Screening Libraries of Amphiphilic Janus Dendrimers Based on Natural Phenolic Acids to Discover Monodisperse Unilamellar Dendrimersomes

Article abstract of DOI:10.1021/acs.biomac.8b01405

Natural, including plant, and synthetic phenolic acids are employed as building blocks for the synthesis of constitutional isomeric libraries of self-assembling dendrons and dendrimers that are the simplest examples of programmed synthetic macromolecules.

Full text of DOI:10.1021/acs.biomac.8b01405

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Article
Screening Libraries of Amphiphilic Janus Dendrimers Based on Natural
Phenolic Acids to Discover Monodisperse Unilamellar Dendrimersomes
Irene Buzzacchera, Qi Xiao, Hong Han, Khosrow Rahimi, Shangda Li, Nina Yu. Kostina, B.
Jelle Toebes, Samantha E Wilner, Martin Moeller, Cesar Rodriguez-Emmenegger, Tobias
Baumgart, Daniela A. Wilson, Christopher James Wilson, Michael L. Klein, and Virgil Percec
Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 25 Oct 2018
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Screening Libraries of Amphiphilic Janus Dendrimers Based on Natural Phenolic
Acids to Discover Monodisperse Unilamellar Dendrimersomes
Irene Buzzacchera,†,‡,‖ Qi Xiao, Hong Han, Khosrow Rahimi, Shangda Li, Nina Yu. Kostina, B.
†,§
†
‡,⊥
†
‡,⊥
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#
†
‡,⊥
Cesar Rodriguez-Emmenegger,‡,⊥ Tobias
Jelle Toebes, Samantha E Wilner, Martin Möller,
†
#
‖
§
†,
Baumgart, Daniela A. Wilson, Christopher J. Wilson, Michael L. Klein and Virgil Percec *
†Roy & Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia,
Pennsylvania 19104-6323, United States
‡DWI−Leibniz Institute for Interactive Materials, RWTH Aachen University, 52074 Aachen, Germany
‖NovioSense B.V., Transistorweg 5, 6534 AT Nijmegen, The Netherlands
§Institute of Computational Molecular Science, Temple University, Philadelphia, Pennsylvania 19122, United
States
⊥Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, 52074 Aachen, Germany
#Institute of Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, The
Netherlands
*
E-mail: percec@sas.upenn.edu
ABSTRACT: Natural, including plant, and synthetic phenolic acids are employed as building blocks for the
synthesis of constitutional isomeric libraries of self-assembling dendrons and dendrimers that are the simplest
examples of programmed synthetic macromolecules. Amphiphilic Janus dendrimers are synthesized from a
diversity of building blocks including natural phenolic acids. They self-assemble in water or buffer into vesicular
dendrimersomes employed as biological membrane mimics, hybrid and synthetic cells. These dendrimersomes
are predominantly unilamellar or multilamellar vesicles with size and polydispersity that is predicted by their
primary structure. However, in numerous cases unilamellar dendrimersomes completely free of multilamellar
assemblies are desirable. Here we report the synthesis and structural analysis of a library containing 13
amphiphilic Janus dendrimers containing linear and branched alkyl chains on their hydrophobic part. They were
prepared by an optimized iterative modular synthesis starting from natural phenolic acids. Monodisperse
dendrimersomes were prepared by injection and giant polydisperse by hydration. Both were structurally
characterized to select the molecular design principles that provide unilamellar dendrimersomes in higher yields
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and shorter reaction times than under previously used reaction conditions. These dendrimersomes are expected to
provide important tools for synthetic cell biology, encapsulation and delivery.
INTRODUCTION
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Natural including plant AB phenolic acids are the building blocks employed by our laboratory for the
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iterative synthesis of constitutional isomeric self-assembling dendrons and dendrimers as the simplest
class of Programmed Synthetic Macromolecules.1-12 Phenolic acids are naturally occurring aromatic
compounds having phenol and carboxylic acid functionalities.1–7 Our laboratory elaborated the concept
of Programmed Synthetic Macromolecules, which employs biological principles to achieve a function.
Specifically, the first biological principles that involve primary–secondary–tertiary–quaternary structure
were employed to create a desired function. Programmed Synthetic Macromolecules are monodisperse
self-assembling macromolecules with precise primary structure that includes composition, sequence and
stereochemistry. They undergo, when desired, folding into secondary and tertiary structures and
self-organization into quaternary structures that are responsible for the creation of a defined function.
When the periodic and quasi-periodic assemblies studied exhibit internal order, their structural and
retro-structural analysis allows the elucidation of their structure and the elucidation of the mechanism of
hierarchical supramolecular structure formation by a combination of methods including X-ray
diffraction together with simulation of oriented fiber diffractograms via molecular models, reconstructed
electron-density maps, electron diffraction, transmission electron microscopy (TEM), cryo-TEM,
circular dichroism, and molecular dynamics simulations (Figure 1).
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Figure 1. Accelerated synthesis of constitutional isomeric libraries of quasi-equivalent self-assembling dendrons
and dendrimers and the analysis of their quaternary, tertiary, and secondary structures via structural and
retrostructural analysis of their periodic and quasi-periodic assemblies with internal order.8,9,11,29 Adapted with
permission from ref 29. Copyright 2009 American Chemical Society.
The methodologies elaborated in our laboratory for structural and retrostructural analysis were inspired
by the pioneering work of Watson, Crick, Klug and others13–15 in the field of structural biology and
transplanted into the field of macromolecular self-assembly16–18 Many of the classic problems from
structural biology were solved with this combination of diffraction tools. In the same way by
investigating nine generational17–32 and one deconstruction libraries, our laboratory discovered soft
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matter quasicrystals and Frank-Kasper phases of supramolecular dendrons and dendrimers,17,18,34–38
mediated changes in the chemical reactivity and chemoselectivity,20,39–42 discovered self-repairing
supramolecular electronics,43–45 mimics of transmembrane protein channels analogous to aquaporin,46–54
molecular machines,55–59 chiral supramolecular columns and chiral supramolecular spheres,60–63
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thermodynamically controlled crystallization,64–66 deracemization in the crystal state, a double helix
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69–71
that disregards chirality, as well as a solid-state orientational memory effects.
However, the entire machinery of life including nucleic acids, proteins, photosynthesis, etc., is
compartmentalized, assembled and functions inside a cell, the membrane of which is the least
understood biological system. The main reason for this limited understanding is that cells and cell
membranes are dynamic fluid assemblies that cannot be investigated by the diffraction methodology
elaborated as described above.72–76 Therefore, a fundamental question is how can one generate
Programmed Synthetic Macromolecules that produce biological membrane mimics without the help of
diffraction methods? A typical biological membrane is a fluidic bilayer structure co-assembled from
phospholipids, cholesterol, transmembrane proteins, glycolipids, glycoproteins, and other components.
Once the components of a biological membranes are removed, the host phospholipids alone form
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vesicles named liposomes that are unstable for long time (Figure 2). Stealth liposomes, containing
some phospholipids conjugated to polyethylene glycol or other water-soluble polymers,78–80 and
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amphiphilic block copolymers (their vesicles are named polymersomes) have been used to overcome
the stability problem of phospholipid liposomes (Figure 2). However, both Stealth liposomes and
polymersomes are prepared by complex methods, are polydisperse and cannot be conjugated with
multiple functional groups. In addition, polymersomes have a thicker bilayer than either liposomes or
biological membranes.
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Figure 2. Structures of liposomes, stealth liposomes, polymersomes, dendrimersomes, glycopolymers,
glycodendrimers, glycoliposomes, glycodendrimersomes, sequence-defined glycodendrimersomes. Reprinted
with permission from ref 84. Copyright 2014 National Academy of Sciences USA; from Springer Nature, ref 12.
Copyright 2013 Springer-Verlag Berlin Heidelberg; from ref 96. Copyright 2016 National Academy of Sciences
USA.
Our laboratory entered the field of biological membrane mimics by discovering that amphiphilic Janus
Dendrimers (JDs) made from constitutional isomeric AB and AB natural phenolic acids self-assemble,
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by injection from ethanol into water or buffer, in stable and monodisperse unilamellar and multilamellar
onion-like (Figure 3) vesicles named dendrimersomes (DSs).83–99
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Figure 3. Multilayer onion-like dendrimersomes and glycodendrimersomes.84,90 Reprinted with permission from
ref 84. Copyright 2014 National Academy of Sciences USA.
The dimensions and physical properties of DSs can be predicted when they are prepared by injection but
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not when they are assembled by hydration. Their bilayer is similar in thickness to that of biological
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membranes, and unlike phospholipids and block copolymer amphiphiles, they can be functionalized
with multiple components. Our laboratory next provided models for glycan-decorated cell-membranes.
Previously, glycopolymers, glycodendrimers, and glycoliposomes obtained by complex coassembly,
were the only models for biological glycans (Figure 2). Screening libraries of amphiphilic Janus
glycodendrimers (JGDs) with various primary structures containing plant, bacterial, and human
carbohydrates that bind to sugar-binding proteins (lectins and galectins) led to the discovery of JGDs,
which self-assemble into monodisperse unilamellar and multilamellar (Figure 3) vesicles named
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glycodendrimersomes (GDSs) (Figure 2). Subsequently, twin-twin, single-single and sequence-defined
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JGDs and their corresponding GDSs were synthesized. The next step was to design monodisperse
sequence-defined oligomers containing carbohydrates and inert oligooxyethylene-hydrophilic fragments
in their structure (Figure 4). These sequence-defined oligomers self-assemble into biological membrane
mimics containing various concentrations of carbohydrates (Figure 4).88–95 With these new systems we
could ask the trivial question: What is the minimum concentration of surface carbohydrate that would
allow the glycodendrimer to recognize sugar binding proteins? The shocking finding was that the ability
to bind proteins increased by decreasing the concentration of carbohydrate.89,90 These findings indicated
that as the concentration of carbohydrate decreased, the rate constant of the recognition process
increased! This observation could only be explained with a different rate constant of binding that is
mediated by different carbohydrate morphologies determined by distinct supramolecular arrangements
of carbohydrates on the surface of these biological membrane mimics. However, structure determination
requires diffraction experiments that cannot be performed on fluid vesicles. Fluorescence microscopy is
also not available since the size of dendrimersomes is below the resolution of optical microscopy. While
the methodology to answer this question is being developed we decided to address some other
fundamental problems of biological cell membrane mimics.
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Figure 4. Sequence-defined Janus glycodendrimers self-assemble in glycodendrimersomes and their
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agglutination with human galectins (absorbance and rate constant k). Reprinted with permission from ref 89.
Copyright 2015 American Chemical Society.
In previous studies, JDs with 3,5-substituted dodecyl chains were extensively used including for hybrids
with DSs and bacterial membranes.96 Models for cell fusion and fission were also elaborated.97–99
Dodecyloxy chains on phenolic acids units in the hydrophobic part of the Janus dendrimers were chosen
in these studies as the cell membrane mimics with similar bilayer width to the natural phospholipids
which have 16 or 18 carbons in their alkyl groups. This bilayer width demonstrates that the phenolic
acid fragments are part of the hydrophobic part of the bilayer. The self-assemblies from JDs
containing 3,5-substituted dodecyl chains, one of the routinely employed Janus dendrimers, form
mixtures of unilamellar and multilamellar onion-like DSs. The aim of this report is to deepen the
knowledge on the size and stability of unilamellar DSs self-assembled by JDs synthesized from a fixed
hydrophilic dendron and a variable hydrophobic dendron made from a phenolic acid core substituted
with a diversity of alkyl chains of different length and degree of branching. To understand the effect of
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the number of carbon atoms of the linear alkyl chains or branched alkyl chain, a library containing 13
JDs was synthesized and the factors affecting the self-assembled monodisperse unilamellar DSs were
investigated and are reported here.
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EXPERIMENTAL SECTION
Materials
All reagents were obtained from commercial sources and were used without purification unless
otherwise stated. CH Cl (DCM) was dried over CaH and freshly distilled before use. Tetrahydrofuran
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2
(
THF) for preparation of DSs was distilled with NaHSO and 3,5-di-tert-butyl-4-hydroxytoluene (BHT)
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and restored in dark. Solvents and reagents were deoxygenated when necessary by purging with N2.
Milli-Q water obtained from Milli-Q UV plus with the resistivity 18.2 MΩ·cm was used for the
preparation of phosphate-buffered saline (PBS). PBS (1×) was obtained by dissolving 8 g of NaCl, 0.2 g
of KCl, 1.44 g of Na HPO and 0.24 g of KH PO in 800 mL of Milli-Q water, adjusted to pH = 7.4 and
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diluted to 1,000 mL.
Techniques
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H and C NMR spectra were recorded at 500 MHz and 126 MHz respectively, on a Bruker DRX (500
MHz) NMR spectrometer. All NMR spectra were measured at 23 °C in CDCl . Chemical shifts (δ) are
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reported in ppm and coupling constants (J) are reported in Hertz (Hz). The resonance multiplicities in
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the H NMR spectra are described as “s” (singlet), “d” (doublet), “t” (triplet), and “m” (multiplet).
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Residual protic solvent of CDCl ( H, δ 7.26 ppm; C, δ 77.16 ppm), or tetramethylsilane (TMS) were
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used as the internal reference in the H- and C-NMR spectra. Evolution of the reaction was monitored
by thin-layer chromatography (TLC) using silica gel 60 F254 precoated plates (E. Merck) and
compounds were visualized by UV light with a wavelength of 254 nm. Purifications by flash column
chromatography were performed using flash silica gel from Silicycle (60 Å, 40–63 m) with the
indicated eluent. The purity of the products was determined by a combination of TLC and high-pressure
liquid chromatography (HPLC) was carried out using Shimadzu LC-20AD high-performance liquid
chromatograph pump, a PE Nelson Analytical 900 Series integration data station, a Shimadzu RID-10A
refractive index (RI) detector, a Shimadzu SPD-10A VP (UV-vis) and three AM gel columns (a guard
column, two 500 Å, 10 m columns). THF was used as solvent at the oven temperature of 23 ºC.
Detection was done by UV absorbance at 254 nm. MALDI-TOF mass spectra were performed on a
PerSeptive Biosystem-Voyager-DE (Framingham, MA) mass spectrometer equipped with nitrogen laser
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(
337 nm) and operating in linear mode. Internal calibration was performed using Angiotensin II and
Bombesin as standards. The analytical samples were obtained by mixing the THF solution of the sample
5–10 mg/mL) with the THF solution of the matrix (2,5-dihydroxybenzoic acid, 10 mg/mL) in a 1/5 (v/v)
(
ratio. The prepared solutionof sample and matrix (0.5 L) was loaded on the MALDI plate and allowed
to dry at 23 ºC before the plate was inserted into the vacuum chamber of the MALDI instrument. The
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laser steps and voltages applied were adjusted depending on both the molecular weight and the nature of
each analyzed compound.
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6
Preparation of Monodisperse Dendrimersomes by Injection. A stock solution was prepared by
dissolving the required amount of amphiphilic JD in THF. DSs were then generated by injection of 50
0
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
L of the stock THF solution of JD into 1.00 mL Milli-Q water or PBS, followed by 5 s vortexing.
Eppendorf Research Plus Single Channel Pipette (10 L–100 L) and VWR Signature Ergonomic High
Performance Single-Channel Variable Volume Pipettor (100 L–1000 L) were used for injection into
Milli-Q water or PBS.
Preparation of Polydisperse Giant Dendrimersomes by Hydration. A 200 L (0.2 mL) solution of
−
1
2
JD (10 mg·mL ) in THF was deposited on the top surface of a roughened Teflon sheet (1 cm ), placed
in a 20 mL flat-bottom vial, followed by evaporation of the solvent for 2 h. The Teflon sheet was dried
in vacuo for an additional 12 h. Milli-Q water or PBS (2.0 mL) was added to submerge the film on the
Teflon sheet, and the vial was placed in a 60 °C oven for 12 h for hydration. The sample vial containing
the Teflon sheet was then mixed using a vortex mixer for 30 s. The final concentration of JD of in
−
1
Milli-Q water is 1 mg·mL .
Dynamic Light Scattering (DLS). DLS for the monodisperse vesicles was performed in PBS and in
Milli-Q water with a Malvern Instruments particle sizer (Zetasizer® Nano S, Malvern Instruments, UK)
equipped with 4 mW He-Ne laser 633 nm and avalanche photodiode positioned at 175º to the beam and
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temperature-controlled cuvette holder. Instrument parameters were determined automatically along with
measurement times. Experiments were performed in triplicate.
Cryogenic Transmission Electron Microscopy (cryo-TEM). Cryo-TEM micrographs were taken on a
Carl Zeiss Libra 120 Microscope (Oberkochen, Germany). Cryo-TEM samples were prepared by plunge
freezing of aqueous dispersion on plasma-treated lacey grids. The vitrified specimens were transferred
to a Gatan-910 cryo-holder. The images were recorded at a temperature of –170 °C with an acceleration
voltage of 120 kV.
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Confocal Fluorescence Microscopy. An imaging chamber (5–10 l) containing DSs was formed
between two coverslips (25 × 25 mm, Fisher Scientific) sealed with vacuum grease. DSs were imaged
by confocal fluorescence microscopy (FluoView 300 scanning system configured on an IX81 inverted
microscope platform) with a 60 × 1.2 NA water immersion lens (Olympus, Center Valley, PA). DSs
containing 3,5-C12-RhB were excited at a wavelength of  = 543 nm. 3,5-C12-RhB is a JD with
3
,5-bis(dodecyloxy)benzoate hydrophobic dendrons conjugated to Rhodamine B (RhB) that was
9
7
previously reported to co-assemble with JD with red fluorescense. Laser intensities were adjusted so
that fluorescence signal was not oversaturated. Image processing and analysis were completed with
ImageJ 1.50 software.
Synthesis of Amphiphilic Janus Dendrimers. Accelerated modular synthesis of a library of Janus
dendrimers is shown in Scheme 1. Compounds 1–6 and 3,5-C12-RhB were synthesized as previously
reported.82,97 These alkylation conditions will be later called conventional reaction conditions.
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Scheme 1. Synthesis of a Library of Janus dendrimers
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Synthesis of Methyl 3,5-bis(octyloxy)benzoate at Conventional Reaction Temperature and
Time.30,32,82 1-Bromooctane (5.0 g, 26.2 mmol), 3,5-dihydroxybenzoate (2.0 g, 11.9 mmol, and K CO
2
3
(8.2 g, 60 mmol) were added in a 250 mL round-bottom flask and dissolved in 50 mL of
N,N-dimethylformamide (DMF). The reaction was allowed to stir at 80 ºC under N for 15 h. The
2
mixture was cooled at room temperature and 100 mL of water were added. The aqueous layer was
extracted with DCM for 4 times. The organic phase was dried with Na SO and the solvent was
2
4
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removed. The product was purified by column chromatography on silica gel with a mobile phase
(hexane: DCM = 3:1) to obtain the target ester 4.6 g, Yield: 98%.
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General Optimized Procedure for Alkylation at Higher Reaction Temperature and Shorter
Reaction Time. Alkyl bromide and methyl 3,5-dihydroxybenzoate or methyl 3,4,5-trihydroxybenzoate
(~400 mg), and K CO were added in a 50 mL round-bottom flask. DMF 5 mL was also added. The
2
3
reaction was allowed to stir at 600 rpm and 120 ºC under N for 3.5 to 6.5 h while monitored by
2
conversion by TLC. The mixture was cooled at room temperature and 30 mL of water were added. The
aqueous layer was extracted with DCM 4 times. The organic phase was dried with Na SO and the
2
4
solvent was removed. The product was purified by column chromatography on silica gel with
hexane/ethyl acetate as mobile phase to obtain the target ester.
Methyl 3,5-bis(hexyloxy)benzoate. Reagents: 1-bromohexane (825 mg, 5.0 mmol), methyl
3
,5-dihydroxybenzoate (400 mg, 2.4 mmol), K CO (2.0 g, 14.4 mmol) for 4 h. Eluent for silica column:
2 3
hexane:ethyl acetate = 30:1. Product: 600 mg, Yield: 74%.
Methyl 3,5-bis(heptyloxy)benzoate. Reagents: 1-bromoheptane (752 mg, 4.2 mmol), methyl
3
,5-dihydroxybenzoate (336 mg, 2.0 mmol), K CO (1.6 g, 12 mmol) for 3.5 h. Eluent for silica column:
2 3
hexane:ethyl acetate = 30:1. Product: 576 mg, Yield: 80%.
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Methyl 3,5-bis(nonyloxy)benzoate. Reagents: 1-bromononane (1.0 g, 5 mmol), methyl
5
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1
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3,5-dihydroxybenzoate (400 mg, 2.4 mmol), K CO (2.0 g, 14.4 mmol) for 3.5 h. Eluent for silica
2
3
column: hexane:ethyl acetate = 30:1. Product: 760 mg, Yield: 75%.
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Methyl 3,5-bis(decyloxy)benzoate. Reagents: 1-bromodecane (930 mg, 4.2 mmol), methyl
3
,5-dihydroxybenzoate (336 mg, 2.0 mmol), K CO (1.66 g, 12 mmol) for 3 h. Eluent for silica column:
2 3
hexane:ethyl acetate = 30:1. Product: 710 mg, Yield: 80%.
Methyl 3,5-bis(undecyloxy)benzoate. Reagents: 1-bromoundecane (920 mg, 3.9 mmol), methyl
3
,5-dihydroxybenzoate (312 mg, 1.85 mmol), K CO (1.54 g, 11.1 mmol) for 4.5 h. Eluent for silica
2 3
column: hexane:ethyl acetate = 30:1. Product: 819 mg, Yield: 93%.
Methyl 3,5-bis(dodecyloxy)benzoate. Reagents: 1-bromododecane (933 mg, 3.8 mmol), methyl
3
,5-dihydroxybenzoate (300 mg, 1.8 mmol), K CO (1.54 g, 10.6 mmol) for 4.5 h. Eluent for silica
2 3
column: hexane:ethyl acetate = 30:1. Product: 820 mg, Yield: 91%.
Methyl 3,5-bis(2-ethylhexyloxy)benzoate. Reagents: 2-ethylhexyl bromide (811 mg, 4.2 mmol),
methyl 3,5-dihydroxybenzoate (336 mg, 2.0 mmol), K CO (1.66 g, 12 mmol) for 4.5 h. Eluent for silica
2
3
column: hexane:ethyl acetate = 30:1. Product: 410 mg, Yield: 50%.
Methyl 3,5-bis(3S,7-dimethyloctyloxy)benzoate. Reagents: 1-bromo-3S,7-dimethyloctane (840 mg,
6
8
3
.8 mmol), methyl 3,5-dihydroxybenzoate (300 mg, 1.8 mmol), K CO (1.66 g, 12 mmol) for 4.5 h.
2 3
Eluent for silica column: hexane:ethyl acetate = 30:1. Product: 690 mg, Yield: 85%.
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Methyl 3,5-bis(3,7-dimethyloctyloxy)benzoate. Reagents: 1-bromo-3,7-dimethyloctane (racemic) (840
6
8
mg, 3.8 mmol), methyl 3,5-dihydroxybenzoate (300 mg, 1.8 mmol), K CO (1.66 g, 12 mmol) for 4.5
2
3
h. Eluent for silica column: hexane:ethyl acetate = 30:1. Product: 600 mg, Yield: 74%.
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Methyl 3,4,5-tris(octyloxy)benzoate. Reagents: 1-bromooctane (1.0 g, 5.2 mmol), methyl
3
,4,5-trihydroxybenzoate (300 mg, 1.6 mmol), K CO (1.8 g, 13.2 mmol) for 6.5 h. Eluent for silica
2 3
column: hexane:ethyl acetate = 30:1. Product: 510 mg, Yield: 61%.
Methyl 3,4,5-tris(2-ethylhexyloxy)benzoate. Reagents: 2-ethylhexyl bromide (1.4 g, 7.2 mmol),
methyl 3,4,5-trihydroxybenzoate (400 mg, 2.2 mmol), K CO (1.8 g, 13.2 mmol) for 6.5 h. Eluent for
2
3
silica column: hexane:ethyl acetate = 40:1. Product: 510 mg, Yield: 45%.
Methyl 3,4,5-tris(3,7-dimethyloctyloxy)benzoate. Reagents: 1-bromo-3,7-dimethyloctane (racemic)
(
1.6 g, 7.2 mmol), methyl 3,4,5-trihydroxybenzoate (400 mg, 2.2 mmol), K CO (1.8 g, 13.2 mmol) for
2 3
6
.5 h. Eluent for silica column: hexane:ethyl acetate = 60:1. Product: 640 mg, Yield: 48%.
General Procedure for the Hydrolysis of Esters. The ester and KOH were added in a 50 mL
round-bottom flask together with 10 mL ethanol. The reaction was allowed to stir at 100 ºC for 2 h. The
mixture was evaporated under vacuum. 30 mL of Water (30 mL) was added, and the remaining mixture
was cooled at 0 °C. Hydrochloride acid (2M) was added dropwise until pH < 2. The aqueous layer was
extracted with DCM for 4 times. The organic phase was dried with Na SO and the solvent was
2
4
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removed to obtain the target 3,5-bis(alkyloxy)benzoic acid or 3,4,5-tris(alkyloxy)benzoic acid without
further purification in quantitative yield.
5
6
7
8
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1
1
1
1
1
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General Procedure for Esterification for Janus Dendrimers (JDs). Compound 6,
3
,5-bis(alkyloxy)benzoic acid or 3,4,5-tris(alkyloxy)benzoic acid, and 4-(dimethylamino)pyridinium
4-toluenesulfonate were mixed and dissolved in DCM (10 mL).
(DPTS)100
N,N'-Dicyclohexylcarbodiimide (DCC) was added to the reaction mixture. The mixture was then
allowed to stir at 23 ºC for 12 h. The precipitate was then filtered, and the filtrate was concentrated to
dryness. The crude product was further purified by column chromatography on silica gel with a mobile
phase of (DCM/methanol) to to yield he target JD as a colorless oily liquid.
3
,5-C6. Reagents: Compound 6 (200 mg, 0.15 mmol), 3,5-bis(hexyloxy)benzoic acid (103 mg, 0.32
mmol), DPTS (88 mg, 0.30 mmol), DCC (124 mg, 0.60 mmol). Eluent for silica column:
DCM:methanol = 20:1. Product: 260 mg, Yield: 89%.
3,5-C7. Reagents: Compound 6 (290 mg, 0.20 mmol), 3,5-bis(heptyloxy)benzoic acid (308 mg, 0.80
mmol), DPTS (260 mg, 0.80 mmol), DCC (272 mg, 1.3 mmol). Eluent for silica column:
DCM:methanol = 20:1. Product: 410 mg, Yield: 94%.
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6
3
,5-C8. Reagents: Compound 6 (200 mg, 0.15 mmol), 3,5-bis(otcyloxy)benzoic acid (144 mg, 0.38
mmol), DPTS (112 mg, 0.38 mol), DCC (124 mg, 0.60 mmol). Eluent for silica column: DCM:methanol
20:1. Product: mg, Yield: 83%.
,5-C9. Reagents: Compound 6 (200 mg, 0.15 mmol), 3,5-bis(nonyloxy)benzoic acid (146 mg, 0.36
=
0
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2
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4
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9
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3
mmol), DPTS (132 mg, 0.45 mmol), DCC (155 mg, 0.75 mmol). Eluent for silica column:
DCM:methanol = 20:1. Product: 278 mg, Yield: 89%.
3
,5-C10. Reagents: Compound 6 (220 mg, 0.17 mmol), 3,5-bis(decyloxy)benzoic acid (304 mg, 0.70
mmol), DPTS (206 mg, 0.70 mmol), DCC (210 mg, 1.0 mmol). Eluent for silica column:
DCM:methanol = 40:1. Product: 322 mg, Yield: 88%.
3,5-C11. Reagents: Compound 6 (225 mg, 0.17 mmol), 3,5-bis(undecyloxy)benzoic acid (199 mg, 0.43
mmol), DPTS (151 mg, 0.51 mmol), DCC (177 mg, 0.86 mmol). Eluent for silica column:
DCM:methanol = 20:1. Product: 347 mg, Yield: 92 %.
3
,5-C12. Reagents: Compound 6 (370 mg, 0.28 mmol), 3,5-bis(hexyloxy)benzoic acid (274 mg, 0.56
mmol), DPTS (164 mg, 0.56 mmol), DCC (206 mg, 1.0 mmol). Eluent for silica column:
DCM:methanol = 20:1. Product: 530 mg, Yield: 84%.
3
,5-EH. Reagents: Compound 6 (171 mg, 0.13 mmol), 3,5-bis(2-ethylhexyloxy)benzoic acid (200 mg,
0
.53 mmol), DPTS (156 mg, 0.53 mmol), DCC (161 mg, 0.78 mmol). Eluent for silica column:
DCM:methanol = 40:1. Product: 172 mg, Yield: 85%.
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,5-dm(S). Reagents: Compound 6 (200 mg, 0.20 mmol), 3,5-bis(3S,7-dimethyloctyloxy)benzoic acid
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
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4
4
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4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
(261 mg, 0.6 mmol), DPTS (117 mg, 0,6 mmol), DCC (186 mg, 0,9 mmol). Eluent for silica column:
DCM:methanol = 20:1. Product: 310 mg, Yield: 96%.
0
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3
,5-dm(rac). Reagents: Compound 6 (200 mg, 0.20 mmol), 3,5-bis(3,7-dimethyloctyloxy)benzoic acid
261 mg, 0.6 mmol), DPTS (117 mg, 0,6 mmol), DCC (186 mg, 0,9 mmol). Eluent for silica column:
DCM:methanol = 20:1. Product: 305 mg, Yield: 95%.
,4,5-C8. Reagents: Compound 6 (100 mg, 0.08 mmol), 3,5-bis(otcyloxy)benzoic acid (131 mg, 0.26
mmol), DPTS (68 mg, 0.23 mmol), DCC (78 mg, 0.38 mmol). Eluent for silica column: DCM:methanol
20:1. Product: 162 mg, Yield: 93%.
(
3
=
3,4,5-EH. Reagents: Compound 6 (200 mg, 0.15 mmol), 3,4,5-tris(2-ethylhexyloxy)benzoic acid (193
mg, 0,83 mmol), DPTS (132 mg, 0.45 mmol), DCC (155 mg, 0.75 mmol). Eluent for silica column:
DCM:methanol = 20:1. Product: 326 mg, Yield: 95%.
3
,4,5-dm(rac). Reagents: Compound 6 (200 mg, 0.15 mmol), 3,4,5-tris(3,7-dimethyloctyloxy)benzoic
acid (225 mg, 0.38 mmol), DPTS (132 mg, 0.45 mmol), DCC (155 mg, 0.75 mmol). Eluent for silica
column: DCM:methanol = 20:1. Product: 365 mg, Yield: 93 %.
1
13
Purity by HPLC, H NMR, C NMR, and MALDI-TOF for Previously Unreported Compounds
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1
3
,5-C7. Purity (HPLC): 99%+. H NMR (500 MHz, CDCl ) δ = 7.24 (s, 4 H), 7.09 (d, J = 2.3 Hz,4 H),
3
6.61 (t, J = 2.3 Hz, 2 H), 4.61 (s, 4 H), 4.58 (s, 4 H), 4.22 (m, 4 H), 4.16 (m, 8 H), 3.93 (t, J = 6.5 Hz, 8
H), 3.86 (m, 8 H), 3.79 (m, 4H), 3.71 (m, 12 H), 3.64 (m, 24 H), 3.53 (m, 12 H), 3.36 (s, 6 H), 3.35 (s,
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1
2 H), 1.76 (m, 8 H),1.44 (m, 8 H), 1.38-1.25 (m, 24 H), 0.88 (t, J = 6.9 Hz, 12 H). C NMR (126 MHz,
CDCl ) δ = 165.9, 165.5, 160.3, 152.5, 143.1, 131.1, 124.3, 109.1, 107.8, 106.7, 72.6, 72.0, 70.9, 70.8,
3
70.6, 69.7, 69.0, 68.4, 63.1, 63.0, 59.1, 43.4, 31.9, 29.3, 29.2, 26.1, 22.7, 14.2. MALDI-TOF (m/z):
+
[
M+Na] calcd for C H O Na, 2004; found, 2006.
103 168 36
1
3
,5-C9. Purity (HPLC): 99%+. H NMR (500 MHz, CDCl ) δ = 7.24 (s, 4 H), 7.09 (d, J = 2.3 Hz,4 H),
3
6.61 (t, J = 2.3 Hz, 2 H), 4.61 (s, 4 H), 4.58 (s, 4 H), 4.22 (m, 4 H), 4.17 (m, 8 H), 3.93 (t, J = 6.5 Hz, 8
H), 3.86 (m, 8 H), 3.79 (m, 4H), 3.72 (m, 12 H), 3.64 (m, 24 H), 3.53 (m, 12 H); 3.36 (s, 6 H), 3.35 (s,
1
3
1
2 H), 1.76 (m, 8 H), 1.44 (m, 8 H), 1.36–1.26 (m, 40 H), 0.88 (t, J = 6.9 Hz, 12 H). C NMR (126
MHz, CDCl ) δ = 165.8, 165.4, 160.2, 152.4, 143.0, 131.0, 124.1, 109.0, 107.7, 106.6, 77.4, 77.2, 76.9,
3
7
2.4, 71.9, 70.8, 70.6, 70.5, 70.5, 69.6, 68.9, 68.3, 63.1, 62.9, 58.9, 43.3, 31.8, 29.5, 29.4, 29.2, 29.2,
+
26.0, 22.6, 14.1. MALDI-TOF (m/z): [M+Na] calcd for C H O Na, 2116; found, 2116.
1
11 184 36
1
3
,5-C10. Purity (HPLC): 98%+. H NMR (500 MHz, CDCl ) δ = 7.24 (s, 4 H); 7.08 (d, J = 2.3 Hz,4 H);
3
6
.61 (t, J = 2.3 Hz, 2 H); 4.61 (s, 4 H); 4.58 (s, 4 H); 4.21 (m, 4 H), 4.16 (m, 8 H), 3.92 (t, J = 6.5 Hz, 8
H), 3.85 (m, 8 H), 3.79 (m, 4H), 3.72 (m, 12 H), 3.63 (m, 24 H), 3.53 (m, 12 H); 3.36 (s, 6 H), 3.35 (s,
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2 H), 1.76 (m, 8 H), 1.44 (m, 8 H), 1.35–1.27 (m, 48 H), 0.88 (t, J = 6.9 Hz, 12 H). C NMR (126
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
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4
4
4
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4
4
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MHz, CDCl ) δ = 165.7, 165.3, 160.2, 152.3, 142.9, 130.9, 124.1, 109.0, 107.6, 106.5, 72.4, 71.8, 70.2,
3
7
0.6, 70.5, 70.4, 69.5, 68.9, 68.2, 63.0, 62.8, 58.9, 43.3, 31.8, 29.5, 29.3, 29.2, 29.1, 26.0, 22.6, 14.0.
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8
9
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2
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4
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7
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0
+
MALDI-TOF (m/z): [M+Na] calcd for C H O Na, 2172; found, 2172.
1
15 192 36
3,5-C11. Purity (HPLC): 99%+. H NMR (500 MHz, CDCl ) δ = 7.24 (s, 4 H); 7.08 (d, J = 2.3 Hz,4 H);
3
6
3
3
.61 (t, J = 2.3 Hz, 2 H); 4.61 (s, 4 H); 4.58 (s, 4 H); 4.22 (t, J = 4.5 Hz, 4 H), 4.16 (t, J = 4.5 Hz, 8 H),
.92 (t, J = 6.5 Hz, 8 H), 3.85 (m, 8 H), 3.79 (m, 4H), 3.72 (m, 12 H), 3.65 (m, 24 H), 3.52 (m, 12 H);
13
.36 (s, 6 H), 3.35 (s, 12 H), 1.76 (m, 8 H), 1.42 (m, 8 H), 1.25 (m, 48 H), 0.87 (t, J = 6.9 Hz, 12 H). C
NMR (126 MHz, CDCl ) δ = 166.0, 165.6, 160.4, 152.6, 143.2, 131.2, 124.3, 109.2, 107.9, 106.8, 72.6,
3
7
2
2.1, 71.0, 70.9, 70.7, 70.7, 69.8, 69.1, 68.5, 63.2, 63.2, 59.2, 43.5, 32.1, 29.8, 29.8, 29.6, 29.5, 29.4,
+
6.2, 22.9, 14.3. MALDI-TOF (m/z): [M+K] calcd for C H O K, 2244; found, 2245.
1
19 200 36
1
3
,5-EH. Purity (HPLC): 99%+. H NMR (500 MHz, CDCl ) δ = 7.25 (s, 4 H); 7.11 (d, J = 2.3 Hz,4 H);
3
6.63 (t, J = 2.3 Hz, 2 H); 4.61 (s, 4 H); 4.59 (s, 4 H); 4.22 (m, 4 H), 4.17 (m, 8 H), 3.87–3.82 (m, 16 H),
.79 (m, 4H), 3.72 (m, 12 H), 3.63 (m, 24 H), 3.53 (m, 12 H); 3.36 (s, 6 H), 3.35 (s, 12 H), 1.69 (m, 12
3
1
3
H), 1.54–1.30 (m, 24 H), 0.94–0.88 (m, 24 H). C NMR (126 MHz, CDCl ) δ = 165.9, 165.5, 160.5,
3
1
52.4, 143.0, 131.0, 124.2, 109.1, 107.8, 106.7, 72.5, 72.0, 70.8, 70.7, 70.6, 70.6, 69.7, 69.0, 62.9, 62.8,
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+
5
9.0, 43.4, 39.4, 30.5, 29.1, 23.8, 23.1, 14.1, 11.2. MALDI-TOF (m/z): [M+Na] calcd for
C H O Na, 2160; found, 2162.
1
07 176 36
0
1
2
3
4
5
6
7
8
9
0
1
2
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8
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0
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4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
3
,5-dm(S). Purity (HPLC): 99%+. H NMR (500 MHz, CDCl ) δ = 7.24 (s, 4 H); 7.10 (d, J = 2.3 Hz,4
3
H); 6.62 (t, J = 2.2 Hz, 2 H); 4.61 (s, 4 H); 4.59 (s, 4 H); 4.21 (m, 4 H), 4.17 (m, 8 H), 3.97 (m, 8 H),
3.86 (m, 8 H), 3.79 (m, 4H), 3.72 (m, 12 H), 3.64 (m, 24 H), 3.53 (m, 12 H); 3.36 (s, 6 H), 3.35 (s, 12
H), 1.85–1.62 (m, 12 H), 1.60–1.48 (m, 8 H), 1.36–1.12 (m, 20 H), 0.93 (d, J = 6.6 Hz, 12 H), 0.86 (d, J
1
3
=
6.6 Hz, 24 H). C NMR (126 MHz, CDCl ) δ = 165.9, 165.5, 160.3, 152.4, 143.0, 131.1, 124.2, 109.1,
3
1
07.8, 106.6, 72.5, 72.0, 70.8, 70.7, 70.6, 69.7, 69.0, 66.7, 62.8, 59.0, 43.4, 39.3, 37.3, 36.1, 29.8, 28.0,
+
24.7, 22.7, 22.6, 19.6. MALDI-TOF (m/z): [M+Na] calcd for C H O Na, 2172; found, 2172.
1
15 192 36
1
3
4
3
,5-dm(rac). Purity (HPLC): 99%+. H NMR (500 MHz, CDCl ) δ = 7.25 (s, 4 H); 7.10 (d, J = 2.3 Hz,
3
H); 6.62 (t, J = 2.3 Hz, 2 H); 4.61 (s, 4 H); 4.59 (s, 4 H); 4.21 (m, 4 H), 4.17 (m, 8 H), 3.97 (m, 8 H),
.86 (m, 8 H), 3.79 (m, 4H), 3.72 (m, 12 H), 3.66–3.62 (m, 24 H), 3.52 (m, 12 H); 3.36 (s, 6 H), 3.35 (s,
12 H), 1.85–1.64 (m, 12 H), 1.60–1.48 (m, 8 H), 1.36–1.11 (m, 20 H), 0.93 (d, J=6.6 Hz, 12 H), 0.86 (d,
1
3
J=6.6 Hz, 24 H). C NMR (126 MHz, CDCl ) δ = 165.9, 165.5, 160.3, 152.5, 143.1, 131.1, 124.3,
3
1
2
09.2, 107.9, 106.7, 72.6, 72.0, 70.9, 70.8, 70.6, 69.7, 69.0, 66.8, 63.0, 59.1, 43.4, 39.3, 37.4, 36.2, 29.9,
+
8.0, 24.7, 22.8, 22.7, 19.7. MALDI-TOF (m/z): [M+Na] calcd for C H O Na, 2172; found, 2172.
1
15 192 36
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,4,5-EH. Purity (HPLC): 99%+. H NMR (500 MHz, CDCl ) δ = 7.25 (s, 4 H); 7.20 (s, 4 H); 4.59 (s, 4
3
5
6
7
8
9
1
1
1
1
1
1
1
1
1
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2
2
2
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2
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H); 4.57 (s, 4 H); 4.22 (m, 4 H), 4.17 (m, 8 H), 3.97 (m, 12 H), 3.86 (m, 8 H), 3.79 (m, 4 H), 3.72 (m, 12
H), 3.66–3.62 (m, 24 H); 3.54–3.51 (m, 12 H), 3.36 (s, 6 H), 3.35 (s, 12 H), 1.80 (m, 8 H), 1.73 (m, 4 H),
0
1
2
3
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0
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2
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8
9
0
1
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9
0
1
2
3
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9
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3
4
5
6
7
8
9
0
1
3
1
1
.47 (m, 12 H), 1.28 (m, 48 H), 0.89–0.86 (m, 18 H).
C NMR (126 MHz, CDCl ) δ = 165.9, 165.5,
3
53.2, 152.5, 143.2, 142.9, 124.3, 123.8, 109.2, 107.6, 76.1, 72.6, 72.0, 71.4, 70.9, 70.8, 70.6 69.7, 69.1,
62.7, 59.1, 43.6, 40.7, 39.7, 30.6, 30.5, 29.4, 29.2, 23.9, 23.7, 23.2, 23.2, 14.2, 14.2, 11.3. [M+Na]+
calcd for C H O Na, 2316; found, 2315.
123 208 36
1
3
,4,5-dm(rac). Purity (HPLC): 99%+. H NMR (500 MHz, CDCl ) δ = 7.26 (s, 4 H); 7.23 (s, 4 H); 4.59
3
(s, 4 H); 4.58 (s, 4 H); 4.22 (m, 4 H), 4.17 (m, 8 H), 4.08–3.98 (m, 12 H), 3.86 (m, 8 H), 3.79 (m, 4 H),
.72 (m, 12 H), 3.66–3.62 (m, 24 H); 3.54–3.51 (m, 12 H), 3.36 (s, 6 H), 3.35 (s, 12 H), 1.90–1.78 (m, 6
3
H), 1.70 (m, 6 H), 1.60–1.48 (m, 12 H), 1.37–1.23 (m, 18 H), 1.19–0.11 (m, 18 H), 0.95–0.91 (m, 18 H),
13
0
1
.86 (m, 36 H).
C NMR (126 MHz, CDCl ) δ = 165.8, 165.5, 153.0, 152.5, 143.2, 143.0, 124.3,
3
24.0, 109.2, 108.2, 72.6, 72.0, 71.9, 70.9, 70.8, 70.6, 69.7, 69.1, 67.6, 62.7, 62.6, 59.1, 43.6, 39.5, 39.4,
+
37.6, 37.6, 37.5, 37.5, 36.5, 29.9, 29.8, 28.1, 24.8, 24.8, 22.8, 22.7, 19.6, 19.6. [M+Na] calcd for
C H O Na, 2484; found, 2484.
1
35 232 36
RESULTS and DISCUSSION
Natural Polyphenols and Phenolic acids as Building Blocks for Programmed Synthetic
Macromolecules. Natural phenols, resorcinol, pyrocatechols, pyrogallols, and phenolic acids including
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
-resorcylic acid, protocatechuic acid, and gallic acid (Figure 5) are present in a variety of unrelated
types of fruits, vegetables, cereals, legumes and their derivatives such as coffee, wine and chocolate.1–7
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HO
OH
HO
HO
OH
OH
OH
Pyrocatechol
COOH
OH
Pyrogallol
Phenol
COOH
Resorcinol
COOH
HO
OH
OH
HO
OH
OH
OH
α-Resorcylic Acid
Protocatechuic Acid
Gallic Acid
Figure 5. Natural plant phenols, polyphenols and constitutional isomeric AB and AB phenolic acids.
2
3
Polyphenols and phenolic acids are generated as secondary metabolites during photosynthesis and they
are stored in plant and spice leaves. Their role in plant metabolism is still under investigation. It has
been reported that phenolic acids act as signaling molecules, defense agents, preservatives and are
responsible for the color and taste of fruits and vegetables. They have been intensively studied for their
therapeutic effect on human health. Their main natural function utilizes the antioxidant and free radical
scavenger activities of these molecules (Figure 6).1–7
Figure 6. Functions and applications of natural including plant phenolic acids.
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Even at low concentration they can prevent proteins and other macromolecules from undergoing
oxidative damage by direct quenching of active oxygen species, inhibition of enzymes and chelation of
metal ions. Thanks to their antioxidant activity, a diet rich in phenolic acids has beneficial effects on
ageing and stress-induced disorders, but they are also used as natural additives and preservatives in the
food industry. They find therapeutic use for the treatment of diseases where oxidative processes play an
important role, such as heart diseases, arteriosclerosis, neurodegenerative diseases and some types of
cancer. Lately, they have also been employed in the treatment of Diabetes Mellitus and microbial
infections (Figure 7). Furthermore, the phosphoprotein mussel foot protein Mefp-5 found in mussels
exhibited very strong adhesion to surfaces due to polyphenols. Dopamine building blocks which mimic
Mefp-5 protein containing polyphenols are extensively employed to design mussel-inspired surface
coating materials (Figure 7).101–104
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0
Figure 7. Schematic illustration of Mefp-5 protein from mussels, and dopamine containing amine and catechol
functional groups (broken red square) used to design mussel-inspired surface coatings.100
Synthetic Phenolic Acids as Building Blocks for Programmed Synthetic Macromolecules. In addition
to the natural phenolic acids shown in Figure 5, four additional libraries of constitutional isomeric AB2
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and AB , AB , and AB phenolic acids were designed and investigated in our laboratory for the
3
4
5
synthesis of Programmed Synthetic Macromolecules.25–27,29 Their structures are shown in Figure 8.
COOH
COOH
COOH
COOH
COOH
COOH
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HO
OH
OH HO
OH
OH
OH
COOH
HO
OH
OH HO
OH
OH
OH
COOH
COOH
COOH
COOH
HO
OH
OH HO
OH
OH
OH
HO
OH HO
OH
OH
HO
OH
HO
OH
2
5–27,29
Figure 8. Synthetic constitutional isomeric AB , AB , AB , and AB phenolic acids
designed and
2
3
4
5
employed in the Percec laboratory for the synthesis of libraries of self-assembling dendrons and dendrimers.
Synthesis of the Amphiphilic Janus Dendrimer Library. The unique properties of DSs, the facile and
versatile synthesis of amphiphilic JDs, and their self-assembly has been reported previously.82–86 The
formation of biomimetic vesicles from amphiphilic building blocks follows a complex mechanism that
requires additional experiments. The modular design of many different libraries of molecular building
blocks will contribute to the elucidation of their mechanism of self-assembly. Size, morphology and
mechanical properties of the self-assembled DSs can be predicted based on the molecular structure of
8
5
JDs. During the formation of DSs in polar media, the substitution pattern in the first generation (G1)
dendron in the hydrophobic part of JDs is determinant for the interactions between the medium and the
hydrophilic part of the molecule and for the degree of interdigitation in the hydrophobic bilayer of the
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vesicles. In order to maximize stability and size of DSs it has been shown that the (3,4,5)-G1
substitution in the hydrophilic part containing triethylene glycol of JDs results in a better solvation of
the new-formed vesicles, and the (3,5)-G1 pattern in the hydrophobic side allows more efficient
interdigitation of the alkyl chains as well as tight packing, resulting in tougher membranes compared to
the (3,4)-G1 and (3,4,5)-G1 patterns. A systematic synthesis of JDs with different lengths and degree of
branching of the alkyl chains in the hydrophobic dendron and screening of the effects on size and
stability of DSs have not been performed yet. With this in mind, a new library of 13 amphiphilic JDs has
been prepared by an accelerated iterative modular synthetic approach.
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Scheme 2. Synthesis of 3,5-Substituted Phenolic Acids
0
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0
*
Conventional reaction temperature (80 °C) and time (15 hours).
Scheme 3. Synthesis of 3,4,5-Substituted Phenolic Acids
The convergent synthesis of the JDs has been performed by diversifying the structure the alkyl chains of
the (3,5)- and (3,4,5)-substituted hydrophobic dendrons from phenolic esters, as shown in Schemes 2
and 3. The alkylation of methyl 3,4,5-trihydroxybenzoate and methyl 3,5-bihydroxybenzoate was
performed with 10 different alkyl bromides. The conventional conditions for the alkylation (Scheme 2,
asterisk) is 80 °C and 15 hours, which was adopted from the synthesis of libraries of dendrons,
dendrimers, and Janus dendrimers with the phenolic acid building blocks.9,82 The alkylation reaction has
been optimized according to a previous study.105 The most influential parameters affecting yield and
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Page 29 of 55
Biomacromolecules
1
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reaction time for the alkylation of 3,4,5-trihydroxybenzoate were found to be temperature, equivalents
of the base and stirring rate. Based on this evaluation, an improved synthetic design has been obtained
by ielevating the reaction temperature from 80 °C to 120 °C, increasing the concentration of reagents by
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0% and the amount of base by 20%. By using these optimized conditions, smaller amounts of alkyl
bromides could be used, but more importantly a reduction of about 70% of the reaction time was
obtained. The yield of the newly optimized alkylation reactions ranged between 50 and 90%. The
subsequent hydrolysis of the alkyl-substituted benzoates gave the alkyl-substituted phenolic acids that
were coupled to the (3,4,5)-substituted hydrophilic dendron (compound 6) as shown in Scheme 1.
The length of the linear alkyl chains was varied from 6 to 12 carbons. Three branched molecules were
also employed, two of which being pure enantiomers and one a racemic mixture. As discussed
previously, interdigitation of the alkyl chains is fundamental for the formation of robust membranes and
it is intuitive to think that the longer the chain the more stable the hydrophobic bilayer. The modulation
of chain length was carried out to test the minimum number of carbons required to form stable vesicles.
Furthermore, the introduction of branching points on the alkyl chains helps in understanding what
happens when we introduce a degree of disruption in the packing. Biological membranes are made of
phospholipids that are packed with each other by their hydrophobic chains. The presence of unsaturated
bonds, stereocenters and branching does not destabilize the membranes but are utilized in stabilization
and modulation of physical properties of the membrane such as stabilization of transmembrane proteins
and modulation of the crystallization of the membrane for adaptation to lower temperature environments,
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