Biocompatible, multifunctional, and well-defined OEG-based dendritic platforms for biomedical applications

Article abstract of DOI:10.1039/c3ob40492g

Given the growing importance of drug and gene delivery systems, imaging agents, biosensors, and theranostics, there is a need to develop new multifunctional and biocompatible platforms. Here we synthesized and fully characterized a family of novel multifu

Full text of DOI:10.1039/c3ob40492g

Organic &
Biomolecular Chemistry
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Biocompatible, multifunctional, and well-defined
OEG-based dendritic platforms for biomedical
Cite this: Org. Biomol. Chem., 2013, 11,
4109
applications†
Lorena Simón-Gracia,a Daniel Pulido,b,c Chantal Sevrin,d Christian Grandfils,d
Fernando Albericio*a,b,e,f and Miriam Royo*b,c
Given the growing importance of drug and gene delivery systems, imaging agents, biosensors, and thera-
nostics, there is a need to develop new multifunctional and biocompatible platforms. Here we syn-
thesized and fully characterized a family of novel multifunctional and completely monodisperse dendritic
platforms. Our synthetic methodology, based on compatible protecting groups and the attachment of
monodisperse triethylene glycol units, allows the control of the generation and differentiation of term-
inal groups, thus giving rise to multifunctional and perfectly-defined products. A family of dendrons was
synthesized and four distinct dendritic structures were chosen from the family in order to determine the
effect of the generation and surface groups on their biocompatibility. The stability in serum, cytotoxicity,
and hemocompatibility of these products were studied. Our results indicate that these non-toxic, hemo-
compatible, non-immunogenic, stable and versatile scaffolds may be very interesting candidates for bio-
medical applications.
Received 10th March 2013,
Accepted 26th April 2013
DOI: 10.1039/c3ob40492g
www.rsc.org/obc
thus allowing their use as drug and gene delivery systems,
imaging agents, biosensors, and theranostics.1–4
Introduction
While the number of new drugs approved by several agencies
The composition, surface functionality, and size of these
each year seems to have reached a plateau, complementary macromolecules dictate their toxicity and pharmacokinetic be-
associated technologies, such as in vitro diagnostics, contrast havior. These factors are crucial to allow their use in diverse
agents, and vectors for gene and drug delivery systems, have biomedical applications. The use of polyethylene glycol (PEG)-
met with increasing success. Dendrimers show unique behav- based dendrimers or dendrimers functionalized on their
ior that makes them a promising choice for a wide range of surface with PEG units is a very attractive strategy because it
biomedical applications. These hyperbranched molecules are combines two important aspects: the theoretically well-defined
characterized by a protein-like globular shape and well-defined macromolecular dendrimers and the adoption of well-known
structure, multivalency, and capacity to act as nanocontainers, polymers in drug delivery systems.5–13 However, the polydisper-
sity shown by PEG and the difficulty to prepare highly
branched dendrimers turn these PEG-decorated dendrimers
into polydisperse systems, thereby leading to problems regard-
aInstitute for Research in Biomedicine, Baldiri i Reixac 10, 08028 Barcelona, Spain.
ing purification, synthesis reproducibility and variations in
E-mail: lorenasimongracia@gmail.com, albericio@irbbarcelona.org
their in vivo behavior.14 Here we describe the synthesis of oli-
bCIBER-BBN, Networking Center on Bioengineering, Biomaterials and Nanomedicine,
goethylene glycol (OEG)-based dendrons, prepared and puri-
fied as molecules with an exact molecular weight, thereby
avoiding the aforementioned issues.
Barcelona, Spain
cBarcelona Science Park, Baldiri i Reixac 10, 08028 Barcelona, Spain.
E-mail: dpulido@pcb.ub.cat, mroyo@pcb.ub.cat; Tel: +34934037122
dInterfacultary Research Centre of Biometerials University of Liège, Institute of
Chemistry, Building B&C, Sart-Tillman, Liège, Belgium. E-mail: C.Sevrin@ulg.ac.be,
C.Grandfils@ulg.ac.be
Traditionally, dendrimers are built adopting the growth of a
symmetric core, resulting in isotropic structures with equal
end-groups.15 However, the development of materials with
higher structural complexity and multiple functionalities that
can be precisely modified with diverse molecules (as water
inducing-solubilizers, targeting and imaging ligands and
different drugs) to confer a range of properties to the final
system is very useful and sometimes even necessary for the
eDepartment of Organic Chemistry, University of Barcelona, Martí i Franqués 1-11,
08028 Barcelona, Spain. E-mail: albericio@irbbarcelona.org; Tel: +34934037088
fSchool of Chemistry University of KwaZulu Natal, 4001-Durban, South Africa
†Electronic supplementary information (ESI) available: Purification and charac-
terization methods, dynamic light scattering, ζ-potential, microscopy pictures,
hemocompatibility, HPLC-MS, MALDI-TOF, and NMR spectrometry. See DOI:
10.1039/c3ob40492g
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applications explored.16,17 The chemical strategy that we have of compound 5 was eliminated by hydrogenolysis resulting in
optimized allows the controlled differentiation of dendrons, dendron 6 with a carboxylic acid at the focal point and the
thus giving access to a multifunctional platform. Accordingly, surface amine groups Boc-protected. On the other hand elimi-
the exact molecular weight and multifunctionality of these nation of Boc amino protecting groups of compound 5 by
dendrons provide an exquisite level of structural control that is treatment with 4 M HCl in dioxane gave dendron 7, in which
expected to eliminate the undesirable effects of size and vari- the amino groups are free and the focal point is a benzyl ester.
ation in biomolecule-loading shown by polymer drug delivery The best results in the preparation of 11 were obtained by
systems. Compared with symmetric platforms with all equi- reacting compound 7, with four units of dendron 6 with the
valent functional groups on their surface, our current strategy carboxylic acid activated as a pentafluorophenyl ester. The
offers considerable advantages for the improved control of the multifunctional and well-defined AB16 dendron was obtained
biological applications of these multifunctional nanomater- in a 48% yield and high purity (97%) and was suitable for the
ials. Before these dendrimers are considered for use in diverse derivatization of the carboxylic acid or amino groups.
therapeutic purposes, we studied the effect of their generation
Starting from the first and the second generation (5 and
and surface groups on certain factors that are crucial to deter- 11), a family of dendrons was synthesized (Table 1) by modify-
mine biocompatibility, such as stability in serum, cytotoxicity, ing the surface group and the focal point derivatization,
and hematocompatibility.
thereby demonstrating the versatility of the dendritic platform.
Selective elimination of Boc and benzyl protecting groups was
performed following standard methods (see the Experimental
section). The acetylation of the amino groups of the first gene-
ration dendron 7 was achieved using acetic anhydride and
DIEA in CH2Cl2, and the fully acetylated product 8 was
Results and discussion
Synthesis of dendrons
The dendritic platform was synthesized starting from a diethyl- obtained after 1 h of reaction. Benzyl group elimination
enetriaminepentaacetic acid (DTPA) derivative (Scheme 1). In yielded compound 9. Nevertheless, the acetylation of the
this case, one of the five carboxylic acids of the DTPA-based second generation dendron 13 was performed in water
core (3) is protected with a benzyl group, while the other four because of the poor solubility of the deprotected dendron in
carboxylic acids are protected with a tert-butyl group. This pro- organic solvents. Also, to complete this reaction, the consecu-
tecting scheme allows benzyl group removal preserving the tive addition of an excess of acetic anhydride over two days
other four carboxylic acids protected with the tert-butyl group, was necessary. All the products were obtained as unique com-
or the elimination of the tert-butyl groups while the fifth pounds with exact molecular weight, as demonstrated by
carboxylic acid remains protected. The benzyl-protected MALDI-TOF, HRMS and NMR techniques.
carboxylic group is the focal point for dendron synthesis.
First and second generation dendrons with free or acetyl-
Dendron core 3 was obtained following a three-step pro- ated surface amines (G1-NH2-Bn, G1-Ac-Bn, G2-NH2-Bn and
cedure. Ethanolamine and tert-butyl bromoacetate were G2-Ac-Bn) were chosen from the family as model compounds
reacted to obtain N,N-bis[(tert-butyloxycarbonyl)methyl]-2- of this dendritic platform to evaluate the influence of dendron
hydroxyethylamine, and then its bromoderivative (product 2) generation and terminal groups on the biocompatibility,
was obtained using NBS, attaining 71% of yield after flash stability, and size of the final system. These two surface group
chromatography purification. 2-[Bis-(tert-butyloxycarbonyl- modifications were selected as representative models of this
methyl)amino]ethyl bromide (2) was mixed with glycine benzyl dendron ability to act as: (1) drug conjugated delivery systems
ester, and the reaction was carried out in a biphasic solvent via covalent union between the drug and the dendron (G1-Ac-
system composed of CH3CN and phosphate buffer at pH 8, Bn and G2-Ac-Bn); or (2) drug/gene delivery systems via ionic
affording the dendron core (3) in a 58% yield (92% of purity). interaction between terminal ammonium group dendrons
After removal of tert-butyl groups with HCl–dioxane, the core with drug or gene material (G1-NH2-Bn and G2-NH2-Bn).
dendron with four free carboxylic acids and one carboxylic acid
Size and ζ-potential
protected with a benzyl group was obtained quantitatively (4).
Reaction between 4 and four Boc monoprotected bisamine- The size (hydrodynamic diameter) of second generation
triethylene glycol units (1-(tert-butyloxycarbonyl-amino)-4,7,10- dendrons was measured by dynamic light scattering (Table 2
trioxa-13-tridecanamine) using PyBOP : HOBt as coupling and see also Fig. 1 in ESI†). Due to the threshold of the DLS
reagents system in the presence of DIEA led to the first gene- equipment, the lower concentration used for the measurement
ration dendron (5, Scheme 1) with an AB4 architecture. of the size and zeta-potential was the higher concentration
Product 5 was obtained in a multigram scale (3.5 g) with satis- used in the cytotoxicity and hemocompatibility experiments.
factory yield (72%), excellent purity (99%), and completely Lower dendron concentrations used in these experiments were
monodisperse, as confirmed by HRMS and NMR. The second not optimum for the measurement by DLS. However, in order
generation dendron (11) was prepared by a “semi-convergent” to confirm that the size and zeta-potential were concentration-
synthetic strategy (Scheme 1). To obtain the appropriate den- independent, higher sample concentrations were also used. As
drons necessary for second generation synthesis diverse modi- can be observed in size distribution profiles, G2-Ac-Bn,
fications were carried out for compound 5. The benzyl group containing the acetylated amino-terminal groups, measured
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Scheme 1 Synthesis of first and second generation dendrons. (a) H2, Pd/C; (b) 2,3,4,5,6-pentafluorophenol, EDC, DMAP; (c) dendron 7, DIEA.
4.1 nm. This result is consistent with the previously reported G3-PAMAM dendrimer, which had 32 terminal groups and a
sizes of G2 and G3-PAMAM (poly(amido amine)) higher molecular weight than our dendron (6909 compared
dendrimers.18–24 It is worth noting that the size of the with 6721), was slightly less than G2-Ac-Bn. This difference is
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Table 1 Library of synthesized dendrons
attributable to the superior swelling capacity of the triethylene
Interestingly, when measurements were performed with G2-
glycol branches, thus resulting in a higher hydrodynamic NH2-Bn, the size increased dramatically to about 189.4 nm,
diameter.
and higher size dispersion was also detected. This observation
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also increase the amount of triethylene glycol units and
thus, the “stealth” effect, typical of PEG, prevents interactions
between amide-dendron and hydrolytic components.25
The stability of G2-NH2-Bn could not be determined by this
methodology due to a strong interaction between the dendron
and serum proteins. This results in precipitation of G2-NH2-Bn
with serum proteins when prior to HPLC analysis CH3CN
is added to clean the sample from proteins, removing the
dendrimer from the sample.
Table 2 Size and ζ-potential of second generation dendrons in PBS
Size (nm)
ζ-Potential (mV)
Conc. (mg mL−1
)
G2-NH2-Bn
G2-Ac-Bn G2-NH2-Bn G2-Ac-Bn
10
5
1
189.4 87.7 4.1 0.7
189.6 87.6 3.9 0.9
168.9 61.3 4.4 0.8
41.5 11.5
43.2 9.6
43.8 11.0
5.66 6.4
5.08 5.2
5.61 4.7
highlights that dendron aggregation occurred under our
measurement conditions.
This phenomenon was observed in these experiments
and corroborated by previous studies, where the interaction
between amino-terminated PAMAM dendrimers (G3- and G4-
PAMAM dendrimers containing 32 and 64 primary amines)
and bovine serum albumin was described.26–28 However, the
dendron–albumin interaction was not observed with G2-Ac-Bn
(14), suggesting that this phenomenon is directly associated
with the number of amines exposed on the dendrimer surface.
Therefore, the conjugation of a drug to the dendron amino-
surface groups or the complexation with an oligonucleotide
(forming a dendriplex) will reduce the serum proteins inter-
action resulting in a suitable drug delivery system. Further
future investigations will be conducted toward these issues.
The electrical potential at the dendron surface was deter-
mined by ζ-potential measurements. As expected, at physio-
logical pH, the ζ-potential of G2-NH2-Bn was positive (about
42 mV) while G2-Ac-Bn was practically neutral (about 5 mV)
(Table 2 and see also Scheme 1 in ESI†). These results are
again consistent with previously reported ζ-potential data of
G3-PAMAM dendrimers containing amino terminal groups.24
First generation dendrons were also measured by dynamic
light scattering (data not shown) but their small size (around
1 nm) precluded obtaining an accurate value. Although an
aggregation behavior for G1-NH2-Bn would be expected –
perhaps to a lesser extent than G2-NH2-Bn – it was not
observed under our measurement conditions, indicating that
the aggregation does not take place in systems with few amino
terminal groups.
Cytotoxicity
Cytotoxicity was tested by MTT assay using HT-29 and
SK-BR-3 human tumor cell lines. Cell lines were incubated for
72 h with the different dendrons, and viability was then
measured. SK-BR-3 cell viability was not affected by any of the
compounds tested, even at high dendron doses (100 μM)
(Fig. 2). In the HT-29 cell line, only the highest concentration
of G2-NH2-Bn (100 μM) displayed significant toxicity, but
viability was not reduced at lower doses. On the basis
of these results, and in agreement with the reported
literature,18,19,21,29–32 our data confirm that in vivo toxicity
increases with the number of free amino-terminal groups on
the dendron surface.
Microscopic evaluation of the cells after exposure to the
dendrons corroborated the viability assays. No changes were
observed regarding the morphology of SK-BR-3 cells for any of
the dendrons tested, compared with control cells (Fig. 3).
However, slight morphological differences were observed when
HT-29 cells were treated with G2-NH2-Bn, compared with the
rest of the samples (Fig. 3d).
Stability in serum
Given the potential biomedical applications of these dendritic
platforms, we studied their stability in human serum, cytotoxi-
city and hemocompatibility, in order to evaluate their bio-
compatibility, in particular in the case of intravenous delivery.
Chemical stability in serum was tested for a period of 120 h
(Fig. 1). Comparative results showed degradation of G1-Ac-Bn
and G1-NH2-Bn already after 24 h of incubation; with 85% of
products remaining intact after 120 h. Surprisingly, G2-Ac-Bn
was not degraded after 120 h of incubation. The major stability
of the second generation dendrons could be attributed to a
minor exposure of the amide-dendritic skeleton to the hydro-
lytic serum enzymes. As dendrimer generation increases, the
dendron interior becomes more protected by the surrounding
environment. In addition, increasing the generation would
Fig. 1 Stability of dendrons 7, 8 and 14 (G1-NH2-Bn, G1-Ac-Bn and G2-Ac-
Bn) in human serum at 37 °C. Percentages are referred to the remaining pro-
ducts that are not degraded after the corresponding incubation time.
Fig. 2 Cell viability of HT-29 and SK-BR-3 cell lines treated with dendrons at
72 h. The assay was performed when the cells reached 90% of confluence.
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Fig. 3 Cell morphology observed at 20× using inverse phase microscopy. (a–e) HT-29 cell lines. (f–j) SK-BR-3 cell line. (a) and (f) control cells. (b) and (g) cells incu-
bated with 100 μM of G1-NH2-Bn. (c) and (h) cells incubated with 100 μM of G1-Ac-Bn. (d) and (i) cells incubated with 100 μM of G2-NH2-Bn. (e) and ( j) cells incu-
bated with 100 μM of G2-Ac-Bn. The assay was performed when the cells reached 90% of confluence.
Although no examples of cell viability experiments with
HT-29 or SK-BR3 cells with low generation dendrimers have
been found, some cytotoxicity studies with other cell lines
have been reported.29,33,34 For example, cytotoxicity experi-
Table 3 Percentage of hemolysis after incubation at 37 °C of the dendrons in
whole blood
% Hemolysis
ments using B16F10 melanoma cells were described. Experi- Conc.
ments realized with second generation diaminoethane (DAE)
G1-NH2-Bn
(7)
G1-Ac-Bn
(8)
G2-NH2-Bn
(13)
G2-Ac-Bn
(14)
(μM)
dendrimers displaying also 16 amino terminal groups showed
0.1
1
10
100
1.00 0.56
1.06 0.25
1.38 0.12
1.13 0.00
1.19 0.00 1.44 0.06
1.12 0.00 1.38 0.31
1.44 0.06 1.25 0.06
0.75 0.81 1.19 0.25
1.07 0.37
1.19 0.25
1.18 0.19
1.13 0.00
50% of viability when cells were incubated with 60 μM of the
dendrimer.29 Another study performed with the same cell line
resulted in a reduction of viability to 40% when cells were
incubated with 20 μM of second generation polyamidoamine
(PAMAM) dendrimers containing 16 amino terminal groups.33
Cytotoxicity of lower generations of non-cationic OEG based
dendrimers was also estimated using B16F1 and HaCat cell
lines showing a cell viability around 100% for second gene-
ration dendrimers and reduction of viability below 50% for
B16F1 and 80% for HaCat, respectively, for first generation
dendrimers.34 Regarding these results, our dendrons seem to
be less toxic than other reported dendrimers containing the
same number of cationic groups on their surface; even our
dendrons have higher molecular weight. Indeed, the influence
of this OEG-based scaffold could lead to a reduction of toxicity
compared to other dendrimer platforms.
RBCs in a competitive way.35–37 To evaluate the influence of
plasma proteins in hemolysis, the hemolytic assay was also
performed with RBCs previously washed with PBS to discard
serum proteins. No significant hemolytic activity was observed
for any of the dendrons tested; even avoiding the serum pro-
teins interaction, thereby suggesting that the cationic surface
does not affect the integrity of erythrocytes in this case
(Table 4).
Hemagglutination (erythrocyte aggregation) occurs when
attractive interactions between positively charged molecules
and negatively charged RBCs overcome repulsive forces
between negatively charged cellular surfaces.38 Polycations
in contact with blood can cause hemagglutination,
a
Hemocompatibility
Dendron hemocompatibility was evaluated by studying hemo-
lysis, the morphology of blood cells, complement activation
(C3a), and coagulation activation, through the extrinsic and
intrinsic pathways.
We performed a hemolytic test by incubating whole blood
with dendron samples for 15 min. Following ASTM (Standard
Practice for Assessment of Haemolytic Properties of Materials),
all dendrons were non-hemolytic (hemolysis < 2%) within the
concentrations tested (Table 3). The interaction between poly-
cations and serum proteins can neutralize the positive charges
of polycations and prevent the interaction between them and
Table 4 Percentage of hemolysis after incubation at 37 °C of the dendrons in
blood. The blood was washed with PBS before the incubation
% Hemolysis
Conc.
(μM)
G1-NH2-Bn
(7)
G1-Ac-Bn
(8)
G2-NH2-Bn
(13)
G2-Ac-Bn
(14)
0.1
1
10
100
0.00 0.04
0.02 0.05
0.00 0.03
0.00 0.07
0.00 0.06 0.15 0.07
0.01 0.02 0.07 0.04
0.01 0.07 0.00 0.07
0.05 0.05 0.3 0.06
0.08 0.03
0.23 0.05
0.07 0.08
0.01 0.05
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phenomenon that can induce severe circulatory disorders and
even lethal toxicity.
Therefore, blood morphological changes are a key para-
meter that should be considered.39 Moreover, foreign macro-
molecules can activate white blood cells and platelets and
induce their aggregation, leading to changes in the number
and size distribution of blood cells. Most cationic dendrimers
showed haemotoxic activity because of their positively charged
surface.40 Recent studies have confirmed that large cationic
PAMAM dendrimers (G4, G5, G6 and G7) induce platelet aggre-
gation by disturbing the integrity of cell membranes24 and
altering their morphology.41 Micrographs of blood smear (see
Fig. 2 in ESI†) confirmed the results obtained in hemolysis
tests, since neither morphological changes in cells nor hemag-
glutination were observed. In addition, the count and size dis-
tribution of red blood cells, platelets and white blood cells
Fig. 5 Analysis of the coagulation cascade after incubation with dendrons for
15 min either through (a) an intrinsic pathway (APTT assay), or (b) an extrinsic
pathway (PT assay). Control: blood incubated but not exposed to dendrons.
APTT: activated partial thromboplastin time; PT: prothrombin time.
The effect on hemostasis control was determined by coagu-
were not altered after incubation with dendrons (see Fig. 3 in lation assays, both through the extrinsic pathway (PT assay)
and the intrinsic pathway (APTT assay). Clot formation was
determined after blood incubation with dendrons. The extrin-
ESI†).
This observation supports the notion that our first and
second generation dendrons do not interact significantly with sic coagulation pathway was not affected by any of the den-
drons, while the intrinsic pathway was significantly inhibited
blood cell elements, even displaying amino terminal groups.
at the highest concentrations of G2-NH2-Bn (100 and 10 μM)
Haemoreactivity differences between our dendrons and high
generation cationic PAMAM dendrimers can be associated to (Fig. 5). This inhibition could result from an interaction
between the positively charged G2-NH2-Bn and proteinic
an increasing charge-density effect associated to higher den-
factors associated with the intrinsic coagulation pathway
drimer generations.
The interaction with blood cell elements of both dendri- (several of them with an acidic pI), thus leading to a blocking
or denaturation of these factors.39,43 The lack of reactivity of
mers type with similar or equivalent charge-density should be
the more neutral G2-Ac-Bn supports this assumption.
studied to establish a clear comparison between them.
The complement system is a mechanism of immune
response to eliminate certain pathogens from the body. Acti-
vation of the complement system by systemically administered
drugs can be responsible for hypersensitivity reactions and
anaphylaxis.42
Conclusions
By means of an original chemical strategy, we have successfully
synthesized multifunctional and monodisperse dendritic
Moreover, complement activation mediated by nanoparti-
cles can result in rapid removal of these particles from sys-
temic circulation by mononuclear cells via receptor-mediated
phagocytosis of complement.42 No significant activation of the
complement system was detected in the range of dendron con-
centrations tested compared with untreated blood, even for
the second generation dendron containing amino terminal
groups at high concentrations (100 μM) (Fig. 4).
structures with
a satisfactory yield and purity. Using
triethylene glycol units as dendritic branches, we developed an
oligoethylene glycol-based scaffold, thus avoiding the polydis-
persity problems that arise from the use of large PEG poly-
mers. Consistent with previous studies,18–21,24,29–33,40,41,44–46
the cytotoxicity and hemocompatibility of our compounds
strongly depended on the groups on the dendron surface as
well as on the dendron generation, the second generation cat-
ionic dendron being the most toxic and the least hemocompa-
tible. However, all the first generation dendrons and the
second generation ones with acetylamide surface groups did
not present cytotoxicity or hemoreactivity. Based on our pre-
screening tests, the biocompatibility of our dendritic platforms
highlights them as very interesting candidates for future bio-
medical applications, such as drug delivery systems, diagnosis,
bioassays, and imaging agents.
Experimental section
Materials
Fig. 4 Activation of the complement system after incubation of blood at 37 °C
with the dendrons for 15 min. Complement activation is expressed as a % of
C3a concentration, taking control 2 as 100%. Control +: blood incubated with
Zymosan. Control 1: blood not exposed to dendrons and not incubated. Control
2: blood incubated but not exposed to dendrons.
All solvents, coupling reagents and other reagents were
purchased from commercial suppliers at the highest purity
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available and used without further purification. N-Bromosucci- with CH2Cl2 (50 mL) and all the organic phases were com-
nimide (NBS, from Sigma Aldrich) was purified by crystalliza- bined and washed with brine (100 mL), dried with Na2SO4 and
tion following Purification of Laboratory Chemicals (Armarego, evaporated. The resulting crude product was purified on basic
W. and Chai, C.; Elsevier; 2003). tert-Butyl bromoacetate and alumina (5→30% ethyl acetate in hexane), affording 3.16 g of
ethanolamine were purchased from Sigma Aldrich and 1-(tert- dendron core 3 (4.46 mmol, yield: 58%). 1H NMR (400 MHz,
butyloxycarbonyl-amino)-4,7,10-trioxa-13-tridecanamine (Boc- CDCl3, 25 °C): δ = 1.44 (s, 36 H), 2.82 (s, 8 H), 3.42 (s, 8 H),
TOTA) was purchased from Iris Biotech. McCoy’s 5A medium 3.59 (s, 2 H), 5.12 (s, 2 H), and 7.35 (m, 5 H). 13C NMR
was purchased from Invitrogen and human serum (from (100 MHz, CDCl3, 25 °C): δ = 28.13, 52.25, 52.75, 54.87, 56.02,
human male AB plasma) from Sigma Aldrich. Trypsin-EDTA 65.92, 80.79, 128.17, 128.23, 128.47, 135.87, and 170.60.
0.25% and penicillin/streptomycin were purchased from Bio- Analytical HPLC: 50→100% CH3CN in water over 8 min, tR
=
logical Industries and fetal bovine serum from Lab Clinics. 6.69 min (purity 92% determined at λ = 210 nm). Calcd
MTT, Drabkin’s reagent (cyanmethemoglobin) and bovine mass for C37H61O10N3: 707.4; Mass found by HPLC-MS: 708.4
hemoglobin were purchased from Sigma-Aldrich. The human (M + 1), 354.9 (M + 2)/2.
C3a ELISA Kit for quantification of Human C3a-des-Arg was
from Becton Dickinson (Erembodegem, Belgium). Throm- methyl)-1,4,7-triazaheptane (4). Compound
Synthesis of 4-benzyloxycarbonylmethyl-1,1,7,7-tetra(carboxy-
(2.16 g,
3
borel® S (human thromboplastin, containing calcium for pro- 3.05 mmol, 1 eq.) was placed in a round-bottomed flask and
thrombin time) was acquired from Dade Behring Siemens dissolved in 5 mL of dioxane. 4.0 M HCl in dioxane (20 mL,
(1070 Bruxelles, Belgium). STA®-C.K. Prest® 2 (kaolin, for 80.0 mmol, 26 eq.) was added to the mixture, and the resulting
determination of the activated partial thromboplastin time) solution was stirred overnight at rt. HCl–dioxane was then
was purchased from Roche Diagnostic Belgium (Vilvoorde, removed by evaporation. The resulting crude product was co-
Belgium). Phosphate buffer (PBS) 100 mM was prepared by evaporated twice with dioxane, affording compound 4 quanti-
dissolving one tablet of phosphate salts (Sigma) in 100 mL of tatively as a white solid, which corresponds to the hydrochlo-
Milli-Q water.
ride salt of the tertiary amines (1.99 g). This crude product was
Synthesis of 4-benzyloxycarbonylmethyl-1,1,7,7-tetra(tert- used in the next step without further purification. 1H NMR
butyloxycarbonylmethyl)-1,4,7-triazaheptane (3). KHCO3 was (400 MHz, D2O, 25 °C): δ = 3.13 (t, 3J(H,H) = 4.97 Hz, 4 H), 3.49
3
added (6.18 g, 61.8 mmol, 2.5 eq.) to a solution of tert-butyl (t, J(H,H) = 5.90 Hz, 4 H), 3.69 (s, 2 H), 3.74 (s, 8 H), 5.23 (s,
bromoacetate (8.05 mL, 54.5 mmol, 2.2 eq.) in 50 mL of anhy- 2 H), and 7.45 (m, 5 H). 13C NMR (100 MHz, D2O, 25 °C): δ =
drous DMF. The suspension was cooled at 0 °C, ethanolamine 49.57, 53.41, 54.22, 55.66, 66.72, 128.88, 129.06, 135.35,
(1.48 mL, 24.5 mmol, 1 eq.) was added via a syringe over a 169.29, and 172.27. Analytical HPLC: 5→100% CH3CN in water
5 min period, and the solution was stirred at 0 °C for 30 min. over 8 min, tR = 3.49 min (purity 91% determined at λ =
The reaction mixture was then allowed to warm to room temp- 210 nm). Calcd mass for C21H30O10N3: 484.1931; Calcd mass
erature and stirred for 22 h. After this time, most of the DMF for C21H29O10N3Na: 506.1750; Mass found by MALDI-TOF:
was evaporated under vacuum and 50 mL of saturated 484.2; Mass found by HPLC-MS: 484.2 (M + 1); Mass found by
NaHCO3 was added. The resulting mixture was extracted with HRMS: 484.1920, 506.1746 (M + Na).
diethyl ether (3 × 50 mL), and the combined organic phases
Synthesis of first generation dendron (G1-Boc-Bn, 5). Solid
were washed with 50 mL of brine, dried with Na2SO4, and PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexa-
evaporated to afford the crude product N,N-bis[(tert-butyloxy- fluorophosphate, 7.51 g, 14.4 mmol, 4.8 eq.) and Boc-TOTA
carbonyl)methyl]-2-hydroxyethylamine (1) as an oil (7.41 g). (1-(tert-butyloxycarbonyl-amino)-4,7,10-trioxa-13-tridecanamine,
This crude product was dissolved in CH2Cl2 and solid triphe- 6.62 g, 14.4 mmol, 4.8 eq.) dissolved in 10 mL of CH2Cl2 were
nylphosphine (8.66 g, 33.0 mmol, 1.3 eq.) was added to the added to a solution of compound 4 (2.0 g, 3.1 mmol, 1 eq.) in
solution, which was cooled at 0 °C. After that, solid NBS 200 mL of anhydrous CH2Cl2–DMF (7 : 3, v : v). The pH was
(5.87 g, 33.0 mmol, 1.3 eq.) was added over 5 min and the adjusted to 8 with DIEA (N,N-diisopropylethylamine) and the
mixture was stirred for 2 h at 0 °C. After this time, the solvent reaction mixture was stirred for 1.5 h at rt. After this time, the
was evaporated and the semisolid residue obtained was tritu- solvents were evaporated to dryness. The resulting crude
rated with diethyl ether. The solid was filtered, washed with product was dissolved in 100 mL of CH2Cl2 and washed with
diethylether and discarded, and the ether phase was evapor- 5% NaHCO3 (3 × 100 mL). The organic phase was dried with
ated to afford an oily residue, which was purified by flash Na2SO4 and evaporated. The crude product was transferred to
chromatography on silica (5→30% diethyl ether in hexane), a 50 mL tube, dissolved in CH2Cl2 (5 mL), and hexane (45 mL)
yielding 6.18 g (17.5 mmol, yield: 71%) of 2-[bis-(tert-butyloxy- was added. The mixture was stirred vigorously and centrifuged.
carbonylmethyl)amino]ethyl bromide (2). 50 mL of phosphate The supernatant was discarded and the remaining orange oily
buffer (2 M, pH 8) was added to a mixture of the tosylate salt precipitate was purified by reverse phase chromatography (C18
of glycine benzyl ester (2.60 g, 7.7 mmol, 1 eq.) and bromoderi- column, 50→80% CH3CN in aqueous 20 mM NH4HCO3 solu-
vative 2 (6.14 g, 17.0 mmol, 2.2 eq.) in 50 mL of CH3CN. The tion), affording 3.62 g (2.14 mmol, yield: 72%) of the first gene-
resulting mixture was stirred vigorously for 24 h at rt. Then ration dendron (5). 1H NMR (400 MHz, CDCl3, 25 °C): δ = 1. 43
CH3CN was evaporated and the product was extracted with (s, 36 H), 1.76 (m, 16 H), 2.68 (m, 8 H), 3.18 (m, 16 H), 3.32 (m,
CH2Cl2 (100 mL). The phosphate buffer phase was re-extracted 8 H), 3.42 (s, 2 H), 3.52 (m, 16 H), 3.58 (m, 16 H), 3.62 (m,
4116 | Org. Biomol. Chem., 2013, 11, 4109–4121
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16 H), 5.13 (s, 2 H), 7.35 (m, 5 H), and 7.66 (bs, NH). 13C NMR
Synthesis of dendron 8 (G1-Ac-Bn). Dendron 7 (60 mg,
(100 MHz, CDCl3, 25 °C): δ = 28.27, 29.30, 29.50, 36.80, 38.22, 0.046 mmol, 1 eq.) was dissolved in CH2Cl2 and mixed with
52.31, 53.30, 55.29, 58.91, 66.29, 69.08, 69.24, 69.96, 70.27, acetic anhydride (26.3 μL, 0.28 mmol, 6 eq.) and DIEA (97 μL,
70.29, 78.63, 128.11, 128.28, 128.45, 135.23, 155.88, 170.52, 0.56 mmol, 12 eq.). After stirring for 1 h, the mixture was evap-
and 171.11. Analytical HPLC: 5→100% CH3CN in water over orated and the crude product was purified by reverse phase
8 min, tR = 7.02 min (purity 99% determined at λ = 210 nm). chromatography (C18 column, 25→60% CH3CN in aqueous
Calcd mass for C81H149O26N11: 1692.0670; Calcd mass for 20 mM NH4HCO3 solution), affording the waxy dendron 8
1
C81H148O26N11Na: 1714.0489; Calcd mass for C81H148O26N11K: (37 mg, 0.025 mmol, yield: 51%). H NMR (500 MHz, CDCl3,
1730.0228; Mass found by MALDI-TOF: 1693.1 (M + 1), 1715.1 25 °C): δ = 1.77 (m, 16 H), 1.95 (s, 12 H), 2.97 (bs, 4 H), 3.10
(M + Na) 1731.1 (M + K); Mass found by HPLC-MS: 1694.2 (bs, 4 H), 3.31 (m, 16 H), 3.38 (s, 2 H), 3.50 (t, 3J(H,H) =
(M + 1), 847.4 (M + 2)/2; Mass found by HRMS: 1692.0672, 6.02 Hz, 8 H), 3.53–3.60 (m, 32 H), 3.60–3.65 (m, 16 H), 5.15 (s,
1714.0483 (M + Na), 1730.0114 (M + K).
Synthesis of dendron (G1-Boc-COOH). Compound
2 H), 7.34 (m, 5 H), and 7.98 (bs, NH). 13C NMR (125 MHz,
CDCl3, 25 °C): δ = 23.35, 28.90, 29.30, 37.04, 37.90, 51.14,
6
5
(200 mg, 0.12 mmol, 1 eq.) was dissolved in 5 mL of MeOH, 51.79, 57.65, 67.04, 69.15, 69.91, 69.98, 70.06, 70.35, 70.41,
and 10% Pd/C (20.2 mg, 10% w/w) was added to this solution. 70.44, 128.44, 128.64, 128.69, 135.00, 169.42, and 170.39.
The resulting suspension was stirred for 1 h under an H2 Analytical HPLC: 5→100% CH3CN in water over 8 min, tR
=
atmosphere at rt. The catalyst was removed by filtration 4.26 min (purity 97% determined at λ = 210 nm). Calcd
through Celite, and the solvent was evaporated to afford mass for C69H125O22N11: 1459.9001; Calcd mass for
dendron 6 as a waxy solid (176 mg, 0.11 mmol, yield: 92%). 1H C69H124O22N11Na: 1481.8820; Mass found by MALDI-TOF:
NMR (400 MHz, CDCl3, 25 °C): δ = 1.43 (s, 36 H), 1.77 (m, 16 1460.9 (M + 1), 1482.9 (M + Na); Mass found by HPLC-MS:
H), 2.75 (bs, 8 H), 3.22 (m, 16 H), 3.32 (m, 10 H), 3.53 (m, 16 1461.0 (M + 1), 731.3 (M + 2)/2; Mass found by HRMS:
H), 3.59 (m, 16 H), 3.63 (m, 16 H), and 7.82 (bs, NH). 13C NMR 1459.8999, 1481.8816 (M + Na).
(100 MHz, CDCl3, 25 °C): δ = 28.40, 29.28, 29.65, 36.99, 38.26,
Synthesis of dendron 9 (G1-Ac-COOH). Dendron 8 (31 mg,
52.88, 53.59, 58.98, 69.30, 69.98, 70.05, 70.35, 70.39, 78.86, 0.021 mmol, 1 eq.) was dissolved in 5 mL of MeOH, and 10%
139.12, 156.11, and 170.68. Analytical HPLC: 5→100% CH3CN Pd/C (3 mg, 10% w/w) was added to the solution. The resulting
in water over 8 min, tR = 6.45 min (purity 97% determined at suspension was stirred for 1 h under an H2 atmosphere at rt.
λ = 210 nm). Calcd mass for C74H143O26N11: 1602.0206; Calcd The catalyst was removed by filtration through Celite, and the
mass for C74H142O26N11Na: 1624.0025; Mass found by MALDI- solvent was evaporated to afford dendron 9 as a waxy solid
1
TOF: 1625.3 (M + Na); Mass found by HPLC-MS: 1604.2 (M + (23 mg, 0.017 mmol, yield: 84%). H NMR (400 MHz, CDCl3,
1), 802.4 (M + 2)/2; Mass found by HRMS: 1602.0211, 25 °C): δ = 1.77 (m, 16 H), 1.97 (s, 12 H), 3.15 (bs, 4 H), 3.31
1624.0020 (M + Na).
Synthesis of dendron
(m, 16 H), 3.37 (bs, 4 H), 3.50 (t, 3J(H,H) = 5.78 Hz, 8 H),
7
(G1-NH2-Bn). Totally protected 3.53–3.60 (m, 48 H), 4.00 (s, 2 H), and 8.00 (bs, NH). 13C NMR
dendron 5 (200 mg, 0.12 mmol, 1 eq.) was dissolved in 2 mL of (100 MHz, CDCl3, 25 °C): δ = 22.94, 28.81, 29.04, 37.06, 37.75,
dioxane and 4.0 M HCl in dioxane solution was added (5 mL, 51.01, 52.33, 53.40, 57.67, 69.01, 69.67, 69.84, 69.88, 70.24,
20 mmol, 166 eq.). The mixture was vigorously stirred for 1 h 70.31, 169.43, and 171.06. Analytical HPLC: 5→100% CH3CN
at rt and the resulting suspension was evaporated to obtain in water over 8 min, tR = 3.61 min (purity 99% determined at
the semi-solid product 7. The product was dissolved in 1 mL λ = 210 nm). Calcd mass for C62H119O22N11: 1369.8531; Calcd
of 20 mM NH4HCO3 and the salts were removed by reverse mass for C62H118O22N11Na: 1391.8351; Mass found by MALDI-
phase chromatography using a PoraPak column and 20% of TOF: 1370.9 (M + 1), 1392.9 (M + Na); Mass found by
CH3CN in water as an eluent, obtaining 134 mg of dendron 7 HPLC-MS: 1370.9 (M + 1), 686.1 (M + 2)/2; Mass found by
1
(0.10 mmol, yield: 87%). H NMR (400 MHz, D2O, 25 °C): δ = HRMS: 1369.8502, 1391.8330 (M + Na).
3
1.80 (m, 8 H), 1.98 (m, 8 H), 3.09 (m, 4 H), 3.13 (t, J(H,H) =
Synthesis of dendron 10 (G1-NH2-COOH). Dendron 6
3
7.07 Hz, 8 H), 3.27 (t, J(H,H) = 6.90 Hz, 8 H), 3.41 (m, 12 H), (55 mg, 0.034 mmol, 1 eq.) was dissolved in 2 mL of dioxane
3.56 (t, J(H,H) = 6.41 Hz, 8 H), 3.69 (m, 40 H), 4.35 (s, 2 H), and 4.0 M HCl in dioxane solution was added (5 mL, 20 mmol,
3
5.37 (s, 2 H), and 7.49 (m, 5 H). 13C NMR (100 MHz, D2O, 588 eq.). The mixture was vigorously stirred for 1 h at rt and
25 °C): δ = 13C NMR (100 MHz, D2O, 25 °C): δ = 26.73, 28.60, the resulting suspension was evaporated, obtaining the semi-
36.57, 37.89, 50.76, 53.30, 53.48, 57.89, 68.49, 68.55, 68.71, solid product 10. The product was dissolved in 1 mL of 20 mM
69.56, 69.62, 69.76, 69.79, 128.49, 129.14, 129.21, 135.05, NH4HCO3 and the salts were removed by reverse phase
168.02, and 172.31. Analytical HPLC: 5→100% CH3CN chromatography using a PoraPak column and 20% of CH3CN
in water over 8 min, tR = 3.44 min (purity 92% determined in water as an eluent, obtaining 34 mg of dendron 10
1
at
λ
=
210 nm). Calcd mass for C61H117O18N11: (0.028 mmol, yield: 83%). H NMR (400 MHz, D2O, 25 °C): δ =
3
1291.8573; Calcd mass for C61H116O18N11Na: 1314.8470; 1.83 (m, 8 H), 1.97 (m, 8 H), 3.13 (t, J(H,H) = 6.64 Hz, 8 H),
Mass found by MALDI-TOF: 1292.9 (M + 1), 1314.9 (M + Na); 3.23 (bs, 4 H), 3.33 (t, J(H,H) = 5.98 Hz, 8 H), 3.45 (bs, 4 H),
3
3
Mass found by HPLC-MS: 1294.0 (M + 1), 647.1 (M + 2)/2, 3.59 (t, J(H,H) = 6.07 Hz, 8 H), 3.65–3.73 (m, 40 H), 3.77 (bs,
431.8 (M + 3)/3, 324.0 (M + 4)/4; Mass found by HRMS: 2 H), and 3.97 (s, 8 H). 13C NMR (100 MHz, D2O, 25 °C): δ =
1291.8570.
29.84, 31.56, 39.94, 40.97, 53.81, 55.83, 56.94, 60.08, 71.59,
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71.61, 72.66, 72.73, 72.87, 72.89, 170.70, and 175.59. Analytical HPLC: 5→100% CH3CN in water over 8 min, tR = 7.21 min
HPLC: 5→100% CH3CN in water over 8 min, tR = 3.04 min (purity 95% at λ = 210 nm). Calcd mass for C350H675O118N55:
(purity 98% determined at λ = 210 nm). Calcd mass for 7537.8509; Calcd mass for C350H674O118N55Na: 7559.8328;
C54H111O18N11: 1201.8109; Calcd mass for C54H110O18N11Na: Calcd mass for C350H673O118N55Na2: 7581.8148; Calcd mass
1223.7928; Mass found by MALDI-TOF: 1202.8 (M + 1), 1224.8 for
C
350H672O118N55Na3: 7597.7498; Calcd mass for
350H671O118N55Na4: 7619.7317; Mass found by HPLC-MS:
2514.9 (M + 3)/3, 1886.6 (M + 4)/4, 1509.6 (M + 5)/5; Mass
(M + Na); Mass found by HPLC-MS: 1202.6 (M + 1), 602.2
(M + 2)/2; Mass found by HRMS: 1201.8104.
C
Synthesis of second generation dendron (G2-Boc-Bn, found by HRMS: 7559.8138 (M + Na), 7581.8126 (M + 2Na),
11). 2,3,4,5,6-Pentafluorophenol (96.6 mg, 0.53 mmol, 1.5 eq.), 7597.7803 (M + 3Na), 7619.7565 (M + 4Na).
EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 100.6 mg,
Synthesis of dendron 13 (G2-NH2-Bn). Compound 11
0.53 mmol, 1.5 eq.), and DMAP (4-dimethylaminopyridine, (45 mg, 0.0059 mmol, 1 eq.) was dissolved in 3 mL of dioxane,
21.4 mg, 0.18 mmol, 0.5 eq.) were added to a solution of and 4.0 M HCl in dioxane solution was added (2 mL, 1356
dendron 6 (600 mg, 0.35 mmol, 1 eq.) in anhydrous CH2Cl2. eq.). The mixture was vigorously stirred for 2 h at rt, and the
The solution was then stirred for 3 h at rt. Dendron 7 resulting suspension was evaporated to obtain the solid
(103.1 mg, 0.08 mmol, 0.22 eq.) dissolved in a minimal product 13. The product was dissolved in 1 mL of 20 mM
amount of CH2Cl2 was then added to the resulting active ester, NH4HCO3, and the salts were removed by reverse phase chrom-
and the pH was adjusted to 8 with DIEA. The mixture was atography using a PoraPak column and 20% of CH3CN
stirred overnight at rt. After that, the solution was washed in water as an eluent, obtaining 28 mg of dendron 13
three times with 5% NaHCO3 (3 × 50 mL), and the organic (0.0047 mmol, yield: 79%). 1H NMR (400 MHz, D2O, 25 °C): δ =
phase was dried with Na2SO4 and evaporated. The crude 1.83 (m, 48 H), 1.98 (m, 32 H), 3.14 (t, 3J(H,H) = 6.92 Hz, 32 H),
3
product was transferred to a 50 mL tube and dissolved in 5 mL 3.22 (bs, 16 H), 3.32 (t, J(H,H) = 6.95 Hz, 54 H), 3.39 (bs, 16
of CH2Cl2, and 45 mL of hexane was added. The mixture was H), 3.57 (m, 80 H), 3.65–3.75 (m, 208 H), 4.03 (s, 2 H), 5.31 (s,
stirred vigorously and centrifuged. The supernatant was dis- 2 H), and 7.49 (m, 5 H). 13C NMR (100 MHz, D2O, 25 °C): δ =
carded, and the remaining orange oily precipitate was purified 26.69, 28.46, 28.57, 36.59, 36.86, 37.84, 51.07, 53.03, 57.87,
by reverse phase chromatography (C18 column, 60→90% 68.46, 68.51, 69.54, 69.59, 69.73, 69.75, 129.11, 166.53 and
CH3CN in aqueous 20 mM NH4HCO3 solution), affording 171.35. Analytical HPLC: 5→100% CH3CN in water over 8 min,
second generation dendron 11 (292.3 mg, 0.038 mmol, yield: tR = 3.27 min (purity 98% determined at λ = 210 nm). Calcd
1
48%). H NMR (400 MHz, CDCl3, 25 °C): δ = 1.43 (s, 144 H), mass for
C
277H553O86N55
:
6027.0589; Calcd mass for
1.77 (m, 80 H), 2.66 (bs, 40 H), 3.18 (m, 80 H), 3.32 (m, 48 H),
C277H552O86N55Na: 6050.0482; Mass found by MALDI-TOF:
3.42 (s, 2 H), 3.53 (m, 80 H), 3.59 (m, 80 H), 3.63 (m, 80 H), 6043.6 (M + NH4+), 6060.2 (M + Na); Mass found by HPLC-MS:
5.13 (s, 2 H), 7.35 (m, 5 H), and 7.79 (bs, NH). 13C NMR 1509.06 (M + 4)/4, 603.98 (M + 10)/10, 549.22 (M + 11)/11,
(100 MHz, CDCl3, 25 °C): δ = 28.39, 29.33, 29.47, 29.54, 29.64, 503.50 (M + 12)/12; Mass found by HRMS: 6027.0877.
36.76, 38.29, 53.08, 59.07, 66.42, 69.03, 69.10, 69.17, 69.32,
Synthesis of dendron 14 (G2-Ac-Bn). Dendron 13 (60 mg,
70.02, 70.05, 70.07, 70.33, 70.37, 70.40, 78.81, 156.04, 170.75, 0.01 mmol, 1 eq.) was dissolved in 5 mL of water, and acetic
and 170.85. Analytical HPLC: 5→100% CH3CN in water anhydride (76 μL, 0.8 mmol, 80 eq.) was added. Solid NaHCO3
over 8 min, tR = 7.04 min (purity 97% determined at λ = was added to the solution to achieve pH 6–7. The mixture was
210 nm). Calcd mass for
Calcd mass for C357H680O118N55Na: 7649.8798; Calcd mass added. The pH was again increased to 6–7 with NaHCO3. After
for 357H679O118N55Na2: 7671.8617; Calcd mass for 24 h, 80 eq. more of acetic anhydride was added to the mixture
357H678O118N55Na3: 7692.8358; Mass found by MALDI-TOF: and the pH was adjusted to 6–7. The solution was then stirred
C357H681O118N55: 7627.8978; stirred for 24 h, and 80 eq. more of acetic anhydride were
C
C
7643.7; Mass found by HPLC-MS: 1273.0 (M + 6)/6, 1091.2 for a further 15 h and freeze-dried. The crude product was pur-
(M + 7)/7, 955.0 (M + 8)/8; Mass found by HRMS: 7627.8948, ified by reverse phase chromatography (C18 column, 30→70%
7649.8713 (M
(M + 3Na).
+
Na), 7671.8518 (M
+
2Na), 7692.8318 CH3CN in aqueous 20 mM NH4HCO3 solution), affording the
dendron 14 (32.4 mg, 0.0048 mmol, yield: 48%). 1H NMR
Synthesis of dendron 12 (G2-Boc-COOH). Dendron 11 (400 MHz, D2O, 25 °C): δ = 1.78 (m, 80 H), 1.98 (s, 48 H), 2.67
(57 mg, 0.0075 mmol, 1 eq.) was dissolved in 5 mL of MeOH, (s, 40 H), 3.19 (s, 2 H), 3.26 (m, 128 H), 3.56 (m, 80 H),
and 10% Pd/C (14 mg, 20% w/w) was added. The resulting sus- 3.61–3.71 (m, 160 H), 5.22 (s, 2 H), and 7.43 (m, 5 H). 13C NMR
pension was stirred for 2 h under an H2 atmosphere at rt. The (100 MHz, D2O, 25 °C) δ: 22.0, 28.42, 28.74, 28.87, 36.40, 36.64,
catalyst was removed by filtration through Celite, and the 52.83, 53.17, 57.96, 58.72, 67.05, 68.58, 68.57, 68.70, 69.54,
solvent was evaporated, affording dendron 12 as a waxy solid 69.62, 69.79, 69.80, 128.37, 128.87, 129.05, 135.77, 160.39,
1
(39 mg, 0.0052 mmol, yield: 69%). H NMR (400 MHz, CDCl3, 173.20, 173.26, and 173.96. Analytical HPLC: 5→100% CH3CN
25 °C): δ = 1.43 (s, 144 H), 1.77 (m, 80 H), 2.70 (bs, 40 H), 3.21 in water over 8 min, tR = 3.98 min (purity 88% determined at
(m, 80 H), 3.31 (m, 50 H), 3.53 (m, 80 H), 3.59 (m, 80 H), 3.63 λ = 210 nm). Calcd mass for C309H585O102N55: 6699.2280; Calcd
(m, 80 H), and 7.81 (bs, NH). 13C NMR (100 MHz, CDCl3, mass for
C309H584O102N55Na: 6721.2099; Mass found by
25 °C): δ = 28.43, 29.25, 29.67, 37.06, 38.35, 58.39, 69.17, 69.37, MALDI-TOF: 6718.2 (M + Na); Mass found by HPLC-MS: 2235.4
70.05, 70.10, 70.39, 70.43, 78.86, 156.09, and 170.59. Analytical (M + 3)/3, 1676.9 (M + 4)/4, 1341.6 (M + 5)/5, 1118.0 (M + 6)/6,
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958.5 (M + 7)/7, 838.8 (M + 8)/8; Mass found by HRMS: Cell culture
6699.2392, 6721.2303 (M + Na).
HT-29 and SK-BR-3 cell lines were cultured in McCoy’s 5A
medium supplemented with 10% (v/v) fetal calf serum
and 100 mg mL−1 of penicillin and streptomycin. Cells were
subcultured routinely using 0.02% (w/v) trypsin-EDTA.
Synthesis of dendron 15 (G2-NH2-COOH). Dendron 12
(32 mg, 0.0042 mmol, 1 eq.) was dissolved in 1 mL of dioxane,
and 4 M HCl in dioxane solution was added (1 mL, 0.25 mmol,
60 eq.). The solution was stirred for 2 h and evaporated to
dryness. The resulting crude product was dissolved in water
and freeze-dried. Solid product 15 was obtained quantitatively
as a hydrochloride salt (24.7 mg, 0.0041 mmol, yield: 99%).
1H NMR (400 MHz, D2O, 25 °C): δ = 1.83 (m, 48 H), 1.97 (m, 32
H), 3.13 (t, 3J(H,H) = 6.79 Hz, 32 H), 3.32 (m, 88 H), 3.58 (t,
3J(H,H) = 6.17 Hz, 32 H), 3.70 (m, 208 H), 3.74–3.86 (bs, 48 H),
and 3.89 (s, 2 H). 13C NMR (100 MHz, D2O, 25 °C): δ = 26.62,
28.41, 36.65, 37.75, 51.68, 51.99, 57.21, 68.38, 68.41, 69.46,
69.51, 69.66, 69.68, and 168.91. HPLC: 5→100% CH3CN in
water over 8 min, tR = 3.17 min (purity 85% determined at λ =
210 nm). Calcd mass for C270H547O86N55: 5937.0115; Calcd
Antiproliferative activity via MTT assay
HT-29 and SK-BR-3 cell lines were seeded in standard 96-well
plates at a density of 5 × 103 and 104 cells per well respectively
and grown for 24 h in McCoy’s 5A culture medium. When the
HT-29 and SK-BR-3 cell monolayers reached 90% of conflu-
ence they were treated for 72 h with 100 μM, 10 μM, 1 μM, or
0.1 μM of the different dendron solutions in a fresh culture
medium. A control experiment was also performed by incubat-
ing the cells with PBS instead of dendrons. Viable cell density
was then assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide) assay. Briefly, both treated
and untreated cells were washed thoroughly with PBS and
incubated in MTT solution (0.5 mg mL−1 MTT in PBS, 100 μL
per well of a 96-well plate) for 2 h at 37 °C in a humidified
atmosphere of 5% CO2. Dehydrogenase activity in the cells
reduces MTT, forming a purple formazan salt, which was
eluted from the cells using isopropanol (100 μL per well of a
96-well plate). After dissolution of the formazan salt, the
optical density at 540 nm was measured using a plate reading
spectrophotometer (ELx800 Bio-Tek Instruments, Inc). The
percentage of viability was expressed taking the control experi-
ment as 100% of viability.
mass for
C270H545O86N55Na2: 5981.9832; Mass found by
MALDI-TOF: 5980.0 (M + 2Na); Mass found by HPLC-MS: 743.5
(M + 8)/8, 660.9 (M + 9)/9, 594.9 (M + 10)/10, 540.9 (M + 11)/11,
495.9 (M + 12)/12, 457.9 (M + 13)/13. Mass found by HRMS:
5937.0063.
Dynamic light scattering (DLS) and ζ-potential experiments
For dendron size determination, DLS measurements were per-
formed on a Brookhaven Instruments 200 SM laser light scat-
tering goniometer using an He–Ne 125 mW633 nm laser.
Single scans of 10 min exposure were performed, and particle
sizes were estimated using the CONTIN multiple-pass method
of data analysis at angles of 60°, 90°, and 120°. Measurements
for ζ-potentials were made with a Malvern ZetaSizer Nano ZS
instrument. Dendrons dissolved in PBS at different concen-
trations (10, 5, and 1 mg mL−1) and passed through a 0.2 μm
filter were placed in a specific cuvette, and certain parameters
of the refractive index and the absorption coefficient of the
material and the viscosity of the solvent were introduced into
the software.
Microscopy
The HT-29 and SK-BR-3 cell lines were seeded in 24-well plates
at a density of 104 and 5 × 104 cells per well respectively and
grown for 24 h in McCoy’s 5A culture medium. Cells were incu-
bated with 100 μM of the different dendron solutions for 72 h.
Cell morphology was assessed using a Nikon TE200 inverse
phase microscopy (Japan) equipped with a 40× objective Nikon
LWD 0.52 NA (Japan).
Stability experiments in serum
Blood sample collection
Stability assays in human serum were carried out by incu-
bation of dendrons (dendrons 7, 8 and 13) with serum (diluted
9 : 1 in HBBS buffer) at 37 °C. Compounds were used at a final
concentration of 0.8 mg mL−1 for dendrons 7 and 8 (G1-NH2-
Bn and G1-Ac-Bn) and 1.25 mg mL−1 for dendron 14 (G2-Ac-
Bn). Aliquots of 60 μL were periodically taken at 0 to 120 h and
mixed with 200 μL of CH3CN in order to precipitate the serum
proteins. The mixture was cooled to 4 °C and centrifuged at
13 000 rpm for 15 min at 4 °C. The supernatant was passed
through a 0.45 μm filter, and 20 μL was injected in an analyti-
The studies involving donor blood were approved by the Ethics
Committee of the Medical Faculty of Liège. Human blood was
obtained from the Red Cross Transfusion, University Hospital
of Liège. Blood was collected from healthy donors in 4.5 mL
tubes containing 3.2% sodium citrate as an anticoagulant.
Experiments were done 15 min after collection.
Blood smears for the control of the RBC (red blood cell)
morphology
cal HPLC system using a C18 reverse phase column and Dendrons were dissolved in PBS at a range of concentrations,
CH3CN–water 0.05% of TFA as eluents. The gradient used was and a volume of sample solution was diluted in 9 volumes of
5→100% of CH3CN in water over 8 min. Two blank samples whole blood, to obtain final concentrations of 100, 10, 1, and
were also performed in parallel following the same procedure 0.1 μM of dendron solutions. Samples were incubated for
reported above. In the first blank sample, water was used 15 min at 37 °C under lateral agitation (250 rpm). After blood
instead of human serum, whereas in the second blank the incubation, 5 μL of the blood was withdrawn and spread on a
serum solution did not contain dendrons.
microscopy glass slide. Blood cells were observed with an
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Organic & Biomolecular Chemistry
Olympus Provis microscope at 20× and 50× magnification in the specific activators of coagulation (thromboplastin). Pro-
transmission mode. At least two representative pictures were thrombin time (PT), to evaluate the extrinsic pathway, and acti-
acquired per sample with a digital camera (VisiCam (5 mega- vated partial thromboplastin time (APTT), to evaluate the
pixels), VWR International).
intrinsic pathway, were measured directly with a Dade Behring
Coagulation Timer analyzer (BCT) (Siemens Healthcare Diag-
nostics NV/SA, Belgium). Kaolin reagent was used as a positive
Hemolytic test
Dendron solutions and blood were prepared and incubated as control and PBS as a negative control. Clotting time was
described above. The hemolytic test was performed following measured for each sample, and coagulation capacity was
Standard Practice for Assessment of Haemolytic Properties expressed as a percentage, taking the value of standard human
of Materials (ASTM designation
F 756-00). Briefly, after plasma (Dade Behring/Siemens) as 100%. Two independent
incubation, the samples were centrifuged at 600g for 5 min experiments were performed, and measurements were done in
at rt, and supernatants were collected and mixed with the duplicate.
cyanmethemoglobin reagent. The hemoglobin released was
measured at 540 nm in a microplate reader (Anthos HT III,
type 12600, Anthos, Salzburg, AU). A calibration curve was
Acknowledgements
established using bovine hemoglobin as the standard.
Saponine (0.8 mg mL−1) and PBS were used as positive and
negative controls, respectively. Hemolysis was expressed as the
percentage of hemoglobin released to total content, taking the
positive control as 100% of hemolysis. Three independent
experiments (with distinct blood donor samples) were per-
formed, and the tests were done in triplicate. In view to assess
the influence of plasma protein on the hemolytic action of
the dendrons this test was also performed using washed RBCs
(4% RBC suspension) instead of whole blood.
This work was financially supported by Spanish Government
(MICINN: CTQ2008-00177(MR) and CTQ2009-07758(FA)
and EUI2008-00174(FA); MINECO: SAF2011- 30508-C02-01
(MR) and the CIBER-BBN-Instituto de Salud Carlos III:
CB06_01_0074 (FA)) and the Generalitat de Catalunya
(2009SGR 1024).
Notes and references
Count and size distribution of RBCs, platelets and white blood
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