Synthesis of Amino Terminal Clicked Dendrimers. Approaches to the Application as a Biomarker

Article abstract of DOI:10.1021/acs.joc.9b01369

Herein, we present an easy and efficient synthesis of amino terminal dendrons, combining protection/deprotection reactions with copper-catalyzed azide alkyne cycloaddition in a convergent way. This new approach affords dendrons in gram scale with excellent yields and easy purification. By choosing the appropriate azido-functionalized core, these dendrons lead to a more efficient and controlled convergent synthesis of dendrimers with different sizes and shapes and multivalence. The amino terminal dendrimers were analyzed by diffusion-ordered spectroscopy experiments. The observed dendrimer size is in excellent correlation with the expected size and shape by molecular dynamic simulations. The construction of these kinds of nanostructures, in a simple and efficient way, opens new opportunities for biomedical applications. Moreover, by choosing the appropriate core, these versatile macromolecules become an excellent fluorescent biomarker.

Full text of DOI:10.1021/acs.joc.9b01369

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Article
Synthesis of Amino Terminal Clicked dendrimers.
Approaches to the application as a biomarker
Noemi Molina, Francisco Nájera Albendín, Juan A Guadix, Jose
M Perez-Pomares, Yolanda Vida, and Ezequiel Perez-Inestrosa
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b01369 • Publication Date (Web): 16 Jul 2019
Downloaded from pubs.acs.org on July 20, 2019
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The Journal of Organic Chemistry
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Synthesis of Amino Terminal Clicked dendrimers. Approaches to the
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Noemi Molina, Francisco Nájera,†,‡ Juan A. Guadix, Jose M. Perez-Pomares,,‡ Yolanda
Vida,*
†
,‡
,‡
,
†,‡
, †,‡
and Ezequiel Perez-Inestrosa*
†
Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Málaga - IBIMA, Campus de Teatinos
s/n, 29071 Málaga, Spain
‡
Centro Andaluz de Nanomedicina y Biotecnología (BIONAND), Junta de Andalucía, Universidad de Málaga, C/
Severo Ochoa 35, 29590 Campanillas (Málaga), Spain

Departamento de Biología Animal, Facultad de Ciencias, Universidad de Málaga - IBIMA, Campus de Teatinos s/n,
9071 Málaga, Spain
2
KEYWORDS. Dendrons, Dendrimers, Click reactions, DOSY experiments and Molecular Dynamic Simulations.
ABSTRACT: Herein we present an easy and efficient synthesis of amino terminal dendrons combining
protection/deprotection reactions with copper-catalyzed azide alkyne cycloaddition in a convergent way. This new
approach affords dendrons in gram scale with excellent yields and easy purification. By choosing the appropriate azido
functionalized core, those dendrons lead to more efficient and controlled convergent synthesis of dendrimers with different
size, shape and multivalence. The amino terminal dendrimers were analyzed by Diffusion-Ordered Spectroscopy
experiments. The observed dendrimer size is in excellent correlation with the expected size and shape by Molecular
Dynamic Simulations. The construction of these kinds of nanostructures in a simple and efficient way, opens new
opportunities for biomedical applications. Moreover, by choosing the appropriate core, this versatile macromolecules
becomes an excellent fluorescent biomarker.
INTRODUCTION
molecule, for example in emulating the carrier protein in
the hapten-carrier conjugate involved in allergic
reactions.
The design and synthesis of improved biocompatible
polymeric materials is an important topic nowadays due to
their widespread use in biomedicine. In particular,
dendrimers are excellent candidates for many biomedical
applications, especially where linear homologs are not
6–8
The most suitable and available amino terminal
9
dendrimers are Vögtle`s polypropylenimine (PPI),
10
Denkewalter`s
polyamidoamine (PAMAM),
poly(L-lysine)
(PLL),
Tomalia`s
1
11,12
effective.
Dendrimers
are
highly
branched
and more recently a
macromolecules with an extremely orderly structure. Their
synthetic procedures, based on a stepwise growth, afford
well defined and monodisperse compounds. The
structures of the core, branch multiplicities and branch
segment length have a significant effect on the overall
structure and properties of the dendrimer, defining the
number and density of functional groups at the periphery,
modification proposed by Malkoch from Hult´s polyester
(bis-MPA). While these dendrimers are characterized by
13
their symmetrical branching, PLL differs in their
1
4
asymmetry.
Nevertheless,
limitations
regarding
synthesis, quality or stability of those dendrimers have
been reported. The most extended used is PAMAN, the
first commercially-available even for high generations.
However, the stability and quality of PAMAM dendrimers
2–5
usually increasing exponentially with each generation.
1
5
These attributes, together with their synthetic versatility,
make dendrimers excellent scaffolds for many biomedical
have been previously questioned. Moreover, their
synthesis requires long reaction times and large excess of
monomers to reach full conversions. For instance, defects
are reported to be present in their structure due to retro-
1
applications.
For biomedical purposes, nearly all dendrimers are
modified by a conjugation of their terminal groups with at
least one type of ligand. Amino terminal dendrimers are an
excellent alternative, since this functional group reacts
easily with a great number of bioactive molecules. Another
important key for some bioapplications is stability,
especially where the dendrimer plays the role of a carrier
1
Michael additions and intramolecular lactam formation.
Similarly, PPI dendrimer growth can be limited by retro-
Michael reactions or intramolecular amine cyclization. It
has been reported that for the fifth generation, only 29% of
1
the dendrimer will be defect-free. Another aspect to take
into account for certain applications is the high basicity of
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PAMAM and PPI. The conformation of both dendrimers is amide formation. All reactions with already well stablished
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strongly affected by low pH due to electrostatic repulsion
of the protonated internal tertiary amines. On the other
protocols, excellent yields (above 85%), easy purification
procedures (no chromatography is needed), cheap
reactants and carried out on a gram scale. The coupling of
those alkyne-bearing dendrons to azide cores using click
chemistry gives as a results different dendrimers in one
step. The goal of this synthetic strategy relays in the
versatility of the methodology. With a third generation
dendron it is possible the construction of dendrimers with
different size, shape or amount of amino terminal groups
by choosing the appropriate core. By changing the
multiplicity and functionality of the core, we can design
and prepare stable amino terminal dendrimers with a
specific ability, size or shape for a particular application.
The easy and efficient synthesis of the dendrimer was
carried out in one step, using click chemistry to couple
alkyne-bearing dendrons to azide cores. The dendrimers
were completely characterized and Diffusion-Ordered
Spectroscopy (DOSY) experiments were used to determine
the sizes and shape of the dendrimers. Results are in good
agreement with calculated values. As a proof of concept, a
fluorescent core was chosen to prepare a luminescent
dendrimer whose abilities as biosensor has been proved,
labeling bacteria cells with a characteristic fluorescence
which can be used for two-photon excitation microscopy.
1
hand, polyester (bis-MPA) dendrimers can be synthetized
and purified more efficiently and also post-functionalized
1
3
to display terminal amino groups up to generation five.
However, the lack of stability due to the reported
hydrolysis of the ester bond may be a problem for certain
applications in which the steadiness of the dendrimer is
1
3
required.
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We previously reported a new class of amino terminal
dendrimer constructed through amide linkages. Using a
divergent approach, this novel polyamide dendrimer
(BAPAD) was synthetized based on a 3,3´-diaminopivalic
1
6
acid scaffold. Their excellent stability allowed the use of
such dendrimers for different bioapplications.17,18 However,
despite the versatility of the system, the maximum
generation of the amino terminal dendrimer obtained was
generation 3, which contains 16 peripheral amino
1
6,19
groups.
This could be explained by a dramatic decrease
in the reactivity of the azide groups caused by a
backfolding of these surface groups. This effect has been
previously observed and is more pronounced when the
synthesis of the dendrimer is performed in a divergent
1
9
way. All mentioned dendrimers were synthetized
following a divergent approach, and chromatographic
procedures were required in all cases. Although some of
the purification procedures are well established, they are
tedious and time consuming.
RESULTS AND DISCUSSION
Motivated by the efficiency and versatility of the copper-
mediated click transformation, we designed a divergent
synthetic route for new dendrons based on 3,3´-
diazidopivalic acid as building block. The azide groups
permit the growth of the dendrimers by CuAAC reactions.
The final step in the dendrimer synthesis consists of the
coupling between an alkyne functionalized dendron and
an azido-bearing central core.
The convergent approach for dendrimer synthesis was
introduced by Hawker and Fréchet.20 This methodology
proceeds from the surface of the dendrimer inward to form
a dendron that reacts with a suitable core to complete the
synthesis. The advantage of the convergent approach is
that only a limited number of active sites are present in
each reaction, reducing the structural defects of the
product. However, this methodology is used to form
relatively lower generation dendrimers because steric
hindrance can make the coupling of large dendrons with
Synthesis of Dendrons
The propargyl-functionalized dendrons (dGn) were
synthetized following the procedure shown in Scheme 1.
3,3´-diazidopivalic acid was prepared under previously
1
6
described procedures. The protection of the carboxylic
acid function as benzylic ester gives as result the monomer
used in the synthesis.
21
small cores difficult. To increase coupling yields for the
convergent approach, the 1,3-dipolar cycloaddition
22
reactions results an excellent candidate. The copper-
To obtain the first generation dendron (dG1, Scheme 1),
azide groups were reduced. According to a convergent
approach, these resulting groups will be placed on the
surface of the final dendrimer. After protection of those
amino groups and deprotection of the carboxylic acid, the
compound was reacted with propargyl amine to yield dG1
in excellent yields. From this point, the growth of dendrons
involves three iterative steps: (i) “click” reaction between
dGn and the monomer (ii) hydrogenation reaction for
carboxylic acid deprotection and (iii) amide formation via
propargyl amine reaction.
catalyzed azide-alkyne “click” cycloaddition (CuAAC)
requires mild reaction conditions and simple work-up
procedures, tolerates a wide range of functional groups,
including aqueous media, and results in 1,4-regiospecific
23
1
,2,3-triazole formation in high yields. All these features
made Click chemistry an excellent tool in the construction
of a large variety of dendritic structures with high level of
efficiency, and have been used to improve traditionally
24–27
synthetic processes.
Herein we present a simple, convenient and efficient
method for the synthesis of stable and water soluble amino
terminal dendrimer taking advantage of Click reactions.
Different generation dendrons have been prepared
combining protection/deprotection reactions and copper-
catalyzed azide alkyne cycloaddition, in a convergent way.
To increase dendron generation, 3 synthetic steps are
involved: click reaction, deprotection (hydrogenation) and
For the first step, a general procedure for the reaction
between azides and alkynes was used. A mixture of azide,
alkyne, CuSO
alcohol (2:1) was stirred at room temperature. The crude
was recovered in CH Cl and washed with an
ammonia/brine mixture to eliminate the copper excess.
4
and sodium ascorbate in water/tert-butyl
2
2
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dendrons. These reactions were monitorized by IR and
The final products were precipitated with hexane to yield
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pure dendrons with more than 90% yields. A slight excess
of the alkyne derivative with respect to the azide was used
in order to ensure the reaction of all branches of the
NMR spectroscopies.
Scheme 1. Synthesis of dG1-dG3 dendrons
N3
N3
NHBoc NHBoc
NHBoc NHBoc
i
ii
iii
iv
CO2Bn
dG1
NH
O
CO2Bn
BocHNBocHN
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0
BocHNBocHN
BocHN
BocHN
BocHN
BocHN
O
NH
N3
N3
N3
N3
N
HN
O
O
O
N
BocHN
HN
HN
BocHN
BocHN
N
N
BocHN
CO2Bn
H2/cat.
CO2Bn
N
N
dG1
N
N
N
H2/cat.
N
N
O
N
H
NH2
NH2
N
N
NH
NH
N
O
N
dG2
O
N
N
N
N
NH
NH
N
N
O
O
N
dG3
N
NH
N
O
3
Reagents and conditions: i) 1) PPh , THF, 2) H
2
O, 98%; ii) (Boc)
2
O, acetone:H
2
O, 96%; iii) H
2
, Pd(OH)
2
, MeOH, quantitative;
iv) Propargylamine, CDI, CH CN, 83%.
3
1
The complete reaction of the azide moieties is confirmed
by the IR spectra. Figure 1 (insets) shows the IR spectra of
the resulting compounds of click reactions between
benzyl-3,3´-diazidopivaloate with dG1 (namely dG2-
Figure 1). Additionally, in both cases the H-NMR spectra
indicate the complete reaction of the alkyne moiety, since
the peak around 3.8 ppm, corresponding to the adjacent
methylene (b for dG1, Figure 1a), are shifted to lower field
(around 4.27 ppm, b and b´, Figure 1b and 1c). The
hydrogenation reaction for carboxylic acid deprotection
resulted quantitative for all cases. Finally, products
obtained after reaction with propargyl amine were
precipitated to yield pure dendrons with excellent yields.
It should be noted that no chromatography is need for
purification.
2 2
CO Bn) and dG2 (namely dG3-CO Bn). The
disappearance of the intense azide associated signal at
-1
2
085 cm indicates in both cases the complete reaction of
azide groups. The formation of the 1,2,3-triazole ring in
1
clicked compounds was confirmed by their H-NMR
spectra, appearing new peaks between 7.7-8.0 ppm where
triazole proton resonance peaks are observed (d and d´ in
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O
a)
b)
a
b
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N
H
BocHN
c
b
a, c
BocHN
0
.9
O
1.7
2.0
4.3
O
a
b
d
e
N
H
OBn
N
N
N
BocHN
.
BocHN
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d
.
b
a
e
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.7
1.9
4.8
3.3
2.0
2.0 2.0
4.0
8.1
O
c)
O
O
e´
d´
a
b
d
b´
OBn
e
N
N
N
N
N
N
H
H
N
N
BocHN
BocHN
a
b, b´
.
d, d´
e, e´
.
1
.3
3.8
5.9
4.3
7.4
2.0
13.0
12.9
17.1
8
.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
ppm
Figure 1. NMR spectra in DMSO-d
diazidopivaloate (blue) and dG2-CO Bn (black) and c) IR spectra of benzyl-3,3´-diazidopivaloate (blue) and dG3-CO
6 2 2
of a) dG1, b) dG2-CO Bn and c) dG3-CO Bn. Inset: b) IR spectra of benzyl-3,3´-
2
2
Bn (black)
Scheme 2. Synthesis of GnEDANH
2
dendrimers
H2N
O
H2N
NH2
NH2
N
N
N
N3
N3
O
N
H
N
dG1
O
HN
G1EDANH2
Deprotection
N
N
N
H
H2N
H2N
N
N
N
N
N
O
N
N3
N3
H
N
N
N
N
N
N
O
H
dG2-NHBoc
G2EDANH2
NH
Deprotection
O
O
N
N
N
NH
N
N
N
NH2
NH2
H2N
H2N
O
N
N
N
N3
HN
N3
N
O
HN
H
O
N
N
N
dG3-NHBoc
N
N
Deprotection
N
N
H
N
NH2
NH2
N
O
N
N
N
N
N
H
N
N
N
O
NH
H2N
H2N
N
H
O
N
N
G3EDANH2
NH2
NH2
O
Reagents and conditions: CuSO
for amino deprotection.
4
, sodium ascorbate, water/tert-butyl alcohol (2:1) for click coupling; HCl 4M in dioxane, THF
Synthesis of Dendrimers
evaluated using 1,2-diazidoethane as core. This compound
was prepared according to
procedure, and coupled with generation 1 (dG1), 2 (dG2)
and 3 (dG3) dendrons (Scheme 2).
a previously reported
For an effective connection between the propargyl focal
point of dendrons and azide-functionalized cores, alkane,
aromatic and fluorescent moieties were designed in order
to achieve dendrimers with different functionalities, size
and number of terminal amino groups. In this sense 1,2-
diazidoethane, 1,3,5-tris(azidomethyl)benzene and N-(3-
azidopropyl)-4-((3-azidopropyl)amino)-1,8-naphthalimide
were prepared. The efficiency of this methodology for the
construction of different generation dendrimers were
28
Click reactions afford the corresponding dendrimers in
excellent yields (92%, 80% and 93% for generations 1, 2 and
3). The deprotection reactions of the terminal amino
groups were carried out under acidic conditions, yielding
G1EDANH
2
, G2EDANH
2
and G3EDANH2, respectively in an
almost quantitative way for all cases (Scheme 2). The
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known.29 Tri-azidomethyl benzene was synthetized
complete click reactions of alkyne dendrons with 1,2-
1
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1
1
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30
diazidoethane to yield G1EDANHBoc, G2EDANHBoc and
G3EDANHBoc were confirmed by the IR spectra (inset,
Figure 2).
following previously described procedures.
N-(3-
azidopropyl)-4-((3-azidopropyl)amino)-1,8-naphthalimide
was prepared by coupling 3-azidopropylamine (obtained
from 3-Bromopropylamine) with 4-bromo-1,8-naphthalic
anhydride in DMSO, heating at 80ºC overnight.
The formation of two news 1,2,3-triazole rings in
1
dendrimers was confirmed by H-NMR (Figure 2) by the
Click reactions with dG3 were carried out under the
conditions described earlier, and the reactions were
monitored in a similar way as for the previous dendrimers,
in terms of the disappearance of the azide signal in IR
(ensuring reaction of all branches of the core, Figures S78
and S79, ESI) and the generation of new signals in H-NMR
(Figures S52 and S62, ESI). Both dendrimers were obtained
with good yields and deprotection reactions of the
appearance of triazole proton, d´´ and the adjacent
methylene, f in Figure 2. Additionally, the peaks around 3.8
ppm in dG1, dG2 and dG3 spectra corresponding to the
adjacent methylenes of the alkyne moiety (Figures S11, S20
and S29, ESI, respectively), are shifted to lower field
0
1
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3
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9
0
1
(
around 4.27 ppm, b, b´ and b´´ in Figure 2) in dendrimers.
In a similar way, dG3 was coupled to the other azido-cores
2 2
terminal amino groups yielded G33ABNH and G3NaphNH
in an almost quantitative way (Scheme 3).
yielding after amino deprotection G33ABNH and
(Scheme 3). Tri-substituted benzene was
2
G3NaphNH
2
chosen to introduce a rigid core in the molecule. A
fluorescent naphthalimide moiety was selected in order to
introduce a functional core in the molecule, since the
potential applications of luminescent dendrimer are well
O
a)
b)
b
a
d´´
N
f
N
N
H
BocHN
N
f
BocHN
2
d´´
b
a
1
.6
1.7
3.4
4.1
4.1
8.0
37.4
6.1
O
O
b´
d´´
a
b
d
f
e
N
N
N
N
N
N
N
H
H
N
BocHN
BocHN
2
b, b´
a
d, d´´
f
e
1
.6
3.6 6.4
6.3
4.8 3.8 4.2 12.9
16.0
76.1
18.0
c)
d´´
N
O
b´´
f
O
O
e´
b´
d´
N
a
b
d
e
N
N
H
N
N
N
N
H
H
N
N
BocHN
BocHN
N
N
2
a
b, b´, b´´
e, e´
d, d´, d´´
f
3
.4
7.8
14.7
12.2
6.5
3.6 23.4 27.4
5.0 4.5
ppm
32.0
159.3 39.9
1.5 1.0
9
.0
8.5
8.0
7.5
7.0
6.0
5.5
4.0
3.5
3.0
2.5
2.0
6
Figure 2. NMR spectra in DMSO-d of a) G1EDANHBoc, b) G2EDANHBoc and c) G3EDANHBoc. Inset: IR spectra of 1,2-
diazidoethane (red) and a) G1EDANHBoc (black), b) G2EDANHBoc and c) G3EDANHBoc (black)
All amino terminal dendrimers were purified by size
exclusion chromatography, and their structures confirmed
by NMR (Figures S36-S38, S42-S44, S48-S50, S55-57 and
S65-S67, ESI). As previously reported, characterization of
dendrimers with high molecular weights and charges by
X
protecting groups (in Gn NHBoc derivatives) and the high
degree of positive charges (in Gn NH derivatives) during
X 2
measurements.
31,32
MALDI-TOF is extremely difficult.
No mass spectrum
could be obtained for described dendrimers, probably as a
result of the lability of the tert-butyloxycarbonyl (BOC)
Scheme 3. Synthesis of G33ABNH and G3NaphNH dendrimers
2 2
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NH2
NH2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
NH
NH2
O
NH2
NH2
NH2
N
N
N
HN
H2N
H N
N
N
H2N
H N
2
N
2
N3
O
2
O
H2N
HN
NH
NH
O
NH
H2N
O
HN
O
N
O
N
N
N
N
O
N
N
N3
N
N
N
N
N
N
N
N
N
NH2
HN
H2N
O
N
N
O
HN
G3NaphNH2
H
R
N3
2
O
N
N
H
H2N
N
N
NH2
N
NH
O
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N3
N
N3
N
N
NH
N
N
O
dG3
N
N
N
N
N
NH
Deprotection
Deprotection
N
N
N
O
N
NH
N
N
N
N
N
O
2
O
N
O
G33ABNH2
N
R
HN
2
N
N
N
R
Reagents and conditions: CuSO
4
, sodium ascorbate, water/tert-butyl alcohol (2:1) for click coupling; HCl 4M in dioxane, THF
for amino deprotection.
As an alternative, in order to characterize the molecular
weight and ensure the homogeneity of each generation of
dendrimers, we used polyacrylamide gel electrophoresis
PAGE). Due to the structural similarity between
dendrimers and basic proteins, the former can migrate on
electrophoresis gels and be stained by reagents commonly
used in PAGE.
Figure 3 shows the electropherogram of the amino
terminal dendrimers G2EDANH and
, obtained on a 20% polyacrylamide gel.
has not been included in the analysis since its
molecular weight is relatively small for the standards used.
It can be clearly seen in Figure 3 that the bands of all
dendrimers (lanes 1, 2, 3 and 4) appear in the gel according
to their molecular weight (Table 1) and in all cases
resembles the homogeneity of the sample.
Lane 5: Polypeptide SDS-PAGE Standards (BIO-RAD). The
injected sample solution was 20 µg for each dendrimer. All
dendrimers and Polypeptide SDS-PAGE Standards were
stained uniformly with Coomassie Blue G-250 stain
(
33,34
Amino terminal dendrimers were examined by diffusion
NMR techniques. DOSY (diffusion-ordered spectroscopy)
experiments were carried out observing that the decays of
all signals are monoexponential, resulting in a linear
Stejskal-Tanner plot, which proves that the dendrimers
were monodisperse (Figures S68-S72, ESI). Diffusion
coefficients (D) were also determined and used to estimate
the size of all dendrimers in solution, by calculating the
2
2 2
, G3EDANH , G33ABNH
G3NaphNH
G1EDANH
2
2
3
5
h
hydrodynamic radius (R ) using the Stokes-Einstein
equation (Table 1).
3
6,37
Larger structures diffuse more slowly, showing smaller
diffusion constants. As expected, the dendrimer radius
increases with generation in dendrimers with the same
core (GnEDANH
comparing generation 3 dendrimers, probably due to a
folding of the structure. G3NaphNH shows a slightly higher
diffusion constant than G3EDANH , which can be translated
into a smaller size of the dendrimer. However, the
inclusion in the structure of a third dendron, which implies
a considerable increase in molecular weight, does not
translate into a significant increase in the size of the
2
). This correlation is not so clear when
2
2
dendrimer (Table 1). It is important to note that G3-NH
2
dendrimers are built from different cores, whose structures
and multiplicities influence not only the number of amino
terminal groups but also the shape, morphology and size
of the final dendrimers.
Figure 3. Electrophoresis of amine-terminated NH
dendrimers. Electrophoresis was performed on 20%
polyacrylamide gel for 160 min at 125 V. Lane 1: G2EDANH
Lane 2: G3EDANH ; Lane 3: G33ABNH ; Lane 4: G3NaphNH
2
Molecular Dynamic Simulations
To obtain some information about the structure of these
dendrimers, molecular models were created and simulated
2
;
;
2
2
2
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in water as explicit solvent using molecular dynamics
analyzed and several properties calculated (Table 1), such
as the Radius of gyration (R ), the aspect ratio and their
asphericities.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
Figure 4). These compounds were built with three
g
different residues: the respective core for each dendrimer
(COR), the branched repeating fragment (REP), and the
terminal ends (TAM), respectively (Figure S82, ESI). The
equilibrated structures of these molecules have also been
Table 1. Data of prepared dendrimers. Diffusion coefficients (D) and hydrodynamic radius (R
h
) determined by NMR
experiments. Radius of gyration (R ), aspect ratios (I /I and I /I ) and asphericities (δ) calculated by MDS
g
z
x
z
y
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Dendrimer
G1EDANH
2
G2EDANH
2
G3EDANH
2
G33ABNH
2
G3NaphNH
2
Molecular Formula
C
18
H
34
N
4
12
O
2
C
50
H
86
N
8
32
O
6
C
114
H
190
N
72
O
14
C
177
H
288
N
108
O
21
128 200 74 16
C H N O
Terminal -NH
2
groups
16
2793.26
1.93·10
24
4264.99
1.72·10
11.65
16
3031.51
-1
M. W. (gmol )
450.55
3.80·10
5.27
1231.46
3.01·10
6.66
2
−1
-10
-10
-10
-10
-10
D (m s )
2.19·10
9.15
R
R
I
h
(Å)
(Å)
10.38
g
4.17 ± 0.06
1.22 ± 0.07
1.62 ± 0.10
6.79 ± 0.09
1.20 ± 0.03
2.71 ± 0.16
10.51 ± 0.15
1.08 ± 0.03
3.79 ± 0.23
11.37 ± 0.16
1.27 ± 0.02
2.35 ± 0.12
9.93 ± 0.14
1.08 ± 0.03
2.85 ± 0.19
x
/I
/I
y
I
x
z

0.019 ± 0.004
0.066 ± 0.006
0.103 ± 0.006
0.052 ± 0.005
0.073 ± 0.007
We found that the size of these molecules for the
series increases with each generation, as
shown in Figure S85, ESI. The maximum density is found
to be close to the core of the dendrimers, decaying toward
the edge. Second and third generation GnEDANH
2
GnEDANH
2
quantified from the DOSY experiments, and their values
are in good agreement with the calculated radius of
dendrimers have a plateau corresponding with the
distribution of the repetitive unit (REP) and decaying
slowly toward the end of the molecule. This shows a region
with low atom mobility, high localization and therefore
with a dense dendrimer shell pattern. The number of
terminal monomers (TAM) doubles with each generation,
so the terminal amine groups extend over the molecule,
always with increasing density toward the outer region of
the dendrimer, but with a higher degree of terminal
monomer back-folding when the generation increases
gyration (R
for these compounds can be inferred from the relation
between R and the number of the dendrimer’s atoms (N)
g f
) (Table 1). The fractal dimensionality (d ) value
g
and has a value of 1.74 (Figure S83, ESI). This indicates that
these dendrimer generations do not form perfect spheres
since the fractal dimension for a perfectly spherical smooth
5
surface is 3.
The values of the three principal moments of inertia (I
in decreasing order) can give information about the
structural characteristics of these dendrimers. The ratios
/I ) and (I /I ) are measures of the dendrimer’s ellipsoid
shape eccentricity. GnEDANH dendrimers showed I /I and
ratios between 1.22-1.08 and 1.62-3.79 respectively
Table 1). The increase in the gap between the I /I and I /I
x y
, I ,
I
z
(
Figure S85, ESI).
(
I
x
y
x
z
To get more insight into how different cores affect the
properties of these dendrimers, we have analyzed the
calculated parameters for the G3-NH generations. The R
2 g
of these dendrimers are similar and their values are within
1 Å and reproduce the experimental values (Table 1). The
x y x z
ellipsoid shape eccentricity displays I /I and I /I ratios
2
x
y
I
x
/I
z
(
x
y
x
z
values implies that the ellipsoidal shape is favored when
the generation increases. This is corroborated by their
asphericity values (Figure S84, ESI).
between 1.08, 1.08, 1.27 and 3.79, 2.85, 2.35 for the EDA,
Napth and 3AB cores, respectively (Table 1).
The atoms distribution within the dendrimers can be
described using radial density profiles. Those
corresponding to GnEDANH dendrimer generations are
2
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2 2 2 2 2
Figure 4. Snapshots from Molecular Dynamic Simulations of G1EDANH , G2EDANH , G3EDANH , G33ABNH and G3NaphNH ; (to
simplify the picture, carbon atoms are depicted in cyan, oxygen atoms in red, nitrogen atoms in blue and hydrogens atoms are
omitted)
The gap decrease between the I
x
/I
y
and I
x
/I
z
values implies
growths, an ellipsoidal shape is favored when the
that a more globular structure is favored when the number
of atoms in the dendrimers increases, this is validated by
the asphericity values (Table 1, TableS1 and Figure S86,
ESI).
generation of GnEDANH2 increases.
Luminescent properties of G3NaphNH
2
G3NaphNH
2
exhibits an intense fluorescence emission
-5
centered at 550 nm in aqueous solution (10 M, Figure 5a)
with an excited state lifetime of 7.5 ns (em = 550 nm).
Although it has a quantum yield of 26 %, this value is
unexpectedly considering an aqueous solution. Excitation
and emission spectra acquired under two-photon
excitation (TPE) conditions were also recorded (Figure 5a).
The emission observed using an excitation wavelength of
2
The radial density profiles for the G3-NH dendrimers are
shown in Figure S87 (ESI). In these profiles, density is
maxima around the core of the dendrimers. For EDA core-
dendrimers, the density shows a plateau with a good
correlation with the distribution of the repetitive unit REP
and implies a dense dendrimer pattern. For 3AB and Napth
core-dendrimers this plateau is smaller. The terminal
amine groups are extended from the middle of the
molecules toward the outside region of the dendrimer,
except for the molecule with the 3AB core, which presents
a high degree of back-folding and the TAM residue can be
found along all the molecule (Figure S87, ESI). From all
these dendrimers, the molecule with the Napth core has
the least back-folding (Figure 4).
8
80 nm coincides with the one obtained in the one-photon
excitation (OPE) regime. When comparing these results
with a model chromophore, similar results are observed.
Emission and excitation spectra of N-(2-aminoethyl)-4-((2-
aminoethyl)amino)-1,8-naphthalimide resembles the ones
of the dendrimer Figure S80 (ESI), but possess a lower
2
quantum yield (15%). G3NaphNH becomes therefore an
excellent tool for bioapplications where luminescence is
required. The molecule combines the excellent properties
of amino terminal dendrimers with a chromophore widely
Comparing the family GnEDANH2 with others amino-
5
38
16
terminal dendrimers (PAMAM, PPI and BAPAD ), the
dendrimers of equivalent generation are similar in size, but
differs in shape. While for PAMAM, PPI and BAPAD
dendrimers are spheroids in shape when the generation
3
9
supported by its excellent photophysical properties.
To evaluate the use of G3NaphNH as a multichannel
fluorescent marker for biological samples, a strain of E. coli
2
bacteria was employed as a model target. The amino
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terminal groups of G3NaphNH
to bacterial surface, owing to their capacity for hydrogen
2
provide an effective binder
the dendrimer to the bacterial wall. To further confirm that
the origin of this fluorescence derives from the intrinsic
emission of the naphthalimide core of the dendrimer, and
to rule out biological sample autofluorescence and
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
40
bonds and electrostatic interaction. After incubation
with a solution of the dendrimer, G3NaphNH effectively
labeled the E. coli cells as can be seen in Figure 5 (10 M)
2
-4
41
background noise, specific controls were set up. In
-4
and Figure S81 (ESI) [5·10 M (500 μM)]. The outer surface
of some bacteria shows an intense fluorescence under both
OPE or TPE conditions, thus confirming the adhesion of
untreated bacteria no autofluorescence is observed under
OPE or TPE conditions (the capture settings and image
processing are identical).
a)
c )
d )
e )
1
1
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
c )
d )
e )
2
2
2
b)
Figure 5. a) Excitation (dotted line) and emission (solid line) spectra of G3NaphNH
2
upon one-photon (black) or two-photon (red)
excitation (450 nm and 880 nm respectively), in aqueous solutions. b) Growth curves for E. coli (bacteria were cultured in LB
medium in the presence of different concentrations of G3NaphNH (10 µM, blue; 100 µM, pink) or without G3NaphNH (red).
Confocal micrographs of E. coli incubated for 8 h with (upper row; c , d and e ) or without (bottom row; c , d and e
, c ) recorded emission with one photon excitation (exc = 450 nm; collected through 500-600 nm); d , d ) bright field images; e
) recorded emission with two photon excitation (exc = 880 nm; collected through 500-550 nm). Scale Bars: 5 m
2
2
1
1
2
2
2
2
) G3NaphNH
2
:
,
c
1
2
1
2
1
e
2
The dynamics of bacterial growth was monitored in liquid
LB medium originally inoculated with 4
the shape and morphology of the final dendrimers,
obtaining ellipsoidal shape from dendrimers with an alkyl
core and a more globular shape in dendrimers with
aromatic cores. We demonstrate that our effective
dendron can be combined with suitable cores of different
multiplicity, thus providing a powerful tool for the easy
assembly of amino terminal dendrimers, with the desired
molecular weight, shape and number of amino terminal
groups. The chemical stability of these aliphatic-
imidazole-amide dendrimers make them excellent
candidates for biomedical applications. Moreover,
inherent fluorescent dendrimer can be obtained in good
yields, completely aqueous soluble and with the amino
terminal groups intact. We also demonstrate the
application in bioimaging of such dendrimers using both
OPE and TPE conditions.
6
x
10 E. coli and
incubated in the presence (10 or 100 μM) or absence of
. No significant E. coli growth delay was
G3NaphNH
2
recorded with increasing G3NaphNH
to 100 μM (Figure 5b).
2
concentration from 10
CONCLUSIONS
A new family of stable amino terminal dendrimers has
been obtained combining protection/deprotection
reactions and efficient CuAAC. In our convergent design,
dG3 dendron was prepared alternating click reactions,
deprotections reactions, and amide formation, resulting in
an effective synthesis where dG3 can be obtained in large
quantities with yields higher than 85% in all steps and with
easy purification procedures. Different dendrimers have
been obtained in one step from our highly versatile
dendron. With this new methodology, it is possible to
increase the number of amino terminal groups by choosing
the appropriate multiplicity of the core. The size and shape
of obtained dendrimers have been evaluated by NMR
techniques. The data observed by DOSY experiments is
well supported by fully atomistic Molecular Dynamic
Simulations. The chemical structure of the core influences
EXPERIMENTAL SECTION
All reactions were performed using commercially available
reagents and solvents from the manufacturer without
further purification. Chemicals were purchased from
Sigma-Aldrich (L (+)-Ascorbic acid sodium salt, 3,3’-
dichloropivalic acid, benzyl bromide, Pearlman’s catalyst,
1,1′-carbonyldiimidazole,
propargylamine,
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tris(bromomethyl)benzene,
anhydride, 1,2-dibromoethane,
4-bromo-1,8-naphthalic
3-bromopropylamine
Synthesis of 3,3´-diazidopivalic acid. Sodium azide (7.50
g, 115 mmol, 4 eq) was added to a solution of 3,3’-
dichloropivalic acid(5.00 g, 29 mmol, 1 eq) in DMF/H O 9:1
(20 mL). The resulting solution was heated in a heat block
at 80°C overnight. The solvent was removed under vacuum
and ethyl acetate (50 mL) was added to promote the
precipitation of the remaining sodium azide. The solution
was left in the refrigerator overnight. Then the mixture was
filtered and the solvent was removed under vacuum to
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
hydrobromide), VWR Chemicals (sodium azide) or Alfa
Aesar (HCl 4M in dioxane, triphenylphosphine, di-tert-
butyl decarbonate) and used without further purification,
unless otherwise indicated. Solvents were purchased from
VWR Chemicals and Panreac. H2O was purified with a
Mili-Q purification system from Millipore. Unless
otherwise stated, all reactions were performed in air.
Column chromatography and TLC were performed on
silica gel 60 (0.040-0.063 mm) using UV light and/or stains
2
obtain the product (4.80 g, 26.1 mmol, 90%) as a colorless
1
oil. H-NMR (400 MHz, CDCl
3
) δ ppm: 3.63 (d, J = 12.3 Hz,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
TM
13
1
to visualize the products. Sephadex G-10 pre-packed
2 H), 3.52 (d, J = 12.3 Hz, 2 H), 1.27 (s, 3 H). C{ H} NMR (100
1
columns were used to purify the final dendrimers. H and
MHz, CDCl3) δ: 179.8, 54.6, 47.6, 19.4. HRMS calcd. for
1
3
–
C NMR spectra were measured in the indicated
C
5
H
7
N
6
O
2
– 183.0625 [M – H] , found 183.0626.
deuterated solvent at 25 °C on a Bruker Ascend 400 MHz
spectrometer. Proton chemical shifts (δ) are reported with
the solvent resonance employed as the internal standard
Synthesis of benzyl-3,3´-diazidopivaloate. To a solution
of 3,3´-diazidopivalic acid (5.00 g, 27 mmol, 1 eq) in DMF
(20 mL) was added sodium carbonate (4.29 g, 40.5 mmol,
(
CDCl
3 6 2 4
δ 7.26, DMSO-d δ 2.50, D O δ 4.79, MeOD-d δ
1
.5 eq) and benzyl bromide (4.8 mL, 40.5 mmol, 1.5 eq). The
3
.31). Data are reported as follows: chemical shift,
mixture was stirred for overnight at room temperature.
Then, n-hexane (100 mL) and water (50 mL) were added
and the phases were separated. The organic layer was
washed with water (3 × 50mL), dried over MgSO anh. and
4
the solvent was removed under vacuum. Purification was
performed by silica gel column chromatography (n-
multiplicity, coupling constants (Hz) and integration.
Carbon chemical shifts are reported in ppm with the
solvent resonance as the internal standard (CDCl
DMSO-d δ 39.52, MeOD-d δ 49.00). The HRMS
Electrospray Ionization Time of Flight, ESI-TOF) mass
3
δ 77.16,
6
4
(
spectra (MS) were performed on a High Resolution Mass
Spectrometer Orbitrap, Q-Exactive (Thermo Fisher
Scientific, Waltham, MA, USA), in either positive or
negative ion mode. Infrared (IR) spectra were recorded
using a Jasco FT/IR-4100 spectrophotometer at ambient
temperature. Hydrogenation reactions were carried out
under hydrogen atmosphere (50 bar) using a Mini-Reactor
hexane/ethyl acetate, 9:1 v/v) to obtain the product (6.67 g,
1
2
4.3 mmol, 90%) as a colorless oil. H NMR (400 MHz,
CDCl
3
) δ: 7.42 – 7.32 (m, 5 H), 5.19 (s, 2 H), 3.63 (d, J = 12.2
13
1
Hz, 2 H), 3.52 (d, J = 12.2 Hz, 2 H) 1.24 (s, 3 H). C{ H} NMR
(
5
100 MHz, CDCl
3
) δ: 173.1, 135.4, 128.8, 128.6, 128.3, 67.3,
+
4.9, 47.8, 19.4. HRMS calcd. for C12
H
14
N
6
O
2
Na 297.1070
+
[
M + Na] , found 297.1072.
Synthesis of benzyl-3,3´-diaminopivaloate. Benzyl-
,3´-diazidopivaloate (2.00 g, 7.30 mmol, 1 eq) was
from
Erie-Autoclave
Engineers.
Luminescence
measurements were performed using an Edinburgh
Instruments FLS920 spectrometer equipped with a 450W
Xenon lamp (Xe900) as continuous excitation source for
stationary state measurements and Picoquant PLS-450 and
PLS-500 pulsed LED diodes as pulsed excitation source for
time-resolved measurements.
3
dissolved in THF (20 mL) and placed in an ice bath.
Triphenylphosphine (10.2 g, 38.6 mmol, 5.3 eq) was
dissolved in THF (10 mL) and added dropwise to the
previous solution. The mixture was left under reflux in a
heat block overnight. Afterwards, 1.5 mL of water were
added and the reaction was left under reflux for another
day. Then, THF was removed under vacuum and the
product was dissolved in HCl 1M (10 mL). The aqueous
phase was washed with dichloromethane (5 × 30 mL). The
water was later removed under vacuum to obtain the
desired compound (2.11 g, 7.15 mmol, 98%) as a colorless
General procedure for click reactions. Azido-
compound (1 eq), alkyne (1.1 eq per azido group), Copper
(II) Sulphate 5-hydrate (0.01 eq per azido group) and L (+)-
Ascorbic acid Sodium salt (0.1 eq per azido group) were
dissolved in a tert-butanol/water 1:2 mixture. The mixture
was stirred at room temperature for one week. Afterwards,
the solvent was removed using rotatory evaporation. NH
aq. (50 mL) and dichloromethane (50 mL) were added and
the phases were separated. The aqueous phase was
3
1
solid; mp 160–162 °C. H NMR (400 MHz, D
2
O) δ: 7.56 - 7.43
(
m, 5 H), 5.35 (s, 2 H), 3.45 (d, J = 13.7 Hz, 2 H), 3.29 (d, J =
13
1
1
3.7 Hz, 2 H), 1.49 (s, 3 H). C{ H} NMR (100 MHz, MeOD-
extracted with CH
layers were washed with NH
organic layer was dried over MgSO
2
Cl
2
(3 × 30 mL). The combined organic
aq./Brine 1:1 (3 × 80 mL). The
anh. and the solvent
d
4
) δ: 173.2, 136.3, 129.63, 129.61, 129.59, 69.4, 45.0, 44.34,
3
+
+
19.4. HRMS calcd. for C12
H
19
N
2
O
2
223.1441 [M + H] , found
4
223.1441.
was removed by rotary evaporation. The product was
purified by precipitation in n-hexane.
Synthesis
of
benzyl-3,3´-bis(tert-
butoxycarbonyl)aminopivaloate. To an ice-cooled
solution of benzyl-3,3´-diaminopivaloate (2.07 g, 7.00
mmol, 1 eq) in H O/acetone 1:1 (20 mL), NaOH 1M was
2
added dropwise until pH>10 was achieved. Di-tert-butyl
dicarbonate (3.05 mg, 15.40 mmol, 2 eq) was then added
and the reaction was stirred overnight at room
temperature. The product was extracted using
dichloromethane (5 × 30mL). The organic phase was dried
General procedure for deprotection of amines. The
compounds were dissolved in THF (10 mL) and the
solution was cooled in an ice-water bath. HCl 4M in
dioxane (10 mL) was added dropwise and the mixture was
stirred overnight. Afterwards, the solvent was evaporated
under vacuum. The compounds were purified by sephadex
column.
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Page 11 of 17
The Journal of Organic Chemistry
with MgSO
4
anh. and the solvent was removed under
(100 mg, 0.71 mmol, 0.3 eq). Hydrogenation took place in a
hydrogenation reactor at room temperature and 50 bar
hydrogen pressure. After five hours, the catalyst was
removed by filtration through MeOH-pre-wetted Celite.
The solvent was removed under vacuum to obtain the
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
vacuum to obtain the product (2.84 mg, 6.72 mmol, 96%)
as a colorless oil. H NMR (400 MHz, CDCl
(m, 5 H), 5.14 (s, 2 H), 3.48 (dd, J = 14.4, 8.6 Hz, 2 H), 3.12
(dd, J = 14.4, 5.2 Hz, 2 H), 1.43 (s, 18 H), 1.14 (s, 3 H). C{ H}
NMR (100 MHz, CDCl ) δ: 175.2, 156.7, 135.8, 128.7, 128.4,
128.0, 79.4, 66.7, 48.9, 43.5, 28.4, 19.0. HRMS calcd. for
1
3
) δ: 7.39-7.30
1
3
1
3
product (1.61 mg, 1.71 mmol, 98%) as a solid (compound
1
decomposes above 210 °C). H-NMR (400 MHz, DMSO-d
6
)
+
+
C
22
H
34
N
2
O
6
Na 445.2309 [M + Na] , found 445.2309.
δ ppm: 7.92 (s, 2 H), 4.51 (d, J = 13.7 Hz, 2 H), 4.35 (d, J = 13.7
Hz, 2 H), 4.27 (s, 4 H), 3.16-3.04 (m, 8 H), 1.35 (s, 36 H), 0.96
Synthesis
of 3,3´-bis(tert-
1
3
1
6
(s, 6 H), 0.81 (s, 3 H). C{ H} NMR (100 MHz, DMSO-d ) δ
butoxycarbonyl)aminopivalic acid. To a solution of
benzyl-3,3´-(tert-butoxycarbonyl)aminopivaloate (400 mg,
0.95 mmol, 1 eq) in methanol (10 mL), Pearlman’s catalyst
(100 mg, 0.71 mmol, 0.7 eq) is added. After hydrogenation
for two hours, the catalyst was removed by filtration
through MeOH-pre-wetted Celite. The solvent was
removed under vacuum to obtain the desired compound
ppm: 174.2, 174.1, 156.2, 144.6, 124.0, 77.9, 54.3, 48.5, 47.7,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
4
9
4.4, 34.6, 28.1, 19.1, 18.5. HRMS calcd. for C41
45.5134 [M + Na] , found: 945.5124.
H
70
N
12
O
12Na
+
2
Synthesis of compound dG2. A solution of dG2-CO H
(2.78 g, 3.02 mmol, 1 eq) in anhydrous acetonitrile (15 mL)
was added to a solution of CDI (734 mg, 4.53 mmol, 1.5 eq)
in anhydrous acetonitrile (45 mL) and the mixture was
stirred at room temperature for one hour. Afterwards,
propargylamine (0.3 mL, 4.53 mmol, 1.5 eq) was added and
the stirring mixture was left overnight at room
temperature. The solvent was removed under vacuum. The
residue was dissolved in dichloromethane (50 mL) and
washed with HCl 0.05M (5 × 50 mL). The combined organic
1
(309 mg, 0.93 mmol, 98%) as a colorless oil. H NMR (400
MHz, MeOD-d
Hz, 2 H), 1.43 (s, 18 H) 1.07 (s, 3 H). C{ H} NMR (100 MHz,
DMSO-d ) δ: 180.0, 158.7, 80.2, 46.7, 45.5, 28.7, 19.6. HRMS
calcd. for C15
55.1838.
Synthesis of dG1.
4
) δ: 3.25 (d, J = 14.2 Hz, 2 H), 3.17 (d, J = 14.2
1
3
1
6
+
+
28 2 6
H N O Na 355.1840 [M + Na] , found
3
A
solution of 3,3´-(tert-
phase was dried with MgSO
4
anh., filtered and
butoxycarbonyl)aminopivalic acid (1.70 g, 5.11 mmol, 1eq)
in anhydrous acetonitrile (5 mL) was added to a solution of
concentrated under reduced pressure to obtain the
product (2.46 g, 2.57 mmol, 85 %) as a colorless solid
1
,1′-carbonyldiimidazole (CDI) (1.3 g, 7.66 mmol, 1.5 eq) in
1
(
compound decomposes above 105 °C). H-NMR (400
anhydrous acetonitrile (15 mL) and the mixture was stirred
at room temperature for one hour. Afterwards,
propargylamine (0.7 mL, 10.22 mmol, 2 eq) was added and
the stirring mixture was left overnight at room
temperature. The solvent was removed under vacuum. The
residue was dissolved in dichloromethane (50 mL) and
washed with HCl 0.05M (3 × 50 mL). The combined organic
6
MHz, DMSO-d ) δ ppm: 7.22 (s, 2 H), 4.66 (d, J = 14.1 Hz, 2
H), 4.52 (d, J = 14.1 Hz, 2 H), 4.27 (d, J = 5.1 Hz, 4 H), 3.83
(
3
d, J = 2.7 Hz, 2 H), 3.12-3.07 (m, 8 H), 1.76 (s, 1 H), 1.36 (s,
1
3
1
6 H), 0.96 (s, 6 H), 0.93 (s, 3 H). C{ H} NMR (100 MHz,
) δ ppm: 174.1, 171.3, 156.1, 144.9, 123.9, 78.0, 73.1,
DMSO-d
6
6
7.0, 53.9, 48.0, 47.6, 44.4, 34.6, 28.1, 18.4, 17.4. HRMS calcd.
+
+
for C44
H
73
N
13
O
11Na 982.5450 [M + Na] , found: 982.5447.
phase was dried with MgSO
concentrated under reduced pressure to obtain the
4
anh., filtered and
2
Synthesis of dG3-CO Bn. This compound was obtained
product (1.57 g, 4.24 mmol, 83 %) as a colorless solid; mp
from benzyl-3,3´-diazidopivaloate (320 mg, 1.17 mmol, 1
eq), dG2 (2.47 g, 2.58 mmol, 2.2 eq), copper (II) sulphate 5-
hydrate (6 mg, 0.02 mmol, 0.02 eq) and L(+)-ascorbic acid
sodium salt (24 mg, 0.12 mmol, 0.1 eq) in tert-
butanol/water 1:2 (27 mL) to obtain the desired product
1
7
0–71 °C. H-NMR (400 MHz, DMSO-d
6
) δ ppm: 3.80 (dd, J
=
3
4.8, 2.1 Hz, 2 H), 3.14-3.01 (m, 5 H), 1.37 (s, 18 H), 0.95 (s,
H). C{ H} NMR (100 MHz, DMSO-d ) δ ppm: 173.9, 156.1,
6
1
3
1
8
1.3, 78.0, 72.7, 47.6, 44.3, 28.4, 28.2, 18.5. HRMS calcd. for
+
+
C
18
H
31
N
3
O
5
Na 392.2161 [M + Na] , found 392.2164.
(2.31 g, 1.05 mmol, 90%) as a colorless solid (compound
1
decomposes above 149 °C). H-NMR (400 MHz, DMSO-d
6
)
Synthesis of dG2-CO
2
Bn. This compound was obtained
δ ppm: 7.94-7.75 (m, 6 H), 7.36-7.32 (m, 5 H), 5.10 (s, 2 H),
.80 (d, J = 14.2 Hz, 2 H), 4.65 (d, J = 14.2 Hz, 4 H), 4.51 (d,
from benzyl-3,3´-diazidopivaloate (418 mg, 1.53 mmol, 1
eq), dG1 (1.23 g, 3.37 mmol, 2.2 eq), Copper (II) Sulphate 5-
hydrate (10 mg, 0.04 mmol, 0.02 eq) and L(+)-ascorbic acid
sodium salt (32 mg, 0.16 mmol, 0.1 eq) in tert-
butanol/water 1:2 (10 mL) to obtain the product (1.34 g, 1.32
4
J = 14.0 Hz, 4 H), 4.44 (d, J = 13.7 Hz, 2 H), 4.31-4.18 (m, 12
H), 3.18-3.04 (m, 16 H), 1.35 (s, 72 H), 0.96-0.93 (m, 21 H).
13
1
6
C{ H} NMR (100 MHz, DMSO-d ) δ ppm: 174.2, 171.8, 171.4,
1
156.1, 144.9, 144.2, 135.3, 128.4, 128.1, 128.0, 126.4, 124.0, 78.0,
mmol, 87%) as a colorless powder; mp 111–113 °C. H-NMR
66.8, 53.8, 53.5, 48.4, 47.9, 47.6, 44.4, 34.8, 34.6, 28.1, 18.4,
(
400 MHz, DMSO-d
6
) δ ppm: 7.79 (s, 2 H), 7.40-7.32 (m, 5
+
17.8, 17.6. HRMS calcd. for C100
+ Na] , found: 2216.2263.
H
160
N
32
O
24Na 2216.2181 [M
H), 5.09 (s, 2 H), ), 4.72 (d, J = 14.2 Hz, 2 H), ), 4.59 (d, J =
+
1
1
4.1 Hz, 2 H), 4.27 (d, J = 4.8 Hz, 4 H), 3.16-3.04 (m, 8 H),
.35 (s, 36 H), 1.00 (s, 3 H), 0.96 (s, 6 H). C{ H} NMR (100
) δ ppm: 174.1, 171.8, 156.1, 145.0, 135.3, 128.4,
13
1
Synthesis of dG3-CO
0.54 mmol, 1 eq) in MeOH (10 mL) is added to Pd(OH)
2
H. A solution of dG3-CO
2
Bn (1.18 g,
(23
MHz, DMSO-d
6
2
1
1
28.1, 128.0, 124.2, 77.9, 66.8, 53.4, 48.3, 47.6, 44.4, 34.6, 28.1,
8.4, 17.7. HRMS calcd. for C48
mg, 0.16 mmol, 0.3 eq). After hydrogenating for 6 days, the
catalyst was removed by filtration through MeOH-pre-
wetted Celite. The solvent was removed under vacuum to
obtain the product (966 mg, 0.46 mmol, 85%) as a solid
+
+
H
77
N
12
O
12 1013.5784 [M + H] ,
found 1013.5781.
Synthesis of dG2-CO
g, 1.74 mmol, 1 eq) in MeOH (10 mL) was added to Pd(OH)
2
H. A solution of dG2-CO
2
Bn (1.76
1
(compound decomposes above 230 °C). H-NMR (400
2
6
MHz, DMSO-d ) δ ppm: 8.18-7.54 (m, 6 H), 4.72-4.44 (m,
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 12 of 17
1
0
2 H), 4.37-4.22 (m, 12 H), 3.16-2.96 (m, 16 H), 1.36 (s, 72 H), 12 H), 3.24-3.00 (m, 16 H), 1.35 (s, 72 H), 0.96 (s, 12 H), 0.93
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
1
13
1
.96 (s, 12 H), 0.95 (s, 6 H), 0.81 (s, 3 H). C{ H} NMR (100
) δ ppm: 174.3, 174.2, 171.1, 156.2, 145.0, 144.5,
6
(s, 6 H). C{ H} NMR (100 MHz, DMSO-d ) δ ppm: 174.1,
171.4, 156.1, 144.9, 144.6, 124.0, 123.4, 78.0, 53.8, 49.0, 47.9,
47.6, 44.4, 34.8, 34.6, 28.1, 18.4, 17.5.
MHz, DMSO-d
123.9, 123.9, 77.9, 54.7, 53.9, 48.7, 47.6, 47.5, 44.4, 34.6, 34.5,
28.1, 19.0, 18.5, 17.6. HRMS calcd. for C93
2102.1735 [M – H] , found: 2102.1636.
6
-
H
153
N
32
O
24
2
Synthesis of G2EDANH . This compound was obtained
-
from G2EDANHBoc (400 mg, 0.20 mmol) to obtain the
1
Synthesis of dG3. A solution of dG3-CO
2
H (1.10 g, 0.52
product (299 mg, 0.20 mmol, 98 %) as a colorless solid. H-
NMR (400 MHz, D O) δ ppm: 7.78-7.66 (m, 6 H), 5.02 (s, 4
H), 4.58-4.43 (m, 12 H), 4.31 (s, 8 H), 3.33 (d, J = 13.2 Hz, 8
mmol, 1 eq) in anhydrous acetonitrile (10 mL) was added to
a solution of CDI (126 mg, 0.78 mmol, 1.5 eq) in anhydrous
acetonitrile (10 mL) and the mixture was stirred at room
temperature for one hour. Afterwards, propargylamine (52
µL, 0.78 mmol, 1.5 eq) was added and the stirring mixture
was left overnight at room temperature. The solvent was
removed under vacuum and the residue was dissolved in
dichloromethane (30 mL) and washed with HCl 0.05M (5 ×
30mL). The combined organic phase were dried with
2
13
1
H), 3.15 (d, J = 13.5 Hz, 8 H), 1.41 (s, 12 H), 1.13 (s, 6 H). C{ H}
NMR (100 MHz, D O) δ ppm: 173.2, 172.4, 143.6, 143.1, 124.2,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
123.8, 54.1, 49.2, 47.9, 44.3, 43.4, 34.0, 33.8, 16.7, 15.8.
Synthesis of G3EDANHBoc. This compound was obtained
from dG3 (179 mg, 0.084 mmol, 2.2 eq), 1,2-diazidoethane
(
4 mg, 0.04 mmol, 1 eq), copper (II) sulphate 5-hydrate (0.2
mg, 0.001 mmol, 0.02 eq) and L(+)-ascorbic acid sodium
salt (2 mg, 0.01 mmol, 0.1 eq) in tert-butanol/water 1:2 (6
mL) to obtain the desired product (163 mg, 0.04 mmol, 93
%) as a colorless solid (compound decomposes above 145
4
MgSO anh., filtered and concentrated under reduced
pressure to obtain the product (723 mg, 0.34 mmol, 65 %)
as a colorless solid (compound decomposes above 146 °C).
1
H-NMR (400 MHz, DMSO-d
.71-4.53 (m, 12 H), 4.39-4.15 (m, 12 H), 3.83 (s, 2 H), 3.20-
.95 (m, 16 H), 1,74 (s, 1 H), 1.35 (s, 72 H), 0.96-0.93 (m, 21
6
) δ ppm: 7.93-7.54 (m, 6 H),
1
6
°C). H-NMR (400 MHz, DMSO-d ) δ ppm: 7.97-7.77 (m, 14
4
2
H), 4.90 (s, 4 H), 4.68-4.57 (m, 24 H), 4.43-4.16 (m, 28 H),
3.22-2.98 (m, 32 H), 1.34 (s, 144 H), 1.08-0.88 (m, 42 H).
C{ H} NMR (100 MHz, DMSO-d ) δ ppm: 174.2, 171.48,
6
171.49, 156.2, 145.1, 145.0, 144.9, 124.2, 124.1, 124.0,78.0, 53.8,
51.4, 48.4, 47.9, 47.7, 47.4, 44.4, 34.6, 34.59, 34.57,28.1, 18.5,
1
3
1
H). C{ H} NMR (100 MHz, DMSO-d
1
5
1
6
) δ ppm: 174.2, 171.5,
13
1
71.4, 156.2, 144.9, 144.2, 124.2, 124.0, 78.0, 73.1, 53.88, 53.92,
3.8, 51.4, 48.0, 47.9, 47.6, 44.4, 34.6, 28.6, 28.2, 18.5, 17.6,
7.4. HRMS calcd. for C96H N O23 2142.2287 [M + 2 H] ,
159 33
2+
2+
1
7.7, 17.6.
found: 1071.1137.
2
Synthesis of G3EDANH . This compound was obtained
Synthesis of 1,2-diazidoethane. 1,2-diazidoethane was
synthesized as described in literature. H-NMR (400
MHz, CDCl
CDCl ) δ ppm: 50.7.
from G3EDANHBoc (109 mg, 0.025 mmol) to obtain the
product (82 mg, 0.025, 98 %) as a colorless solid. H-NMR
2
8 1
1
1
3
1
3
) δ ppm: 3.46 (s, 4 H). C{ H} NMR (100 MHz,
2
(400 MHz, D O) δ ppm: 8.04-7.62 (m, 14 H), 5.01 (s, 4 H),
3
4.66-4.20 (m, 44 H), 3.85-3.56 (m, 8 H), 3.44-3.09 (m, 32 H),
1
3
1
Synthesis of G1EDANHBoc. dG1 (500 mg, 1.36 mmol, 2.2
eq), 1,2-diazidoethane (69 mg, 0.62 mmol, 1 eq), copper (II)
sulphate 5-hydrate (3 mg, 0.01 mmol, 0.02 eq) and L(+)-
ascorbic acid sodium salt (12 mg, 0.06 mmol, 0.1 eq) in tert-
butanol/water 1:2 (18 mL) to obtain the product (485 mg,
0
1.45 (s, 24 H), 1.16 (s, 12 H), 1.00 (s, 6 H). C{ H} NMR (100
MHz, D O) δ ppm: 174.0, 173.0, 172.4, 143.2, 143.1, 143.0,
2
123.93, 123.90, 123.88, 54.7, 54.2, 49.2, 48.1, 44.2, 44.0, 43.2,
34.0, 33.83, 33.77, 16.5, 16.4, 15.9.
Synthesis of 1,3,5-tris(azidomethyl)benzene. 1,3,5-
1
.57 mmol, 92 %) as a colorless oil. H-NMR (400 MHz,
30
tris(azidomethyl)benzene was synthesized as described.
DMSO-d
6
) δ ppm: 7.76 (s, 2 H), 4.82 (s, 4 H), 4.24 (d, J = 5.2
1
H-NMR (400 MHz, CDCl
6H).
3
) δ ppm: 7.25 (s, 3 H), 4.40 (s,
Hz, 4 H), 3.18-2.98 (m, 8 H), 1.36 (s, 36 H), 0.96 (s, 6 H).
1
3
1
C{ H} NMR (100 MHz, DMSO-d
6
) δ ppm: 174.1, 156.1, 145.3,
22.9, 78.0, 48.9, 47.6, 44.4, 34.6, 28.1, 18.4. HRMS calcd. for
Synthesis of G33ABNHBoc. This compound was obtained
from dG3 (179 mg, 0.084 mmol, 3.3 eq), 1,3,5-
tris(azidomethyl)benzene (6 mg, 0.025mmol, 1 eq), copper
(II) sulphate 5-hydrate (0.2 mg, 0.001 mmol, 0.03 eq) and
L(+)-ascorbic acid sodium salt (3 mg, 0.01 mmol, 0.3 eq) in
tert-butanol/water 1:2 (6 mL) to obtain the product (98 mg,
1
C
+
+
38
H
66
N
12
O
10Na 873.4923 [M + Na] , found: 873.4921.
. This compound was obtained
Synthesis of G1EDANH
from G1EDANHBoc (450 mg, 0.53 mmol) to obtain the
desired product (309 mg, 0.52 mmol, 98%) as a colorless
solid. H-NMR (400 MHz, D
2
1
2
O) δ ppm: 7.85 (s, 2 H), 4.94
s, 4 H), 4.49 (s, 4 H), 3.34 (d, J = 13.5 Hz, 4 H), 3.15 (d, J =
0.015 mmol, 59 %) as a colorless solid (compound
(
1
decomposes above 140 °C). H-NMR (400 MHz, DMSO-d
6
)
1
3
1
1
3.5 Hz, 4 H), 1.42 (s, 6 H). C{ H} NMR (100 MHz, D
ppm: 173.1, 143.7, 123.2, 49.2, 44.2, 43.3, 33.8, 16.6.
Synthesis of G2EDANHBoc. dG2 (1.09 g, 1.14 mmol, 2.2 eq),
,2-diazidoethane (58 mg, 0.52 mmol, 1 eq), copper (II)
2
O) δ
δ ppm: 7.97-7.77 (m, 24 H), 5.63-5.56 (m, 6 H), 4.77-4.45
m, 36 H), 4.39-4.14 (m, 42H), 3.22-2.93 (m, 48 H), 1.34 (s,
(
1
3
1
216 H), 1.06-0.88 (m, 54 H). C{ H} NMR (100 MHz, DMSO-
6
d ) δ ppm: 174.2, 172.4, 171.4, 156.2, 145.1, 144.9, 144.6, 137.1,
1
sulphate 5-hydrate (3 mg, 0.01 mmol, 0.02 eq) and L(+)-
ascorbic acid sodium salt (10 mg, 0.06 mmol, 0.1 eq) in tert-
butanol/water 1:2 (18 mL) to obtain the product (845 mg,
127.5, 124.04, 123.95, 123.4, 78.0, 53.8, 48.4, 47.9, 47.8, 47.7,
47.6, 47.4, 44.4, 35.9, 34.9, 34.6, 28.1, 18.5, 18.0, 17.7.
2
Synthesis of G33ABNH . This compound was obtained
from G33ABNHBoc (50 mg, 0.008mmol) to obtain the
0
.42 mmol, 80 %) as a colorless solid (compound
1
decomposes above 140 °C). H-NMR (400 MHz, DMSO-d
6
)
product (40 mg, 0.008 mmol, 98%) as a colorless solid in a
δ ppm: 8.55-7.72 (m, 6 H), 4.90 (d, J = 14.7 Hz, 4 H), 4.66
d, J = 13.9 Hz, 4 H), 4.51 (d, J = 14.1 Hz, 4 H), 4.30-4.14 (m,
1
quantitative way. H-NMR (400 MHz, D
2
O) δ ppm: 8.10-
(
7.62 (m, 21 H), 7.26 (s, 3 H), 5.65 (m, 6 H), 4.67-4.18 (m, 66
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The Journal of Organic Chemistry
1
H), 3.71-3.52 (m, 6H), 3.47 (d, J = 13.6 Hz, 24 H), 3.23 (d, J =
3.5 Hz, 24 H), 3.14-2.93 (m, 6 H), 1.50 (s, 36 H), 1.16 (s, 18
(H
2
O): λmax nm (ε): 259 (4709), 284 (4125), 447 (3209). H-
O) δ ppm: 8.05-7.62 (m, 19 H), 4.65-4.23
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
NMR (400 MHz, D
2
13
1
2
H) 1.01 (s, 9 H). C{ H} NMR (100 MHz, D O) δ ppm: 172.3,
(m, 44 H), 3.75 (d, J = 13.6 Hz, 4 H), 3.66 (d, J = 11.6 Hz, 4
H), 3.60-3.49 (m, 4 H), 3.41 (d, J = 13.2 Hz, 16 H), 3.20 (d, J =
10.1 Hz, 16 H), 3.11-2.89 (m, 4 H), 1.46 (s, 24 H), 1.16 (s, 12 H),
172.1, 170.3, 143.0, 142.9, 142.8, 124.3, 124.2, 124.1, 54.2, 49.0,
48.1, 48.0, 47.8, 43.6, 42.7, 34.0, 33.9, 33.8, 16.3, 15.9, 15.8.
1
3
1
2
1.00 (s, 6H). C{ H} NMR (100 MHz, D O) δ ppm: 172.64,
Synthesis of 3-azidopropylamine. 3-Bromopropylamine
hydrobromide (5.00 g, 22.84 mmol, 1 eq) was dissolved in
water (10 mL) and sodium azide was added (4.45 g, 68.52
mmol, 3 eq). The reaction was stirred during three days at
172.57, 172.3, 143.02, 143.05, 143.11, 124.7, 124.5, 124.2, 124.1,
123.9, 54.7, 54.6, 54.2, 53.3, 48., 48.0, 43.8, 42.97, 42.91, 34.0,
33.9, 33.8, 17.0, 16.5, 15.9.
8
0ºC in a heat block. The mixture was cooled in an ice-
DOSY
Nuclear
Magnetic
Resonance
(NMR)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
water bath and ether was added (20 mL). Potassium
hydroxide pellets were added until basic pH was attained.
The organic layer was separated and the aqueous phase
was extracted with ether (3 × 20 mL). The combined
organic layers were dried with MgSO
concentrated to obtain the product (1.14 g, 11.42 mmol, 50
Experiments. The samples were prepared in deuterium
oxide at a concentration between 0.5 and 2 mM (within the
infinite dilution range for similar samples at 0.1–2.1 mM).
The experiments have been performed on a The Bruker
3
7
TM
4
anh. and
Ascend 400 MHz spectrometer, equipped with a 5 mm
PLUS
2
BBFO
probe with H “lock” channel and Z gradient. The
1
%) as a colorless oil. H-NMR (400 MHz, D
2
O) δ ppm: 3.67
spectrometer is also equipped with a control temperature
unit prepared to work at temperatures ranging from 0 °C
to +50 °C. Gradient strength was calibrated by measuring
(
t, J = 7.5 Hz, 2 H), 3.24 (t, J = 6.8 Hz, 2 H), 2.11 (Q, J= 6.8
Hz, 2 H).
2
the diffusion rate of pure water of residual protons in D O.
Synthesis
of
N-(3-azidopropyl)-4-((3-
All experiments were conducted at 300 K. The samples
were allowed to equilibrate for no fewer than 15 min. To
determine the diffusion rates, a 2D sequence using double
stimulated echo for convection compensation and LED
using bipolar gradient pulses for diffusion was used. The
Diffusion coefficients (D) were determined from the slope
of the Stejskal-Tanner plot, which relates it to the signal
azidopropyl)amino)-1,8-naphthalimide. A solution of
4-bromo-1,8-naphthalic anhydride (500 mg, 1.80 mmol, 1
eq) and 3-azidopropylamine (2.7 g, 27 mmol, 15 eq) in
DMSO (2.5 mL) was heated in a heat block at 80ºC
overnight. Then, dichloromethane was added (50 mL) and
the mixture was washed with HCl (3 × 30 mL). The organic
4
layer was dried using MgSO anh. and was concentrated.
퐼
훿
2
2 2
Purification was performed by silica gel column
chromatography (dichloromethane:methanol, 99:1 v/v) to
obtain the product (4.23 g, 1.22 mmol, 68 %) as a yellow
intensity through the equation: ln ( ) = ― 훾 훿 퐺 (∆ ― )퐷,
퐼0 3
where I is the integral of the peak area at a given value of
G, I is the integral of the peak area at a G=0, G is the
0
1
solid (compound decomposes above 171 °C). H-NMR (400
gradient field strength,  is the gyromagnetic ratio,  is the
gradient duration and  is the time between the gradient
pulses. The diffusion coefficients determined were used
to calculate the hydrodynamic radius via the Stokes-
Einstein equation: 푅ℎ =
MHz, CDCl
Hz, 1 H), 8.09 (d, J = 8.1 Hz, 1 H), 7.62 (t, J = 8.1 Hz, 1 H), 6.71
d, J = 8.5 Hz, 1 H), 4.25 (t, J = 7 Hz, 2 H), 3.69-3.49 (m, 4
3
) δ ppm: 8.57 (d, J = 7.3 Hz, 1 H), 8.45 (d, J = 8.4
3
6
(
1
3
1
H), 3.41 (t, J = 7 Hz, 2 H), 2.18-1.93 (m, 4 H). C{ H} NMR
100 MHz, CDCl ) δ ppm: 164.8, 164.2, 149.4, 134.6, 131.4,
퐾 푇
퐵
6휋휂퐷, where K
is the
B
(
3
Boltzmann constant, T is the temperature and η is the
viscosity of the solution (1.0963 cP for D O viscosity).
2
1
29.9, 126.1, 125.1, 123.1, 120.5, 110.6, 104.4, 49.9, 49.6, 41.8,
3
7
-
3
7.7, 28.0, 27.9. HRMS calcd. for C18
H
19
N
8
O
2
379.1631 [M +
+
Molecular Dynamics Simulations. Briefly, we utilized
the AMBER12 MD software package, with the force field
parameters (parm99). For the 1,4-substituted triazole-
based dendrimers we used the parameter described
before, and those not included were transferred from the
H] , found: 379.1625.
42
Synthesis of G3NaphNHBoc. This compound was obtained
from dG3 (179 mg, 0.084 mmol, 2.2 eq), N-(3-azidopropyl)-
4
-((3-azidopropyl)amino)-1,8-naphthalimide (14 mg, 0.04
4
3
mmol, 1 eq), copper (II) sulphate 5-hydrate (0.2 mg, 0.001
mmol, 0.02 eq) and L(+)-ascorbic acid sodium salt (2 mg,
0
obtain the product (107 mg, 0.02 mmol, 58 %) as a yellow
solid (compound decomposes above 126 °C). H-NMR (400
4
4
General Atom Force Field parameters (GAFF). The initial
dendrimer conformations were generated with the
.01 mmol, 0.1 eq) in tert-butanol/water 1:2 (6 mL) to
4
5
Dendrimer Building Tool (DBT) or with AmberTools12
and the LEaP package. The system was minimized and
then heated to 300 K over 40 ps. Simulations were run at
physiological pH (7.4) in NPT ensemble at 300 K and 1 atm
for 20 ns. The cutoff for non-bonded interactions was 9 Å.
Time steps of 2 fs were taken with implementation of the
1
MHz, DMSO-d
6
) δ ppm: 8.14-7.59 (m, 19 H), 4.77-4.04 (m,
5
2
6 H), 3.52 (s, 2H), 3.21-2.94 (m, 34 H), 2.34-2.15 (m, 2 H),
13
1
.01-1.83 (m, 2 H) 1.35 (s, 144 H), 1.08-0.88 (m, 42 H). C{ H}
) δ ppm: 174.2, 172.4, 171.5, 163.9,
63.1, 156.2, 150.7, 145.1, 145.0, 144.9, 134.3, 130.8, 129.5, 129.0,
28.9, 128.3, 124.3, 124.0, 121.9, 120.3, 107.9, 103.8, 78.0, 53.9,
NMR (100 MHz, DMSO-d
1
1
6
4
6
SHAKE routine. Dendrimers were equilibrated for 2 ns
and starting from these configurations, production runs of
20 ns trajectories were performed under an NPT ensemble.
5
3
3.5, 48.9, 48.6, 48.4, 48.0, 47.7, 44.4, 37.0, 35.6, 34.9, 34.6,
4.6, 28.2, 27.2, 27.2, 18.5, 17.7, 17.6.
Trajectory analyses were performed using the Amber
modules ptraj and cpptraj. Snapshots from the trajectories
within this paper were created with VMD software.
Further details of the Molecular Dynamics Simulations are
described in the Electronic Supporting Information (ESI).
Synthesis of G3NaphNH
from G3NaphNHBoc (67 mg, 0.014mmol) to obtain the
product (50 mg, 0.014 mmol, 98 %) as a yellow solid. UV
2
. This compound was obtained
47
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The Journal of Organic Chemistry
Page 14 of 17
Luminescent Microscopy experiments with E. coli
bacteria incubated with G3NaphNH . Bacteria were grown
This work was supported by the Spanish Ministerio de
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Economía, Industria y Competitividad (CTQ2016-75870-P),
Instituto de Salud Carlos III (ISCIII; RETIC ARADYAL
RD16/0006/0012), Consejería de Salud, Junta de Andalucía
2
in 10 mL of LB Broth at 37ºC in a rocking incubator (18
hours). Then, culture contents were split into four 15 mL
vials, centrifuged (5000g, 5 minutes), and washed again in
5 mL PBS. After an additional centrifugation (5000 g, 5
minutes), bacterial pellets were either resuspended in 3 mL
(
PI0250-2016) and FEDER founds. N.M. holds a FPU grant of
MECD (FPU15/00641). J.A.G. acknowledges financial support
from Plan Propio-Universidad de Málaga.
-4
-4
2
PBS with G3NaphNH (10 M or 5·10 M dilution) or
ACKNOWLEDGMENT
resuspended in 3 mL PBS alone. A 8 h incubation step in a
rocking incubator followed (4ºC). Then, both samples were
centrifuged (5000 g, 5 minutes) and washed twice in 3 mL
PBS. Finally, each bacterial sample was resuspended in 100
µL PBS. Bacterial cultures were analyzed using a Leica SP5
MP confocal microscope equipped with Spectraphysics
MaiTai HP pulse IR laser for multiphoton excitation and a
HCX PL APO lambda blue 63x NA 1.40 oil immersion
objective lens was used. Brightfield and confocal images
were acquired using 405 nm excitation with emissions
detected with a spectral PMT detector set to 500-600 nm.
Multiphoton images were acquired sequentially with
excitation at 720 nm and detection between 500-550 nm
with an external HyD non-descanned detector.
We gratefully acknowledge the computer resources provided
by the SCBI (Supercomputing and Bioinformatics Center) of
the University of Malaga. We are indebted to Dr. Luis Díaz-
Martínez and Dr. Diego Romero from Departamento de
Microbiología, Facultad de Ciencias, Universidad de Málaga
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
Spain) for their excellent technical assistance and help with
the bacterial assays.
ABBREVIATIONS
Boc, tButoxycarbonyl; MALDI-TOF matrix-assisted laser
desorption ionization-time of flight.
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AUTHOR INFORMATION
Corresponding Author
*yolvida@uma.es, *inestrosa@uma.es, Tel.: +34-952137565
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
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The Journal of Organic Chemistry
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9
BocHNBocHN
BocHNBocHN
BocHN
BocHN
O
NH
N
HN
O
O
N
h (OPE) Luminescent Microscopy
E. coli bacteria
BocHN
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N3
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Multivalent
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1
7
ACS Paragon Plus Environment