1.24 (m, 32H, -CH3), 1.99 (s, 8H, -C
H
), 2.43–2.55
2-C-N-),
(m, 32H, -CO-C
H
2-CH
2-C-CH), 2.71 (m, 12H, -CO-C 2-CH2-C-N-), 2.96 (m, 12H, -CO-CH2-CH
H
3.42 (s, 2H, -C-CH2-O-CH2-C-N-), 4.11 (s, 12H, CH3-C-CH2-O-CO-CH2-CH2-C-N-), 4.17–4.26 (m, 16H,
CH3-C-CH2-O-CO-CH2-CH2-C-CH), 4.49–4.66 (m, 30H, -N-CH2-CH2-O-, -C-CH2-O-CH2-C-N-),
7.48–7.80 (m, 7H, -C-C
17.29, 17.62, 17.72, 20.9, 29.69, 33.14, 43.60, 45.22, 46.33, 48.68, 48.98, 62.95, 63.14,
64.82 (-C-CH2-O-CH2-C-N-N-N-CH2- H2-), 64.97, 65.14, 65.28, 65.80, 69.39, 77.21, 82.30, 121.91,
H
-N-) ppm.
13C{1H}-NMR (300 MHz, CDCl3):
δ = ppm 14.29,
C
121.98, 122.38, 146.18, 146.25, 146.29, 171.12, 172.01, 172.05 172.13, 172.15, 172.18. MALDI-MS:
m/z = 10,000–11,000 (theoretical mass = 10,456).
Amine functionalization: tert-butyl (2-hydroxyethyl)carbamate (17). Di-tert-butyl dicarbonate (BOC2O)
(7.86 g, 0.036 mol) was added slowly in a portionwise fashion to a stirred solution of ethanolamine
◦
(2.0 g, 0.033 mol) in DCM and triethylamine (5 mL), at 0 C. The mixture was allowed to react overnight,
at room temperature. The crude mixture was washed with an aqueous potassium carbonate solution,
followed by an aqueous hydrochloric acid solution, brine, and finally water. The organic phase was
isolated, dried with magnesium sulfate, and the solvent evaporated to yield the product as a colourless
1
oil (3.70 g, 0.023 mol, 70% yield). H-NMR (400 MHz, CDCl3):
δ
= 1.47 (s, 9H, -C-(CH3)3), 3.26 (s, 2H,
-) ppm. 13C{1H}-NMR (300 MHz, CDCl3):
= 28.6
(s, -C-(CH3)3), 43.4 (s, -CH2-NH-), 62.8 (s, -CH2-OH), 79.9 (C-(CH3)3), 157.1 (-NH-CO-O-) ppm.
-CH2-NH-), 3.67 (s, 2H, -CH2-OH), 5.04 (s, 1H, -N
H
δ
2-((tert-Butoxycarbonyl)amino)ethyl methanesulfonate (18). Methanesulfonyl chloride (9.42 g, 0.0822 mol)
was added very slowly in a dropwise fashion to a stirred solution of (17) (12.8713 g, 0.0798 mol) in
anhydrous DCM (500 mL) and triethylamine (20 mL) at 0 ◦C, under nitrogen. The reaction was left
stirring, under nitrogen at room temperature overnight. The crude mixture was then washed with
water (2
sulfate and the solvent evaporated to yield the product as a yellow oil (17.95 g, 0.075 mol, 94% yield).
1H-NMR (400 MHz, CDCl3):
= 1.44 (s, 9H, -C-(CH3)3), 3.03 (s, 3H, -S-CH3), 3.46 (m, 2H, -CH2-NH-),
4.28 (m, 2H, -C 2-O-), 4.92 (s, 1H, -N = 28.6 ( -C-( H3)3),
-) ppm. 13C{1H}-NMR (300 MHz, CDCl3):
37.6 (-CH2-NH-), 40.2 (-S-CH3), 69.1 (-CH2-O-), 80.2 (-C-(CH3)3) 158 (-NH-CO-O-) ppm.
×
250 mL) and brine (2
×
250 mL). The organic layer was isolated, dried with magnesium
δ
H
H
δ
C

Molecules 2017, 22, 868
13 of 18
tert-Butyl (2-azidoethyl)carbamate (19): Sodium azide (27.30 g, 0.4199 mol) was added to a stirred solution
of (18) (19.98 g, 0.0835 mol) in DMF (100 mL) and water (100 mL), under nitrogen. The reaction was left
◦
stirring, at 80 C overnight, under nitrogen. The crude product mixture was extracted with ethyl acetate
(3
and brine (2
evaporated to yield the product as a yellow oil (9.93 g, 0.0533 mol, 64% yield). H-NMR (400 MHz,
CDCl3): = 1.44 (s, 9H, -C-(CH3)3), 3.28 (m, 2H, -CH2-NH-), 3.40 (m, 2H, N3-CH2-), 4.82 (s, 1H,
-N = 28.3 (-C-( H3)3), 40.0 (- H2-NH-), 51.2 (- H2-N3),
-) ppm. 13C{1H}-NMR (300 MHz, CDCl3):
79.8 (-C-(CH3)3), 155.70 (-NH-CO-O-) ppm.
×
100 mL), the organic layers were isolated and combined and washed with water (3
×
100 mL)
×
100 mL). The organic layer was isolated, dried with magnesium sulfate and the solvent
1
δ
H
δ
C
C
C
3-Azidopropylamine (20). A solution of sodium azide (3.39 g, 0.05215 mol) in water (11 mL) was
added to a stirred solution of 3-bromopropylamine hydrobromide (3.42 g, 0.01566 mol) in water (8 mL).
The reaction was refluxed overnight, and 2/3 of the solvent was then evaporated and the crude mixture
was placed in an ice bath. Potassium hydroxide (4.2 g, 0.0749 mol) and ether (50 mL) were added,
the aqueous phase was isolated and washed with ether (3
×
35 mL). The organic phases were isolated,
combined and dried with magnesium sulfate to yield the product as a yellow oil (1.38 g, 0.0138 mol,
88% yield). 1H-NMR (400 MHz, CDCl3):
δ = 1.42 (s, 2H, -NH2), 1.72 (q, 2H, -CH2-CH2-CH2-), 2.79 (t, 2H,
NH2-CH2-CH2-CH2-N3), 3.36 (t, 2H, NH2-CH2-CH2-CH2-N3) ppm. 13C{1H}-NMR (300 MHz, CDCl3):
δ = 30.54 (-CH2-CH2-CH2-), 36.13 (N3-CH2-CH2-CH2-NH2), 51.12 (N3-CH2-CH2-CH2-NH2) ppm.
tert-Butyl (3-azidopropyl)carbamate (21). A solution of BOC2O (1.0899 g, 0.004994 mol) in methanol
(6 mL) was added dropwise to a solution of (20) (0.500 g, 0.004994 mol) in triethylamine (3.48 mL) and
methanol (20 mL). The reaction was left stirring, overnight at room temperature. The product mixture
was filtered, and the solvent evaporated. The mixture was purified by column chromatography
1
(chloroform) to yield the product as a yellow oil (0.5457 g, 0.00273 mol, 55% yield). H-NMR (400 MHz,
CDCl3):
3.33 (t, 2H, -CH2-CH2-CH2-N3), 4.75 (s, 1H, -N
(C-( H3)3), 29.87 (-CH2- H2-CH2-), 48.13 (N3-CH2-CH2-
79.74 (-C-(CH3)3), 155.47 (-NH-COO-) ppm.
δ
= 1.41(s, 9H, -C-(CH3)3), 1.74 (q, 2H, -CH2-CH2-CH2-), 3.17 (t, 2H, -CH2-CH2-CH2-N3),
-) ppm. 13C{1H}-NMR (300 MHz, CDCl3):
= 28.39
H2-NH-), 53.93 (N3- H2-CH2-CH2-NH-),
H
δ
C
C
C
C
G0-NHBOC. A solution of CuSO4 5H2O (0.0754 g, 0.000302 mol) in water (2.5 mL) was added
·
to a round bottom flask containing a stirred solution of (0.300 g, 0.00104 mol) and 19
7
(0.8528 g, 0.00458 mol) in THF (2.5 mL). Sodium ascorbate (0.1236 g, 0.000624 mol) was added
and the mixture was allowed to react overnight. The product mixture was evaporated and
purified by column chromatography (acetone) to yield an orange solid (0.9041 g, 0.000875 mol,
97% yield). 1H-NMR (400 MHz, CDCl3):
δ
= 1.36 (s, 9H, -C-(CH3)3), 3.41 (s, 2H, -C-CH2-O-),
3.55 (q, 2H, -N-CH2-CH2-NH-), 4.43 (t, 2H, -N-CH2-CH2-NH-), 4.47 (s, 2H, -C-CH2-O-CH2-),
5.62 (t, 1H, -N -), 7.56 (s, 1H,-C-C = 28.66
-N- ) ppm. 13C{1H}-NMR (300 MHz, CDCl3):
(-C-( H3)3), 40.29 (-CH-N-CH2- H2-), 46.47 (- -CH2-O-CH2-C-N-), 48.52 (-CH-N- H2-CH2-),
65.44 (-C-CH2-O- H2-C-N-), 70.25 (-C- H2-O-CH2-C-N-), 80.21 (- -(CH3)3), 126.40 (- H-),
147.20 (-C-CH-), 154.93 (-CO-) ppm. ESI-MS: m/z = 1056.5 [M + Na+].
H
H
δ
C
C
C
C
C
C
C
C
G1-NHBOC. A solution of CuSO4 5H2O (0.0081 g, 0.00003232 mol) in water (2 mL) was
·
added to a round bottom flask containing a stirred solution of 10 (0.0987 g, 0.0000567 mol)
and 20 (0.1655 g, 0.0006804 mol) in THF (2 mL). Sodium ascorbate (0.0135 g, 0.00006804
mol) was added and the mixture was allowed to react overnight. The product mixture
was evaporated and purified by column chromatography (acetone) to yield a sticky yellow
solid (0.095 g, 0.0000284 mol, 50% yield). 1H-NMR (400 MHz, CD3OD):
δ = 1.09 (s, 3H,
-C-C-CH3), 1.42 (s, 18H, -C-(CH3)3), 2.03 (m, 4H, -N-CH2-CH2-CH2-NH-), 2.68 (t, 4H,
-O-C-CH2-CH2-C-N-), 2.94 (t, 4H, -O-C-CH2-CH2-C-N-), 3.05 (t, 4H, -N-CH2-CH2-CH2-NH-),
3.42 (s, 2H, -C-CH2-O-), 4.09 (q, 4H, CH3-C-CH2-O-), 4.38 (t, 4H, -N-CH2-CH2-CH2-NH-),
4.51 (m, 4H, -CH2-O-CH2-C-CH-N-CH2-), 4.71 (t, 2H, -N-CH2-CH2-O-), 6.69 (t, 2H, -NH-),

Molecules 2017, 22, 868
14 of 18
7.77 (s, 2H, -C
H
-N-CH2-CH2-CH2-), 8.03 (s, 1H, -CH2-O-CH2-C-C
H
-N-) ppm. 13C{1H}-NMR (300 MHz,
CDCl3):
34.22 (-
49.90 (-N-
δ
= 18.08 (
H2-CH2-C-N-), 38.52 (-
H2-CH2-O-), 50.16 (-N-
H2-C-CH3), 70.18 (-C- H2-O-CH2-C-N), 80.12 (-
125.52 (-C-CH2-O-CH2-C- H-N-), 146.45 (-C-CH2-O-CH2-
158.45 (- O-O-C-(CH3)3), 173.43 (-O- O-CH2-), 173.67 (-O-
[M + Na+].
C
H3-C-CH2-), 21.84 (-CH2-
C
H2-C-N-), 28.87 (-C-(
C
H3)3), 31.67 (-
-CH2-O-CH2-C-N), 47.67 (-CH2-
H2-O-), 65.58 (-C-CH2-O- H2-C-N),
-(CH3)3), 123.73 (-C- H-N-CH2-CH2-CH2-),
-CH-N-), 147.43 (- -CH-N-CH2-CH2-CH2-),
O-C-) ppm. MALDI-MS: m/z = 3365.1
C
H2-CH2-NH-),
C
C
C
H2-NH-), 46.55 (-
C
C-CH3),
CH2-CH2-CH2-NH-), 64.45 (-N-CH2-
C
C
66.48 (-
C
C
C
C
C
C
C
C
C
C
G2 NHBOC. A solution of CuSO4 5H2O (0.0059 g, 0.00002349 mol) in water (1 mL) was added
-
·
to a round bottom flask containing a stirred solution of 13 (0.091 g, 0.00001958 mol) and 20
(0.0941 g, 0.0004698 mol) in THF (1 mL). Sodium ascorbate (0.0093 g, 0.00004698 mol) was
added and the mixture was allowed to react overnight. The product mixture was evaporated
and filtered in DCM to remove the salts. The product was precipitated out of DCM using
ether, isolated and dried to yield a viscous yellow oil (0.0429 g, 0.000005463 mol, 28% yield).
1H-NMR (400 MHz, CDCl3):
-N-CH2-CH2-CH2-NH-), 2.69 (t, 12H, -O-C-CH2-CH2-C-N-), 2.97 (t, 12H, -O-C-CH2-CH2-C-N-),
3.10 (m, 8H, -N-CH2-CH2-C 2-NH-), 3.46 (s, 2H, -C-C 2-O-CH2-), 4.10 (m, 12H, CH3-C-C 2-O-C-),
4.36 (t, 8H, -N-CH2-CH2-CH2-NH-), 4.48 (m, 8H, -CH2-CH2-C-N-N-N-CH2-CH2-O-), 4.58 (t, 4H,
-CH2-O-CH2-C-N-N-N-CH2-CH2-O-), 4.65 (s, 2H, -C-CH2-O-CH2-), 5.14 (s, 4H, -N -), 7.47 (s, 4H,
-C -N-CH2-CH2-CH2-NH-), 7.54 (s, 2H, -C-CH2-CH2-C-C -N-CH2-CH2-O-), 7.83 (s, 1H,
-O-CH2-C-C -N-) ppm. = 17.58 (-CH2-C- H3),
13C{1H} NMR (300 MHz, CDCl3):
20.76 (-CO-CH2- H2-C-CH-N-CH2-CH2-O-), 20.81 (- H2-C-CH-N-CH2-CH2-CH2-), 28.36 (-C-( H3)3),
30.62 (-N-CH2- H2-CH2-), 33.17 (-CO- H2-CH2-C-CH-N-CH2-CH2-O-), 33.28 (- H2-CH2-C-CH-
N-CH2-CH2-CH2-), 37.36 (-N-CH2-CH2- H2-), 45.18 (- -CH2-O-CH2-C-N-), 46.26 (-C-CH2-O-CH2-
C-CH-N- H2-CH2-), 46.30 (-CO-CH2-CH2-C-CH-N- H2-CH2-O-), 47.47 (-N- H2-CH2-CH2-),
48.68 (- -CH2-O-CO-CH2-CH2-C-CH-N-CH2-CH2-CH2-), 48.75 (- -CH2-O-CO-CH2-CH2-C-CH-N-
CH2-CH2-O-), 63.05 (-CO-CH2-CH2-C-CH-N-CH2- H2-O-), 63.11 (-C-CH2-O-CH2-C-CH-N-CH2- H2-),
64.75 (-C-CH2-O- H2-C-N-), 65.14 (-C- H2-O-CO-CH2-CH2-C-CH-N-CH2-CH2-CH2-), 69.05 (-C- H2-
O-CH2-C-N-), 70.50 (-C- H2-O-CO-CH2-CH2-C-CH-N-CH2-CH2-O-), 79.32 (- -(CH3)3),
121.67 (-C- H-N-CH2-CH2-CH2-), 122.10 (-CO-CH2-CH2-C- H-N-CH2-CH2-O-), 123.50 (-C-CH2-
O-CH2-C- H-N-), 146.01 (-CO-CH2-CH2- -CH-N-CH2-CH2-O-), 146.15 (- -CH-N-CH2-CH2-CH2-),
149.93 (-C-CH2-O-CH2- -CH-N-), 156.12 (- O-O-C-(CH3)3), 171.96 (- O-CH2-CH2-C-CH-N-CH2-
CH2-O-), 172.01 (- O-CH2-CH2-C-CH-N-CH2-CH2-CH2-), 172.12 (-CO-CH2-CH2-C-CH-N-CH2-CH2-
O- O-), 172.15 (-C-CH2-O-CH2-C-CH-N-CH2-CH2-O-
O-) ppm. MALDI-MS: m/z = 7881.9 [M + Na+].
δ = 1.11 (s, 9H, -O-C-C-CH3), 1.41 (s, 36H, -C-(CH3)3), 2.04 (q, 8H,
H
H
H
H
H
H
H
δ
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
+
G0-NH3 22). TFA (5 mL) was added dropwise to a solution of G0-NHBOC (0.9041 g, 0.000875 mol)
(
in DCM (5 mL). The reaction was left stirring for 5 min, the solvent was evaporated and
the product was dissolved in methanol. The methanol was evaporated, bringing the remaining
TFA with it. This methanol addition and evaporation cycle was then repeated several times
to ensure that all remaining TFA had been evaporated. The product was obtained as a sticky
1
orange solid (0.800 g, 0.000735 mol, 84% yield). H-NMR (400 MHz, CD3OD):
δ = 1.80 (s, 3H,
-NH3), 3.47 (s, 2H, -C-CH2-O-), 3.59 (t, 2H, -N-CH2-CH2-NH3), 4.53 (s, 2H, -C-CH2-O-CH2-),
4.76 (t, 2H, -N-CH2-CH2-NH3), 8.06 (s, 1H, -C-C -N-) ppm.
13C{1H}-NMR (300 MHz,
CD3OD): = 40.30 (-CH-N-CH2- H2-), 46.48 (- -CH2-O-CH2-C-N-), 48.54 (-CH-N- H2-CH2-),
65.46 (-C-CH2-O- H2-C-N-), 70.27 (-C- H2-O-CH2-C-N-), 119.20 (- F3), 126.40 (- H-), 147.22 (- -CH-),
H
δ
C
C
C
C
C
C
C
C
162.95 (-CO-) ppm. ESI-MS: m/z = 633.3 [M − 4TFA + H+].
G1-NH3
+
(23). TFA (0.5 mL) was added dropwise to a solution of G1-NHBOC (0.05 g, 0.000015 mol)
in DCM (0.5 mL). The reaction was left stirring for 5 min, the solvent was evaporated and the
product was dissolved in methanol. The methanol was then evaporated, bringing the remaining TFA
with it. This methanol addition and evaporation cycle was then repeated several times to ensure

Molecules 2017, 22, 868
15 of 18
that all remaining TFA had been evaporated. The product was obtained as a sticky yellow solid
(0.041 g, 0.000012 mol, 80% yield). 1H-NMR (400 MHz, CD3OD):
= 1.10 (s, 3H, -C-CH3), 2.27 (q, 4H,
δ
-N-CH2-CH2-CH2-NH3), 2.69 (t, 4H, -O-C-CH2-CH2-C-N-), 2.95 (t, 4H, -O-C-CH2-CH2-C-N-),
3.00 (t, 4H, -N-CH2-CH2-CH2-NH3), 3.42 (s, 2H, -C-CH2-O-CH2-), 4.08 (s, 4H, CH3-C-CH2-O-),
4.51 (t, 8H, -CH2-O-CH2-C-), 4.71 (t, 2H,-N-CH2-CH2-O-C-, -N-CH2-CH2-CH2-NH3), 7.80 (s, 2H,
-C
= 18.03 (
38.17 (- H2-NH3), 46.55 (-
50.20 (-N- H2-CH2-CH2-NH3-),
66.41 (- H2-C-CH3), 70.15 (-C- H2-O-CH2-C-N), 119.08 (-
125.64 (-C-CH2-O-CH2-C- H-N-), 146.39 (-C-CH2-O-CH2- -CH-N-), 147.69 (-
O-O-C-F3), 173.46 (-O- O-CH2-), 173.69 (-O- O-C-) ppm. MALDI-MS: m/z = 2563.6
H
-N-CH2-CH2-CH2-), 8.04 (s, 1H,-CH2-O-CH2-C-C
H3-C-CH2-), 21.74 (-CH2- H2-C-N-), 29.18 (-
-CH2-O-CH2-C-N-), 47.63 (-CH2-
64.44 (-N-CH2- H2-O-),
-F3), 123.91 (-C-
H
-N-) ppm. 13C{1H}-NMR (300 MHz, CD3OD):
H2-CH2-NH3), 34.14 (- H2-CH2-C-N-),
-CH3), 49.90 (-N- H2-CH2-O-),
65.48 (-C-CH2-O- H2-C-N-),
H-N-CH2-CH2-CH2-),
-CH-N-CH2-CH2-CH2-),
δ
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
162.57 (-
C
C
C
[M − 8TFA + Na+].
+
G2-NH3
(24). TFA (1 mL) was added dropwise to a solution of G2-NHBOC (0.0429 g, 0.000005463 mol)
in DCM (1 mL). The reaction was left stirring for 5 min, the solvent was evaporated and the
product was dissolved in methanol. The methanol was then evaporated, bringing the remaining
TFA with it. This methanol addition and evaporation cycle was repeated several times to
ensure that all remaining TFA had been evaporated. The product was obtained as a sticky
yellow solid (0.034 g, 0.00000421 mol, 77% yield). 1H-NMR (400 MHz, CD3OD):
δ = 1.10
(s, 9H, -O-C-C-CH3), 2.27 (q, 8H, -N-CH2-CH2-CH2-NH3), 2.68 (t, 12H, -O-C-CH2-CH2-C-N-),
2.97 (t, 12H, -O-C-CH2-CH2-C-N-), 3.01 (m, 8H, -N-CH2-CH2-CH2-NH3), 3.41 (s, 2H,
-C-CH2-O-CH2-), 4.08 (m, 12H, CH3-C-CH2-O-C-), 4.50 (m, 8H, -CH2-CH2-C-N-N-N-CH2-CH2-O-,
-N-CH2-CH2-CH2-NH3), 4.66 (t, 4H, -CH2-O-CH2-C-N-N-N-CH2-CH2-O-), 4.70 (s, 2H,
-C-CH2-O-CH2-), 4.88 (s, 12H, -NH3), 7.82 (s, 4H, -C
-C-CH2-CH2-C-C -N-CH2-CH2-O-), 8.07 (s, 1H, -O-CH2-C-C
(300 MHz, CD3OD): 16.55 (-CH2-CH2-CH2-N-N-N-C-CH2-CH2-CO-O-CH2-C-
16.63 (O-CH2-CH2-N-N-N-C-CH2-CH2-CO-O-CH2-C- H3), 20.30 (- H2-C-CH-N-CH2-CH2-CH2-),
20.35 (-CO-CH2- H2-C-CH-N-CH2-CH2-O-), 27.71 (-N-CH2- H2-CH2-), 32.70 (-CO- H2-CH2-C-N-),
36.72 (-N-CH2-CH2- H2-), 45.05 (- -CH2-O-CH2-C-N-), 46.17 (-CO-CH2-CH2-C-CH-N- H2-CH2-O-),
46.19 (-C-CH2-O-CH2-C-CH-N- H2-CH2-), 46.85 (-N- H2-CH2-CH2-), 48.73 (CH3- -CH2-O-CO-),
62.97 (-N-CH2- H2-O-), 64.05 (-C-CH2-O- H2-C-N-), 64.97 (-C- H2-O-CO-CH2-CH2-C-CH-N-CH2-
CH2-CH2-), 65.05 (-C- H2-O-CO-CH2-CH2-C-CH-N-CH2-CH2-O-), 68.75 (-C- H2-O-CH2-C-N-),
118.50 (- F3), 122.48 (-C- H-N-), 146.08 (- -CH-N-), 161.39 (- O-O-CF3), 171.98 (- O-O-CH2-
O-), 172.29 (-C-CH2-O-CH2-C-CH-N-CH2-CH2-
H
-N-CH2-CH2-CH2-NH-), 7.86 (s, 2H,
-N-)ppm. 13C{1H}-NMR
H3),
H
H
δ
=
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C-CH3), 172.24 (-CO-CH2-CH2-C-CH-N-CH2-CH2-O-
O-CO-) ppm.
C
3.3. Determination of MIC
Aqueous solutions with dendrimer concentrations ranging from 0.025–64 mg/L were prepared.
Initial testing was performed with these solutions, with subsequent tests carried out with a more
narrow range around the initially determined MIC, in order to increase accuracy. Mueller Hinton
Broth (MHB) (85 mL) was inoculated with one colony of Escherichia coli (E. coli) ATCC11229 and
incubated with shaking (150 rpm) at 37 ◦C, for 18–24 h. Fresh MHB (85 mL) was then inoculated with
overnight culture (300 µ
L) and incubated for 2 h, with shaking (150 rpm), at 37 ◦C. The OD of the
culture was then measured at 625 nm. The bacterial solution was subsequently diluted in water until
its OD lay in the range of 0.08–0.13, and then 100-fold in 15% MHB/85% H2O. This diluted solution
of bacteria (3 mL) was then added in a 1:1 ratio to the prepared dendrimer solutions, in duplicate.
The initial OD at 625 nm of each dendrimer treated bacteria sample was then measured. Subsequently,
the samples were incubated with shaking (150 rpm) at 37 ◦C, for 18–24 h, when the OD was again
measured. Bacterial controls ensured that each sample contained 5
Experiments were repeated to ensure reproducibility. The OD values of the bacterial solutions taken
×
105 colony forming units (CFU).

Molecules 2017, 22, 868
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18–24 h after dendrimer addition relative to the initial OD were plotted as a function of dendrimer
concentration. The MIC was evaluated as the concentration where the relative OD drops sharply
to zero.
3.4. Determination of MBC
The same dendrimer treated bacterial samples used to evaluate the MIC were also employed to
determine the MBC. After their OD had been measured at 625 nm, 18–24 h after exposure, the samples
were plated on agar. The MBC was determined as the lowest concentration showing no bacterial
growth after incubation for 18–24 h at 37 ◦C.
4. Conclusions
We have developed a synthetic methodology to hydroxide and acetylene terminated dendrimers
that can be easily functionalized with a variety of surface groups including cationic amine moieties for
application as bactericides. The introduction of tetravalent core molecule in the design of dendrimers
reduces the number of generations required to obtain the appropriate number of functional groups at
their periphery. It also reduces the number of steps required in their synthesis, and it may facilitate
their large scale preparation. Availability of terminal acetylene groups allows covalent linking of
any desired functional group using “click” chemistry. We have demonstrated the introduction of
cationic end groups, and have evaluated the potential of these dendrimers as bactericides. However,
the scope of functionalization is not limited, and these dendrimers offer an advantageous platform to
design nanocarriers for varied applications in a diverse range of areas. Our studies suggest that while
designing antimicrobial dendrimers, the dendrimer generation, the functional groups at the periphery
and their permeability (and thus hydrophilicty) plays an important role in enhancing their efficacy.
Acknowledgments: We would like to thank Natural Sciences and Engineering Research Council (NSERC,
Canada), Fonds de Recherche du Qu’ebec Nature et technologies (FRQNT, QC, Canada), and Center for
Self-assembled Chemical Structures (FQRNT, QC, Canada) for financial support. We would like to thank Cristina
Motillo and Tiffany Hua, for their help with bactericidal activity experiments.
Author Contributions: A. Kakkar and T. van de Ven conceived and designed the experiments, E. Ladd performed
majority of the experiments together with N. Li and A. Sheikhi. All the authors contributed to writing of
this manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
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