Construction of a well-defined multifunctional dendrimer for theranostics

Article abstract of DOI:10.1021/ol103019z

A dendrimer-based building block for theranostics was designed. The multifunctional dendrimer is polyamide-based and contains nine azide termini, nine amine termini, and fifty-four terminal acid groups. Orthogonal functionalization of the multifunctional

Full text of DOI:10.1021/ol103019z

ORGANIC
LETTERS
2011
Vol. 13, No. 5
976–979
Construction of a Well-Defined
Multifunctional Dendrimer for Theranostics
†
Catia Ornelas, Ryan Pennell, Leonard F. Liebes, and Marcus Weck*
‡
‡
,†
ꢀ
Molecular Design Institute and Department of Chemistry, New York University,
31 Washington Place, New York, New York 10003, United States, and Cancer Institute,
New York University School of Medicine, Tisch Hospital, 550 First Avenue, New York,
New York 10016, United States
marcus.weck@nyu.edu
Received December 13, 2010
ABSTRACT
A dendrimer-based building block for theranostics was designed. The multifunctional dendrimer is polyamide-based and contains nine azide
termini, nine amine termini, and fifty-four terminal acid groups. Orthogonal functionalization of the multifunctional dendrimer with a near-infrared
(NIR) cyanine dye afforded the final dendrimer that shows fluorescence in the NIR region and no toxicity toward T98G human cells. The synthetic
strategy described here might be promising for fabricating the next generation of materials for theranostics.
Dendrimers are perfectly hyperbranched macromole-
cules that possess a high number of active termini that
define their properties and functions.1-5 They have found
applications in material science as sensors,6,7 nanoreactors,8,9
and green catalyst supports.10 The most promising appli-
cations of dendrimers are in biomaterials where they have
emerged as competitive candidates for drug delivery
applications.5,11-14
Traditionally, dendrimers are synthesized from ABx
monomers, resulting in symmetrical structures with B
terminal groups.15 However, such symmetrical dendritic
structures cannot be employed easily in complex applica-
tions such as theranostics and drug delivery. For example,
materials for theranostics need higher structural complex-
ity because multiple functionalities for water solubility,
targeting, imaging, and drug delivery are required.14 Clearly,
the multifunctionalization of dendrimers is crucial for the
construction of dendrimer-based drug delivery systems.
Multifunctionalization of poly(amidoamine) (PAMAM)
dendrimers, the leading dendrimer scaffold in biomater-
ials, is mainly achieved through a random statistical
† Molecular Design Institute and Department of Chemistry, New York
University.
‡ Cancer Institute, New York University School of Medicine.
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r
10.1021/ol103019z
Published on Web 02/03/2011
2011 American Chemical Society

approach,14,16 i.e. bysuccessivepartial functionalization of
the amine termini, resulting in a stochastic distribution of
products without any degree of control over individual
functional group placement.17 Indeed, Baker and co-work-
ers have recently showed that the random functionaliza-
tion of dendrimers leads to a complex mixture of products
that is far from following a Gaussian distribution and that
in most cases only a small percentage of the dendrimers
have the desired multifunctionality.17 While highly desir-
able, synthetic strategies to selectively multifunctionalize
dendrimers are limited.15,18-26
Our group has reported the synthesis of trifunctional
poly(amide)-based dendrimers and has demonstrated their
orthogonal functionalization by using either the copper-
catalyzed azide alkyne 1,3-dipolar cycloaddition (CuAAC)
or the strain promoted azide alkyne cycloaddition
(SPAAC) combined with Schiff base coupling.27-30 In this
contribution, we describe the basic building blocks for a
dendrimer-based multifunctionalization strategy that has
the potential to be utilized in theranostics and other bio-
materials applications. We report a synthetic strategy
toward multifunctional dendrimers that consists of the
combination of two different dendrons into a Janus-
like18,31-35 dendrimer, followed by the reaction with an
AB6C1 dendron resulting in a well-defined hyperbranched
multifunctional dendrimer.
Among the most promising NIR dyes36,37 are cyanine-
based dyes.38-44 Recently, Gao and co-workers have
shown that when the chlorine atom of the cyanine dyes is
replaced by an amine group, the resulting amino-cyanine
dyes have high quantum yields and large Stokes shifts
(>140 nm).45 Our group has demonstrated that the SRN
1
reaction between the chloride-containing cyanine dye and
functionalized amines is fully orthogonal to azides and
acid groups.46 To increase water solubility, we select here a
cyanine dye that contains two sulfonate groups at the
end of the propyl chains on the nitrogen of the indolenine
ring. Furthermore, the negative charges on the sulfonate
groups should enhance the biocompatibility of the dye-
dendrimer conjugate since it has been demonstrated that
negatively charged dendrimers are less toxic than their
positively charged counterparts.13,14
The dendrons used in our strategy are poly(amide)-
based to ensure biocompatibility and follow the 1f
(2þ1) connectivity pioneered by Newkome.20,26 All peptide
coupling reactions described here were achieved success-
fullyusingHATU asthecouplingagent,andalldendrimers
were purified easily by dialysis using a Spectra-Por MWCO
(molecular weight cut off) 2000 dialysis membrane.
The basic synthesis toward our target dendrimer 7 is
shown in Scheme 1. The dendrimer design is based on
dendron 1 containing nine protected acid groups and one
acidgroupatthe focal pointanddendron2 containingnine
azide groups and one amine at the focal point. Both
dendrons were synthesized according to our previous
report29 (see Supporting Information (SI) for experimental
details). The coupling reaction between 1 and 2 afforded
dendrimer 3 in 93% yield after purification by dialysis
against methanol. The quantitative coupling was proven
To demonstrate our strategy, we report the synthesis of a
dendrimer, bearing fifty-four acids, nine amines, and nine
azide groups, that has the potential to be multifunctiona-
lized in three steps (Scheme 1). As a proof-of-principle, we
demonstrate the orthogonal monofunctionalization by
selectively reacting the nine amine termini with a near-
infrared (NIR) fluorescent dye.
1
by H NMR spectroscopy with the shifting of the CH2-
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COOH protons on 1 from 2.63 to 2.55 ppm as well as the
triplet corresponding to the CH2CH2NH2 protons of 2
from 3.77 to 3.60 ppm upon formation of 3 (the CH2NH2
protons cannot be used to monitor the reaction because
they overlap with the CH2N3 protons). Dendrimer 3 pre-
sents its molecular ion peak (MþH)þ at 3340.5 m/z (calcd
for C153H264N45O38: 3340.0 m/z) in the MALDI-TOF mass
spectrum corroborating the expected structure. The acid
termini on 3 were deprotected at room temperature using
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Org. Lett., Vol. 13, No. 5, 2011
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Scheme 1. Synthesis of Multifunctional Dendrimer 7: Combination of Two Different Dendrons into a “Janus”-Type Dendrimer
Followed by the Reaction with an AB6C1 Dendron
formic acid, resulting in the “Janus”-type dendrimer 4 that
possesses nine acid groups and nine azide groups. Complete
using formic acid. The quantitative deprotection reaction
1
was proven by H and 13C NMR spectroscopies via the
1
deprotection was proven via H NMR spectroscopy by
disappearance of the tBu protons at 1.44 ppm as well as the
disappearance of the COOtBu carbon signals at 174.6 ppm
(CH2COOtBu, protected acids) and at 158.7 ppm (CH2-
NHCOOtBu, protected amines). The resulting dendrimer
7 is fully water-soluble and has on its periphery fifty-four
acid groups, nine amine groups, and nine azide groups,
suitable for further orthogonal functionalization.
Orthogonal functionalization of 7 was carried out by
reacting the nine amine groups of 7 through an SRN1
reaction with the chlorine-cyanine NIR-dye 8 (for synthe-
sis of NIR-dye 8, see SI) (Scheme 2).45-47
The reaction was carried out in DMF at 80 °C for 48 h in
which the initial deep green solution slowly turned to deep
blue suggesting the substitution of the chloride atoms by
the amine groups. The solution was then evaporated to
dryness, and the product was purified by dialysis against
methanol using a Spectra-Por MWCO 2000 dialysis mem-
brane. Dendrimer 9 was obtained as a deep blue waxy
product in 87% yield (Figure 1).
the disappearance of the tBu protons at 1.44 ppm and the
appearance of COOH protons at 8.41 ppm. The structure
of 4wasfurtherconfirmedbymassspectrometry(MALDI-
TOF) showing its molecular ion peak (MþH)þ at
2835.5 m/z (calcd for C117H192N45O38: 2835.4 m/z).
Multifunctional AB6C1 dendron 5, where A = NH2; B =
COOtBu; C = NHBoc (for the synthesis of 5, see SI), was
then coupled to the nine acid groups on 4 yielding dendrimer
6 that contains fifty-four protected acids, nine Boc-protected
amines, and nine azide groups. Again, 6 was purified easily
by dialysis against methanol using a Spectra-Por MWCO
2000 dialysis membrane and isolated as a colorless waxy
1
product in 92% yield. The H NMR spectrum proves
the structure of 6, showing all the expected peaks that
include the PEG linker between 4.0 and 3.59 ppm, CH2N3
at 3.37 ppm, and CH2NHBoc at 3.04 ppm (for NMR
spectra and data, see SI). The acid and the amine groups on
6 were simultaneously deprotected at room temperature
Dendrimer 9 was characterized by NMR spectro-
scopy, mass spectrometry, and UV-vis and fluores-
cence spectrocopies. The 2D COSY NMR spectra of 9
(47) Flanagan, J. H.; Khan, S. H.; Menchen, S.; Soper, S. A.;
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978
Org. Lett., Vol. 13, No. 5, 2011

Scheme 2. Functionalization of Chlorine-Cyanine Dye 8 with
Amine Derivatives
Figure 2. Absorption spectra of dendrimer 9 in five different
solvents.
hydrophilicity of the dendrimer’s periphery and is an
interesting feature taking into account the potential for
biological applications. Dendrimer 9 is fluorescent in the
NIR-region, its emission spectra showing one emission
band at 762 nm with comparable quantum yield (0.062)
to the FDA approved dye, indocyanine green (0.078).
The large Stokes shift (∼140 nm), the emission on the
NIR-region, and the low aggregation in aqueous media are
optimal properties for in vivo optical imaging.
Since our main motivation is the development of a basic
dendrimer building block that can be orthogonally multi-
functionalized for applications in theranostics, it is essential to
assess the cytotoxicity of 9. The toxicity of 9 was evaluated for
T98G human cells that were grown in the presence of different
concentrations of 9. The assay demonstrates that, even after
an incubation time of 72 h, 9is not significantly toxic for T98G
human cells at concentrations up to 50 μM. (see SI p S-35).
In summary, we have designed and synthesized a new
dendrimer-based building block for biomaterials applica-
tions. The well-defined dendrimer is water-soluble and can
be multifunctionalized in a highly controllable and ortho-
gonal fashion. The multifunctional dendrimer is poly-
amide-based and contains nine azide termini, nine amine
termini, and fifty-four terminal acid groups. Orthogo-
nal functionalization of the multifunctional dendrimer
was achieved by the reaction of the amine termini with a
NIR-cyanine dye. The final dye-containing dendrimer
shows fluorescence in the NIR region with a large Stokes
shift and relatively high quantum yields and shows no
toxicity toward T98G human cells. The results obtained
here are promising for the application of these materials in
theranostics. Current efforts in our group are focused on
the attachment of targeting moieties to the azide termini
and cancer drugs to the acid groups on these dendrimers.
Figure 1. Dendrimer 9 containing nine NIR-dyes, nine azide
termini, and fifty-four acid termini.
show the coupling between the CH2NH-dye protons at
2.95 ppm and the cyanine protons at 8.04 ppm sugesting
the proximity of both moieties. The CH2N3 signal at 3.35
ppm and the CH2COOH signal at 2.29 ppm do not show
any coupling with the protons from the NIR-dye demon-
strating that 8 did not react with either the acid or the azide
groups on 9, confirming the orthogonality of the SRN1
reaction and the presence of these functional groups as
dendrimer termini. The MALDI-TOF mass spectra of 9
show its molecular ion peak M2þ at 8037.6 m/z (M2þ
calculated for C765H1091N108Na9O218S18: 8032.6 m/z).
The optical properties of 9 were investigated by UV-
vis spectroscopy in different solvents: phosphate buffer
aqueous solution (PBS), dimethylsulfoxide (DMSO), di-
methylformamide (DMF), ethanol, and methanol (Figure 2).
All UV-vis spectra are characterized by a broad band
around 619-641 nm showing a large blue shift in com-
parison to that of the parent dye 8, which is typical for
the amino-cyanine dyes.45,46 The absorption spectra of 9
also present a narrow absorption band at around 770-
793 nm, suggesting the presence of J-aggregates.38,48 The
intensity of this band is much less significant in PBS than in
other solvents, suggesting that the aggregation phenomena
are lower in aqueous media. This is probably due to the
Acknowledgment. We thank New York University for
financial support of this research.
Supporting Information Available. Detailed synthetic
1
procedures, characterization data, H NMR and mass
spectra for all compounds; emission spectra and toxicity
evaluation of dendrimer 9. This material is available free
of charge via the Internet at http://pubs.acs.org.
(48) Jelly, E. E. Nature 1936, 138, 1009.
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