Synthesis of Heterofunctional Polyester Dendrimers with Internal and External Functionalities as Versatile Multipurpose Platforms

Article abstract of DOI:10.1021/acs.biomac.0c01068

Heterofunctional dendrimers with internal and external representations of functionalities are considered as the ultimate dendritic frameworks. This is reflected by their unprecedented scaffolding, such as precise control over the structure, molecular weig

Full text of DOI:10.1021/acs.biomac.0c01068

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Article
Synthesis of heterofunctional polyester dendrimers with internal
and external functionalities as versatile multipurpose platforms
Sandra García-Gallego, Patrik Stenström, Pablo Mesa-Antunez, Yuning Zhang, and Michael Malkoch
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.0c01068 • Publication Date (Web): 27 Aug 2020
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Synthesis of heterofunctional polyester dendrimers
with internal and external functionalities as versatile
multipurpose platforms
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Sandra García-Gallego, a†§ Patrik Stenström, a§ Pablo Mesa-Antunez, a Yuning Zhang, a
and Michael Malkoch *a
a Department of Fiber and Polymer Technology, School of Chemical Science and
Engineering, KTH Royal Institute of Technology, Teknikringen 56-58, SE-100 44,
Stockholm, Sweden.
E-mail: malkoch@kth.se
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ABSTRACT: Heterofunctional dendrimers with internal and external representation of
functionalities are considered as the ultimate dendritic frameworks. This is reflected by
their unprecedented scaffolding, such as precise control over structure, molecular weight,
number and location of different cargo across the whole dendritic skeleton. Consequently,
these dendrimers with multipurpose character are the pinnacle of precision polymers and
thereof are highly attractive by the scientific community as they can find use in a great
number of cutting-edge applications, especially as discrete unimolecular carriers for
therapeutic exploitation. Unfortunately, most established dendrimer families display
external functionalities but lack internal scaffolding ability, which lead to inherent limitation
for their full potential use as precision carriers. Consequently, we here embark on a novel
synthetic strategy facilitating the introduction of internal functionalization of established
dendrimers. As a Proof-of-Concept, a new class of internally and externally functionalized
multipurpose dendrimers based on the established 2,2-bis(methylol)propionic acid (bis-
MPA) were successfully accomplished by the elegant and simple design of AB2C
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monomers, amalgamated from two traditional AB2 monomers. By utilizing Fluoride-
Promoted Esterification (FPE), straightforward layer-by-layer divergent growth up to the
fourth generation was successful in less than one day reaction time, with a molecular
weight of 15 kDa and displaying 93 reactive groups divided by 45 internal and 48 external
functionalities. The feasibility of post-functionalization through click reactions is
demonstrated, where the fast and effective attachment of drugs, dyes and PEG chains is
achieved, as well as crosslinking into multifunctional hydrogels. The simplicity and
versatility of the presented strategy can easily be transferred to generate a myriad of
functional materials such as polymers, surfaces, nanoparticles or biomolecules.
KEYWORDS. click chemistry, dendrimers, esterification, hydrogels, nanocarriers.
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1. Introduction
The pursuit of multipurpose scaffolds has long been a topic of research in nanomedicine,1-
2 where the ability to transport several cargos such as therapeutic drugs, targeting agents
and fluorescent tags, is highly desirable for cancer therapy, cardiovascular disease and
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infections. For example, actively targeted nanocarriers increase the drug efficiency and
minimize the side-effects;3 or theranostics, which combine therapeutic and diagnostic
actions in a single entity, emerge as a unique tool for predictive, preventive and
personalized medicine.4 Overall, multipurpose scaffolds increase the therapeutic efficacy
by:2 (i) enhancing the water-solubility and biodistribution; (ii) extending the blood
circulation; (iii) improving stability; (iv) providing response to stimuli; (v) enabling an active
or passive targeting; and (vi) enabling an in situ monitoring.
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Despite ongoing nanomedicine research based on nano-based drug delivery systems
(DDS), the journey to clinical approval is arduous.5 Challenging obstacles such as the
ease of preparation, safety and cost of the materials, scalability, batch-to-batch
reproducibility, as well as the stability, biocompatibility and biodegradation of the final
product need to be considered.6-8 Since Doxil®, the first FDA-approved nano-drug,
several formulations for passive and active targeting can be found in the market.9 These
DDS produce biologically reproducible activities, but lack a precise control over the type,
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number and location of the biologically-relevant cargo within the carrier. This would
provide critical information about the therapeutic response of such materials and enable
a personalized treatment.10
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To date, dendrimers are the only polymeric scaffolds capable of providing these features
due to their monodispersity and multivalency,2, 11 besides the influence of the branched
architecture in drug delivery.12-13 Successful examples, at pre-clinical or even clinical
stage, have been reported for dendrimers comprising polylysine (e.g. VivaGel®),14
poly(amidoamine) (PAMAM),15 phosphorous16 and bis-MPA based polyester scaffolds,17
among others. To expand their usefulness, heterofunctional dendrimers (HFDs) were
developed, which contain at least two distinctive functionalities typically attached to the
dendritic surface in a statistical, alternating or block manner, but most often having
dormant interiors.6, 18-19
It is inevitable that heterofunctional dendrimers full potential can only be capitalized on by
displaying functionalities throughout the whole macromolecular skeleton e.g. to increase
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carrying capacity and optimize its potential.2, 11 The complex and tedious synthesis of
dendrimers, however, has led to an extremely low number of reports proposing dendritic
scaffolds with interior and exterior functionalities.6, 20, 21 A viable method to render the
dendritic interior more accessible is to incorporate highly selective and reactive pendant
functional groups during the dendritic growth. This was accomplished by Malkoch et al.,
who converted AB3 monomer tris(hydroxymethyl)aminomethane (Trizma®) to its AB2C
derivative, where the A and B groups were used for dendrimer growth and the C groups
were precisely functionalized through the copper-catalyzed azide-alkyne cycloaddition
(CuAAC) click reaction.22 Unfortunately, Trizma-based dendrimers were found
susceptible toward fast hydrolytical degradation, limiting further exploitation.
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Consequently, novel synthetic strategies that can expand the scaffolding window of
already established dendrimers and in parallel maintain the intrinsic features of the main
building block is logical progression in dendrimer chemistry.16-17 The new multipurpose
HFDs would resemble most of their parent´s properties – e.g. biocompatibility,
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degradability, solubility – while providing increased accessibility and carrying capability of
the typically dormant interior. To accomplish this, traditional building blocks must be
synthetically revisited and re-programmed to include orthogonal chemoselective groups
that are dormant during the dendrimer growth.
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Inspired by the “AB2C approach”, we here unlock a facile strategy to internally and
externally functionalized HFDs synthesized using a new generation AB2C monomers by
combining two identical monomers. As proof-of-concept, the bis-MPA AB2 monomer was
selected – which is simple, versatile and commercially available-,23 and generates
biocompatible and biodegradable dendrimers.17, 23 Nevertheless, the proposed strategy
is a guiding approach which can be translated to other monomer/dendrimer platforms with
other chemistries, where two individual building blocks with orthogonally complementary
functionalities are combined into a single AB2C monomer. Furthermore, the simplicity and
versatility of the AB2C approach could expand the library of high-value precursors with
orthogonal functionalities, including monomers and dendrons as well as derived
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functional materials such as polymers, surfaces, nanoparticles or biomolecules, in a
straightforward way.
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In contrast to the state-of-the-art in internally/externally functionalized HFDs, the current
strategy benefits from the versatility of combining two traditional AB2 building blocks to
generate AB2C monomers, as well as from the advantages of using the Fluoride-
Promoted Esterification (FPE) with imidazolide-activated compounds. FPE chemistry has
earlier been described by Malkoch and coworkers as a powerful synthetic tool to generate
monomers,24 dendrimers24-25 and dendrons26 with high fidelity and functionality. FPE
relies on 1,1'-carbonyldiimidazole (CDI) as an activating agent, where it demonstrates
clear advantages in terms of scalability, widespread utility and green chemistry,27
generating CO2 and imidazole as the only by-products, which can easily be removed
through wash steps; as well as on catalytic Cesium Fluoride (CsF), exhibiting
unprecedented efficacy in the synthesis of complex dendritic polyesters.25 Consequently,
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FPE chemistry was herein explored as a tool in the facile construction of
internally/externally functionalized HFDs.
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2. Experimental section
2.1. Synthetic procedures.
Synthesis and characterization details (1H, 13C-NMR; MALDI-TOF; FTIR; SEC) for the
different monomers, dendrimers and click probes can be found in the Supporting
Information. The most relevant general synthetic procedures are:
Dendrimer synthesis - General esterification procedure. The carboxylic-functionalized
compound is slowly added over a suspension of CDI in EtOAc (1-2 M), while heating the
mixture at 50°C. After 15 min stirring at 50°C, CsF (0.1 or 0.2 eq/OH, for monomers or
dendrimers respectively) and the hydroxyl-functional compound is added and the reaction
proceeds at 50°C. The reaction is monitored by NMR and MALDI-TOF. Upon completion
the mixture is allowed to cool to room temperature, and the excess of imidazolide-
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activated acid is quenched by stirring with water. The mixture is then diluted with EtOAc
and washed repeatedly with aqueous solutions of 10% NaHCO3, 10% NaHSO4 and brine
before being dried with MgSO4, filtered and evaporated. Dendrimers are isolated in 70-
98% yields.
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Dendrimer synthesis - General deprotection procedure. The acetonide-protected
dendrimer is dissolved in MeOH and p-TSA (10 wt.%) is added. The mixture is stirred 1
h at r.t. and then percolated over an Amberlyst A21 column. The solution is concentrated
to dryness and washed thoroughly with DCM to remove methanol traces. Dendrimers are
isolated in 89-98 % yields.
Dendrimer post-functionalization - General procedure for CuAAC click reaction. The
heterofunctional dendrimer and the azide- or alkyne-functional probe are dissolved in
DMSO. The flask is degassed and flushed with argon before adding CuBr and PMDETA.
The flask is once again degassed and sealed. The solution is stirred for 1 h at r.t. under
inert atmosphere. The purification protocol will depend on the nature of the final product.
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Dendrimer post-functionalization - General procedure for thiol-ene click reaction. The
heterofunctional dendrimer and the thiol-functional probe are dissolved in methanol and
the photoinitiatior LAP (1 wt.%) is added. The solution is irradiated for 20 min with 365
nm UV light using a Black-Ray B-100AP UV-lamp, and then the solvent in removed by
rotoevaporation. The purification protocol will depend on the nature of the final product.
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Dendrimer post-functionalization - General procedure for FPE reaction. The
functionalization through Fluoride-Promoted Esterification follows the same procedure as
for dendrimer growth.
2.2. Hydrogels preparation.
Hydrogels preparation - CuAAC crosslinking. The heterofunctional dendrimer and
dialkyne PEG (10 kDa) were dissolved in equal volumes of THF and deionized water
respectively and mixed. Stoichiometric ratios of azides and alkynes were used. The water
solution used to dissolve the PEG contained 0.8 equivalents of sodium ascorbate and 0.8
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equivalents of CuSO4·5H2O per reactive functional group. The total mass of dendrimer
and PEG in the final mixture was 20 wt.%. The mixture was sonicated and 15 µL droplets
were added to a PTFE-plate. The mixture was allowed to gel at room temperature. All
droplets solidified into discs that swelled when submerged in deionized water.
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Hydrogels preparation - Thiol-Ene crosslinking. The heterofunctional dendrimer and
dithiol PEG (10 kDa) were dissolved in a 1:1 ethanol and deionized water solution with
0.5 wt.% LAP at stoichiometric ratios of alkenes and thiols. The total mass of dendrimer
and PEG was 20 wt.%. 15 µL droplets were added to a PTFE-plate that was cured with
365 nm UV light using a Black-Ray B-100AP UV-lamp for 5 min. All droplets solidified into
discs that swelled when submerged in deionized water.
2.3. Cell culture and cytotoxicity assays.
Cell culture. The cell lines (mouse monocyte cell Raw 264.7 and human debris fibroblast
cell (hDF)) were obtained from ATCC (American Tissue Culture Collection) and
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maintained in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine
serum FBS and 100 units mL−1 penicillin plus 100 mg mL−1 streptomycin under 5% CO2
at 37 °C.
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Cytotoxicity assessment. The cytotoxicity produced by the different HFDs was evaluated
through AlamarBlue assay. Cells were cultured into 96-well plates (1×105 cells/well in 100
µL DMEM). After 24 h, media were removed and fresh DMEM containing HFDs samples
were added. Six parallel wells were set for each sample. After 72 h incubation, 10 µL of
AlamarBlue agents were added into each well and fluorescent intensity were measured
4 h later with a plate reader (Tecan Infinite M200 Pro) at the wavelength of 560/590
(excitation/emission) nm.
3. Results and discussion
3.1. Multifunctional dendritic scaffolds preparation
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Synthesis of AB2C bis-MPA monomers. In this context, three bis-MPA derivatives with
different chemical groups available for orthogonal reactions were synthesized. The
targeted C groups (alkynes, azides and alkenes) are known to participate with high
efficiency and selectivity in the CuAAC28 and thiol-ene (TEC)29 click reactions, and be
dormant during FPE growth.24-25 The AB2C monomers 5, 6 and 7 were synthesized using
streamlined strategies (Scheme 1). In brief, bis-MPA undergoes sequential esterification,
starting at one of the hydroxyl groups, with acetonide-protected bis-MPA, followed by a
C-COOH molecule bearing the desired orthogonal moiety C on the other, both assisted
by CDI. Detailed synthetic protocols and characterization (1H, 13C NMR) are described in
the Supporting Information (Figures S1-S4), which confirm the purity of these molecules.
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AB C
monomers
2
O
O
O
O
O
O
O
CDI, C-COOH
EtOAc, 50ºC
O
O
KOH, BnBr,
DMF, 100ºC
R
O
O
O
O
O
OH
HO
O
O
OH
OH
CDI
1 89%
C
HO
OH
OH
Bis-MPA
Pd/C, H2,
EtOAc
3:
4:
R = Bn 78%
EtOAc,
50ºC
R = H
100%
O
DMP, pTSA
acetone
50%
N3
5
: C =
: C =
: C =
O
O
HO
70%
6
7
58%
2
95%
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Scheme 1. Synthetic strategy towards bis-MPA based AB2C monomers. Abbreviations:
Bis-MPA, 2,2-Bis(hydroxymethyl)propionic acid; BnBr, benzyl bromide; DMF,
dimethylformamide; DMP, 2,2-dimethoxypropane; pTSA, p-toluenesulfonic acid; CDI,
1,1´-carbonyldiimidazole; EtOAc, ethyl acetate.
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Synthesis of heterofunctional dendrimers with internal and external functional groups.
Considering the complexity of the dendrimers herein presented, the unified nomenclature
for heterofunctional dendrimers will be employed.23 It is based on the general formula
HFD(ie)-Gb-e-(B)m-i-(C)n, which details: (i) the type of dendrimer (HFD) and location of
the heterofunctionality (ie = internal and external); (ii) the generation of the dendrimer
(Gb); and (iii) the number/type of functional groups in the exterior e-(B)m and the interior
i-(C)n. To verify the feasibility of the proposed strategy, a generation four bis-MPA
dendrimer HFD(ie)-G4-e-(OH)48-i-(C)45 was targeted. This HFD is comparable to the
traditional TMP-G4-(OH)48 dendrimer, exhibiting the same core and peripheral groups,
but differs in the distinct increase in mass due to the larger AB2C monomer and the
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additional 45 internal C groups. A comparison of these bis-MPA dendrimers is highlighted
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in Table 1.
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Table 1. Overview of strategies to fourth generation bis-MPA dendrimers using Fluoride-
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Promoted Esterification.
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Strategy
Properties
Homofunctional25 • 8 steps
• 76% total yield
• 7.5 h
• Mw [482-5356] g·mol-1
• 48 OH groups
Heterofunctional a) • 8 steps
• 51% total yield
• 22 h
• Mw [1071-15150] b) g·mol-1
Mw [1248-17808] c) g·mol-1
• 48 OH and 45 C groups
a) This work; b) Alkyne-HFDs; c) Azide-HFDs
The divergent synthesis starts from a TMP core using FPE protocols (Scheme 2).25
Alkyne-functional AB2C monomer 5 was activated with CDI for 15 min at 50°C and then
reacted in situ with TMP in the presence of the soft inorganic base CsF. After 1 h reaction
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and subsequent washing, the first generation dendrimer HFD(ie)-G1-e-(ac)3-i-(yne)3 8
was isolated in near-quantitative yields. Subsequent deprotection using p-TSA in MeOH
for 1 h at 25ºC led to the bifunctional dendrimer HFD(ie)-G1-e-(OH)6-i-(yne)3 9. The
iterative growth/deprotection steps of HFDs were conducted efficiently without
chromatographic purifications, thereby synthesizing the monodisperse fourth-generation
dendrimer HFD(ie)-G4-e-(OH)48-i-(yne)45 15 with Mw 14188 g·mol-1 (Figure S5-S20).
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C
OH
OH
AB C
2
monomers
O
O
O
HO
HO
O
O
O
O
O
O
O
O
5
6
or
OH
HO
O
O
O
O
O
OH
O
HO
O
C
O
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5
6
O
or
O
O
C
O
TMP
C
5
: C =
: C =
: C =
O
O
N3
[G1]
6
7
[G2]
11
19
[G2]
9
(yne)
(yne)
(az)
OH
24
(yne/ene)
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(az)
OH
7
FPE
Deprotection
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HO
O
HO
HO
HO
C
C
HO
HO
HO
O
O
O
O
HO
O
O
O
OH
OH
O
O
OH
OH
O
O
O
C
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
C
O
OH
OH
O
OH
OH
O
O
O
O
O
O
C
HO
HO
HO
HO
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
C
O
O
O
O
O
O
O
C
O
O
O
C
O
O
O
O
O
O
C
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
C
O
O
O
O
O
O
O
O
O
HO
HO
HO
HO
O
O
O
O
O
O
O
O
O
O
O
O
O
O
C
O
O
O
O
O
O
O
O
OH
OH
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
OH
O
O
O
O
O
O
C
O
O
O
O
O
O
C
O
O
O
O
O
O
O
O
C
O
O
O
O
O
O
O
O
O
O
C
O
O
O
O
O
O
O
O
O
O
O
O
O
O
HO
HO
HO
HO
O
O
O
O
O
O
OH
OH
OH
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
C
O
O
O
O
O
O
O
O
C
C
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
C
O
O
O
O
HO
HO
HO
O
O
OH
OH
OH
O
OH
O
HO
O
O
O
C
C
OH
OH
OH
OH
5
6
or
[G3]
13
[G4]
15
(yne)
[G3]
(yne)
21
26
(yne/ene)
(az)
Homogeneous internal layers
Heterogenous internal layers
Scheme 2. Synthesis of multipurpose bis-MPA dendrimers with homogeneous and
heterogeneous internal layers. Abbreviations: Az, azide; ene, alkene; FPE, Fluoride-
Promoted Esterification; TMP, trimethylolpropane; yne, alkyne.
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The same synthetic sequence was employed to synthesize the analogous family
containing azide groups from AB2C monomer 6. HFDs with internal azides were
successfully isolated up to the third-generation HFD(ie)-G3-i-(N3)21-e-(OH)24 21, but
minor structural imperfections were obtained in the G4 counterpart 22 due to the higher
steric hindrance (Figure S21-S34). The modularity of this strategy was further supported
by combining two different AB2C monomers, 5 and 7, in the dendrimer growth (Scheme
1). Trifunctional dendrimers with heterogeneous internal layers HFD(ie)-G2-e-(OH)12-i-
(yne)3(ene)6 23 and HFD(ie)-G3-e-(OH)24-i-(yne)3(ene)18 25 were isolated in high yields
(Figure S35-S42). The compatibility between the internal alkyne and alkene groups
enabled their selective post-functionalization, as it is later demonstrated. The structure
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1
and purity of all dendrimers was confirmed using MALDI-TOF, H- and 13C-NMR
spectroscopy (Figure 1, Supporting Information).
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Figure 1. Comparison of MALDI-TOF MS spectra of “ traditional ” (homofunctional,
depicted as shadowed lines) and “multilayered” (heterofunctional, depicted with intense
colours) bis-MPA dendrimers with internal alkyne functionalities from the first (G1) to the
fourth (G4) generation and the accumulative reaction time for their synthesis. For
traditional dendrimers, m/z values have been previously reported by our group.25 For
HFDs, experimental m/z values for [M+Na+] are shown in the figure while theoretical m/z
values can be found in the Supporting Information.
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The previously reported Trizma® AB2C monomers were grown up to G3 dendrimers
composed of 21 internal acetylene and 24 peripheral hydroxyl groups with Mw ~7300
g·mol-1.22 However, the protocol herein presented goes beyond state-of-the-art and
delivers G4 dendrimers comprising 45 internal groups, 48 peripheral hydroxyls and Mw
~15000 g·mol-1, including layered dendrimers with different internal groups for the first
time. As previously demonstrated,25 the importance of CsF catalysis is critical, enabling
full conversion of the OH groups within a few hours, despite the steric hindrance of higher
generations. In less than one day of total reaction time, FPE delivers a traditional G6
dendrimer with 192 peripheral hydroxyl groups and Mw 22080ꢀg·mol-1 using traditional
AB2 monomers, or multifunctional G4 dendrimers with up to 93 functional groups, through
the more sterically hindered AB2C monomers.
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2.2. In vitro biocompatibility of multifunctional dendritic scaffolds
Traditional bis-MPA dendrimers are highly biocompatible.30 These novel HFDs, however,
have relatively large interior compartments with hydrophobic pendant groups that may
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cause cytotoxicity, as there is a proven affinity of amphiphilic entities towards cell
membranes.31 In vitro assays demonstrated that bis-MPA HFDs are biocompatible up to
10 µM in fibroblasts (hDF, Figure S54) and higher concentrations in monocytes (RAW
264.7, Figure 2). Unlike similar bis-MPA dendrimers decorated with ammonium groups,32
HFDs’ toxicity did not increase with succeeding dendritic generations, suggesting that the
cytotoxicity is not as a result of the presence of higher concentrations of internal click
groups or peripheral hydroxyl groups. A possible explanation to the low cell viability at
high HFD concentration, 100 µM, could be related to the amphiphilic nature of the
dendrimers, presumably similar among generations.
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Figure 2. Viability of RAW 264.7 cells after 72 h incubation with different HFDs, measured
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2.3. Post-functionalization of multifunctional dendritic scaffolds
To position these dendritic scaffolds in application-driven research, two different routes
were targeted (Figure 3.a). All synthetic protocols and characterization tools (NMR
spectroscopy, MALDI-TOF, Size-Exclusion Chromatography (SEC) and FTIR analysis)
are presented in the Supporting Information.
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Trifunctional
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theranostic
N
a
Cl
6
7
8
9
O
N
O
O
O
O
O
O
O
O
O
O
O
O
O
O
HN
O
H3N
O
O
O
O
O
HN
O
O
O
O
N
N
N
N
O
O
N
O
N
O
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O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
N
H
O
O
O
O
H3N
O
O
O
O
O
O
O
O
O
N
O
N
O
O
N
O
O
O
O
O
O
O
O
O
O
O
O
O
O
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OH
N
O
Bifunctional
dendritic
antibacterial
O
O
N
O
O
O
O
N
OH
O
O
O
O
NH
O
O
O
O
O
O
O
O
O
H3N
O
O
O
S
O
O
O
O
O
S
O
S
S
O
HO
HO
OH
OH
O
O
S
O
O
OH
OH
S
Hydrogel
Trifunctional
dendritic
crosslinker
"Click probes" via CuAAC
via TEC
b
N3
N3
5
5
HO
HO
O
O
O
O
O
O
SH
N
N
N
H
HO
OH
N
O2N
H
Disperse Red 13
Dopamine
Cl
F
H
O
n
O
O
N3
O
5
Dexamethasone
via FPE
PEG
O
O
N
NHBoc
H
HO
Cl
N
N
H
N
Alanine
N3
4-pentenoic acid
Cyanine 5
O
c
d
Mw 62100 g·mol-1
Đ 1.102
+ 3 Cy5
Molar mass (g·mol-1
)
m/z
Figure 3. a. Examples of multipurpose HFDs, with potential applications as antibacterial
agents, theranostic tools and crosslinkers for functional hydrogels. b. Library of “click
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probes” successfully attached to the HFDs via CuAAC, TEC and FPE. c. MALDI-TOF
spectra for HFD(ie)-G3-e-(an)12-i-(yne)3(ene)18 25 before and after reaction with Cy5. d.
SEC spectrum for HFD(ie)-G3-e-(OH)24-i-(N3)21 21 after reaction with PEG-alkyne; the
bimodal nature of the spectrum may be due to physical interaction of individual
dendrimers.
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Evaluation as precision polymer therapeutics. In the first route, selected HFDs were
reacted with a library of “click probes” based on bulky and biologically-relevant substrates
(Figure 3.b), as a mean to showcase the design criteria of new-generation, precision
polymer therapeutics. To test the accessibility of the internal C moieties within the
dendritic scaffolds, several modifications were performed based on click reactions. For
example, HFD(ie)-G3-e-(OH)24-i-(yne)21 13 was reacted via CuAAC with the azide-
bearing dye Disperse Red 13 and the drug dexamethasone. After 1 h at 25 °C, 1H-NMR
spectroscopy and FTIR analysis confirmed the complete conversion of the 21 internal
acetylene groups, even at the most internal layer (Figure S43-44). The alkyne groups in
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the trifunctional HFD(ie)-G3-e-(an)12-i-(yne)3(ene)18 25 were also modified with azide-
functional Cyanine 5 under the same conditions, as confirmed by MALDI-TOF, leading to
a trifunctional potential theranostic agent (Figure 3.a,c, S45). The multivalent presence of
fluorescent tags in the first internal layer could enable the monitoring with enhanced
resolution, without interfering with the drug-loading capacity of the carrier.33 All alkene
groups in HFD(ie)-G3-e-(OH)24-i-(yne)3(ene)18 24 were also easily functionalized with
bulky molecules such as thiol-functional dopamine in less than 20 min of UV-initiated TEC
(Figure S46). The dual functionalization of HFD(ie)-G2-e-(OH)12-i-(N3)9 19 via external
FPE with 4-pentenoic acid and internal CuAAC with modified β-alanine resulted in a
bifunctional cationic dendrimer with potential antibacterial properties (Figure 3.a, S47-52).
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The size of these nanocarriers, which influences the biodistribution and bioavailability
through the Enhanced Permeation and Retention effect,34 can also be tuned through the
attachment of poly(ethylene glycol) (PEG) chains. Azide-functional dendrimers HFD(ie)-
G2-e-(OH)12-i-(N3)9 19 and HFD(ie)-G3-e-(OH)24-i-(N3)21 21 were therefore reacted with
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mPEG-alkyne (5 kg·mol-1) through CuAAC. The Mw values, measured through SEC,
increased approximately 36.6 and 55.4 kg·mol-1 for G2 and G3, respectively, with Đ
values below 1.1 (Figure 3.d, S53). These values correlate to 81% and 53% of the reacted
internal groups, which are considered successful in view of the steric hindrance of the
long PEG chains.
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Evaluation as crosslinkers towards functional hydrogels. In the second route, the HFDs
were tested as sophisticated crosslinkers to generate functional hydrogels. To achieve
this, HFD(ie)-G2-e-(OH)12-i-(N3)9 19 and HFD(ie)-G3-e-(OH)24-i-(N3)21 21 were
crosslinked with dialkyne PEG (10 kg·mol-1) using CuAAC. The gelation process took 30-
60 min to deliver solid networks with hydroxyl groups available for further modification.
Similarly, the trifunctional HFD(ie)-G2-e-(OH)12-i-(yne)3(ene)6 24 and HFD(ie)-G3-e-
(OH)24-i-(yne)3(ene)18 26 were reacted with dithiol PEG (10 kg·mol-1) through TEC. Here,
it required only 5 min of UV irradiation to form solid networks (Figure 3.a) which presented
both alkyne and hydroxy groups available for the attachment of different agents.
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Many of the established dendrimer families lack the capacity of internal functionalities.
Here, we have successfully synthesized heterofunctional dendrimers with internal and
external functionalities based on the bis-MPA building block. This was accomplished by
careful design of novel class of AB2C monomers being amalgamated from two traditional
AB2 building blocks. FPE was demonstrated as a powerful tool that can be used to
streamline the synthesis of complex HFDs by avoiding time-consuming purifications,
being more sustainable from an economic and environmental perspective. Through FPE
chemistry, the functional group density of the dendrimer can be optimized by taking
advantage of the entire dendritic skeleton. This led to fourth generation HFDs with 93
functional groups (45 internal moieties and 48 peripheral hydroxyl groups) and Mw ~15000
g·mol-1 within less than one day of reaction time. The resultant HFDs are biocompatible
and can be used as polymeric “stem-cells” for an array of cutting-edge applications, such
as nanocarriers, theranostic tools or in network formation. The structural perfection of
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