A new poly(propylene imine) dendron as potential convenient building-block in the construction of multifunctional systems

Article abstract of DOI:10.1016/j.tet.2013.01.070

The synthesis of a novel third generation poly(propylene imine) dendron (PPI dendron) is described herein. The dendritic focal point was attached to a tetraethylene glycol spacer that was terminated with a Tmob-protected thiol in order to both avoid any s

Full text of DOI:10.1016/j.tet.2013.01.070

Tetrahedron 69 (2013) 2799e2806
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
A new poly(propylene imine) dendron as potential convenient
building-block in the construction of multifunctional systems
y
y
z
*
Hongmu Pan , Margaret E. Grow , Orla Wilson , Marie-Christine Daniel
Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, MD 21250, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a novel third generation poly(propylene imine) dendron (PPI dendron) is described
herein. The dendritic focal point was attached to a tetraethylene glycol spacer that was terminated with
a Tmob-protected thiol in order to both avoid any side-reaction during functionalization of dendritic
branches as well as enable convenient integration of the dendron into diverse complex systems. The
protonation of the final PPI dendron at pH 4e5 is predicted to induce lysosomal degradation through the
proton-sponge effect, and therefore enable cytosolic release. These features make the prepared dendron
a very convenient building-block for use in the construction of larger systems, such as multifunctional
drug delivery vehicles.
Received 19 November 2012
Received in revised form 16 January 2013
Accepted 22 January 2013
Available online 1 February 2013
Keywords:
Dendron
Poly(propylene imine)
Proton-sponge
Thiol anchoring
Nitrile reduction
Borane dimethyl sulfide
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
one drawback is the fact that they usually display a single type
of termini, and their multifunctionalization remains tedious and
During the past decades, dendrimers have achieved significant
success in a variety of biomedical applications.1e4 For instance,
VivaGelÒ (Starpharma), a topical vaginal microbicide based on
a polylysine dendrimer, is currently undergoing phase II clinical
trials for the prevention of bacterial vaginosis recurrence. Additional
examples are several dendrimer-based products5 that received the
CE mark, primarily for the traumatic or surgically-induced wound
market: Ocuseal (a liquid ocular bandage), and AdherusÔ Dural
Sealant (used in cranial and spine surgeries to prevent cerebrospinal
fluid leaks, and which has also successfully completed a U.S. pivotal
clinical trial). Because of their nanoscale size and their branched
structure, dendrimers allow for a longer circulation time in the body
and they can carry a large payload.1,4,6,7 They also present the ad-
vantage over conventional polymers to be well defined macromol-
ecules with a polydispersity index equal to or close to 1, depending
on the generation.8 This last feature is particularly important in
biomedical applications in order to ensure high batch-to-batch
reproducibility of the therapeutic effects. Although the mono-
dispersity and polyvalence of dendrimers represent great assets,
challenging.9e13 Another limitation is the appearance of defects
when reaching higher generations of dendrimers.
Dendrons differ from dendrimers in their structure by the
presence of a focal point that is usually functionalized differently
than the periphery, thus breaking the symmetry of the molecule.8
This focal point allows for dendrons to be grafted onto other mol-
ecules (dendrons, dendritic cores, polymers.) or surfaces.14e19
This is of particular interest for biomedical applications as den-
drons can be incorporated easily and in a controlled manner in the
preparation of multifunctional systems.20e22 Indeed, optimal bio-
medical devices often require several different properties that are
very challenging, or impossible, to find simultaneously on the same
molecule; therefore, different entities need to be combined to fulfill
the intended effect(s).23e26 Although a variety of poly(amido-
amine) dendrons (PAMAM dendrons)17,22,27e31 and Frechet-type
ꢀ
dendrons20,21,32e34 have been synthesized and utilized in the
preparation of various complex systems, only few poly(propylene
imine) (PPI) dendrons have been described.35,36 Yet, the PPI den-
dritic scaffold presents several distinctive features, such as a more
compact structure,37 an enhanced solubility in a wider range of
solvents due to its less polar interior38 while still displaying water
solubility, and predicted proton-sponge capability. The last feature
has an important role in drug delivery as it is hypothesized to
facilitate endosomal escape of the drug carrier after cell entry
through receptor mediated endocytosis.
* Corresponding author. Tel.: þ1 410 455 5601; fax: þ1 410 455 2608; e-mail
address: mdaniel@umbc.edu (M.-C. Daniel).
y
Contributed equally to the work.
Present address: Materials Science and Engineering, Johns Hopkins University,
z
3400 North Charles Street, Baltimore, MD 21219, USA.
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.01.070

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H. Pan et al. / Tetrahedron 69 (2013) 2799e2806
We report here the design, synthesis, and characterization of
protocol35 for the preparation of G1CN (1) and G2CN (3); however,
for the preparation of G3CN (5), acrylonitrile could not be used as
a solvent due to solubility issues, so methanol was used as the
solvent and 40 equiv of acrylonitrile were added. The presence of
the nitrile groups was evidenced by the distinctive FTIR band at
2248 cmꢀ1. Completion of the reaction was monitored by mass
spectrometry. Addition of acrylonitrile to the phenol group was not
observed, unless the reaction time was prolonged extensively or
the acrylonitrile quantities were increased (data not shown). When
addition to the phenol occurred, the 1H NMR spectrum displayed an
additional pair of aromatic doublets downfield from the peaks of
the unreacted phenol. The 1H NMR and mass spectrometry spectra
of the third generation PPI dendron (G3CN, 5) are shown in Fig. 1.
The NMR signal at 2.8 ppm (in CDCl3) is attributed to the protons of
the eight methylene groups next to the nitrile termini.
a novel dendron comprised of three parts: (1) a PPI dendron with
a phenolic focal point; (2) a tetraethylene glycol (TEG) spacer at the
focal point of the dendron; and (3) a protected thiol group at the
extremity of the spacer. These components were chosen in order to
impart the dendron with specific properties favoring its use in
multifunctional systems for biomedical applications. First, the PPI
and TEG parts are water-soluble entities, thus suitable for biological
media. Second, the proton-sponge capability of PPI is a great asset
in drug delivery, allowing for endo-lysosomal escape. Finally, the
primary amine termini of the dendron can be used in a large variety
of reactions (amide bond formation, nucleophilic substitution,
Michael addition, imine bond formation, (thio)urea bond forma-
tion, etc.) with diverse medically relevant moieties.
The nitrile reduction to primary amines was carried out using
borane dimethyl sulfide as the reducing agent.39 This method readily
reduced all nitrile groups into primary amines without noticeable
side-reactions. To ensure complete reduction, three additions of
5 equiv of BH3$SMe2 per primary amine group were performed over
the course of 8 h, which was followed by stirring at room temper-
ature for about 24 h. After complete reduction, the excess borane
was neutralized by very slow addition of cold methanol. Thus,
B(OMe)3 was formed as a side product and proved to be difficult to
remove from the dendron, with the corresponding signal appearing
in the 1H NMR spectrum at about 3.4 ppm. It is possible that some
2. Results/discussion
2.1. PPI dendron synthesis
Tyramine was chosen as the focal point for the preparation of
the PPI dendron because it displays both a primary amine group,
which can initiate the PPI dendritic growth, and a phenol group
whose reactivity is different enough from the primary amine so
that minimum competition occurs during the Michael addition of
the acrylonitrile. The synthetic route is shown in Scheme 1. The
conditions used for the Michael addition were similar to Voegtle’s
Scheme 1. PPI dendron synthesis.

H. Pan et al. / Tetrahedron 69 (2013) 2799e2806
2801
G3CN was reduced to G3NH2 only after attachment to the spacer
in order to avoid competition between primary amines and the
phenol group as the role of nucleophiles, which would lead to
undesired coupling of the spacer to the dendritic branches.
2.2. Spacer synthesis
In order to facilitate anchoring of the dendron to a variety of
other molecules or surfaces, we attached a spacer to the phenolic
focal point of the G3CN dendron. Tetraethylene glycol (TEG) was
selected as the spacer for a number of reasons: (1) its chemistry is
well established, (2) it is water-soluble, thus facilitating its use in
biological media; (3) it is a monodisperse molecule, unlike high
molecular weight poly(ethylene glycols), thus preserving the
monodispersity of the spacer-dendron construct to be prepared; (4)
other oligo(ethylene glycols) can easily be used in place of TEG by
following the same synthetic protocols as used for TEG.
TEG was functionalized with a bromine group at one end and
a thiol group at the other end to prepare for attachment to the
dendron and to facilitate grafting to a variety of other systems,
respectively. Scheme 2 displays the steps involved in the modifi-
cation of TEG. Monothiolated TEG (HS-TEG-OH, 7) was first pre-
pared as previously described in the literature,43 using
monotosylated TEG (TsO-TEG-OH, 6) as an intermediate followed
by addition of thiourea and basic hydrolysis. Since HS-TEG-OH is
readily prone to oxidation in air over time, (and also to avoid thiol
reactivity during subsequent reactions) the thiol group was pro-
tected. 2,4,6-Trimethoxybenzyl (Tmob) was chosen as the pro-
tective group for its stability under mild acidic conditions, strong
basic conditions, and reductive conditions. Moreover, its depro-
tection can be achieved in high yields using specific conditions (5%
TFA and cation scavenger, vide supra). TmobS-TEG-OH (8) was
prepared in the presence of 10% TFA in dichloromethane and ob-
tained in 46% yield after flash column chromatography. The pres-
ence of the protection is evidenced by the 1H NMR singlet at 6 ppm,
corresponding to the aromatic protons of Tmob group. Next,
TmobS-TEG-Br (10) was synthesized (using TmobS-TEG-OTs (9) as
an intermediate) to provide an excellent leaving group for attach-
ment to G3CN (5) dendron.
Fig. 1. Top: 1H NMR of G3CN PPI dendron in CHCl3; bottom: ESI mass spectrum of
G3CN PPI dendron.
2.3. Coupling of spacer and dendron
B(OMe)3 gets ‘entrapped’ inside the dendron through coordination
by the amines, which renders its removal difficult. We could solve
this problem by refluxing the reduced dendron overnight in fresh
methanol and isolating the dendron into water. Other trials for the
nitrile reduction were performed using various methods and
reducing agents, including CoCl2/NaBH4,35 Raney-Ni,40,41 and
Raney-Co,42 but they either resulted in incomplete reduction of all
eight nitrile groups or in partial degradation of the product. Addi-
tionally, although it needs to be carried out under inert atmosphere,
the reduction using BH3$SMe2 has the advantage over other tech-
niques of not requiring any specialized equipment and of using mild
conditions (room temperature, atmospheric pressure). G1NH2 (2)
and G2NH2 (4) were obtained with high yields (95% and 73%, re-
spectively), after purification by extraction into water, washes with
hexane and diethyl ether, and lyophilization. Dendrons 2 and 4 were
characterized by FTIR, NMR, and mass spectrometry. The stretching
band of the nitrile groups disappears in the FTIR spectra and a broad
band appears at frequencies around 2800e3500 cmꢀ1, indicative of
the primary amino groups. In the 1H NMR spectra, the signal that
corresponded to the methylene groups next to the nitrile functions
(at 2.8 ppm) is shifted upfield to about 1.5 ppm after complete
conversion of the nitriles to primary amines, and the eCH2e formed
next to eNH2 give a signal at 2.7e2.9 ppm depending on the
deuterated solvent (D2O, CD3OD or CDCl3).
The TEG spacer was attached to the PPI dendron through nu-
cleophilic substitution at the phenolic focal point of the dendron
(Scheme 3). Several trials using K2CO3 and DMF resulted in very
long reaction times (about 7 days). On the other hand, complete
coupling was achieved in about 48 h at room temperature using
NaH as the base and THF as the solvent. Furthermore, no depro-
tection of the thiol occurred, as verified in the 1H NMR spectrum
(Fig. S9, Supplementary data) by the singlet at 6 ppm still indicating
an integration of two protons for the two aromatic protons of Tmob
group (relative to the doublets of the phenolic protons, around
7 ppm, also indicating two protons). TmobS-TEG-G3CN (11) was
obtained as viscous light-yellow oil in 74% yield. As a note, the
coupling reaction was also performed with TmobS-TEG-OTs as the
starting material, but required longer reaction times.
After coupling to the TEG spacer, the nitrile termini of the den-
dronwere reduced to primary amines to form dendron 12, following
a similar procedure as when reducing G1CNand G2CN dendrons, i.e.,
using borane dimethyl sulfide (Scheme 3). Again, as shown in the 1H
NMR spectrum of Fig. 2, the Tmob protection remained intact under
these strong reductive conditions. Interestingly, we noticed that
TmobS-TEG-G3NH2 (12) was not only soluble in water and metha-
nol, but also readily dissolved in chloroform, unlike G2NH2; thus, the
presence of the spacer provides the additional advantage of de-
creasing the overall polarity of the dendron, which enables its use in

2802
H. Pan et al. / Tetrahedron 69 (2013) 2799e2806
Scheme 2. Spacer synthesis.
Scheme 3. Synthesis of the TEGylated PPI dendron.
a larger variety of solvents. Mass spectrometry of 12 was carried out
using atmospheric pressure chemical ionization (APCI) because
of difficulties encountered in obtaining a signal using electrospray
ionization (ESI). Because APCI is a slightly harsher technique than
ESI, a few fragmentations occurred and are thus observed in the
spectrum. The lost fragments correspond to (CH2)2NH,
(CH2)2NHþ(CH2)3NH, (CH2)2N[(CH2)2NH]2 and 3ꢁ (CH2)3NH.
2.4. Thiolated PPI dendron
As a control reaction, deprotection of Tmob was attempted on
dendron 11 (Scheme 4). Using 5% TFA in the presence of a cation
scavenger (Et3SiH in this case, but L-cysteine is another possible
scavenger44), complete deprotection was afforded at room tem-
perature overnight, yielding over 90% of HS-TEG-G3CN (13) after

H. Pan et al. / Tetrahedron 69 (2013) 2799e2806
2803
to the Tmob aromatic protons, disappeared, as well as the singlet
from the Tmob methoxy groups (at 3.74 ppm) and the singlet from
Tmob benzylic protons (at 3.68 ppm). The deprotection was further
confirmed by ESI mass spectrometry (Fig. S12, Supplementary
data).
The stability of the thiol group in dendron 13 was investigated:
indeed, HS-TEG-OH is known to oxidize after a few days in air;45
additionally, the presence of trace transition metal (in buffers, re-
agents etc.) can catalyze thiol oxidation.46,47 Thus, after isolation
and characterization, HS-TEG-G3CN (13) was monitored for its
stability under ambient atmosphere for a period of 15 days. No
disulfide formation was noticed by mass spectrometry over this
time period. Furthermore, a control experiment was carried out by
adding iron (III) chloride to an aqueous solution of dendron 13 and
by assessing the oxidation of the thiol group (Supplementary data):
an Ellman’s test revealed that 94% of dendron 13 remained in the
thiol form after 20 min incubation with FeCl3 (the same conditions
led to complete thiol oxidation in Ref. 46). This observed increase in
thiol stability after coupling to the dendron suggests that the
dendron sterically ‘protects’ the thiol from disulfide formation.
2.5. Potential for proton-sponge effect
Similar to polyethyleneimine (PEI) structures,48 the PPI den-
drons presented herein display a number of tertiary amines. PEI has
demonstrated the ability to induce endosomal escape by the ‘pro-
ton-sponge effect’49 due to the protonation of its tertiary amines at
low pH. Thus, we verified the protonation behavior of the tertiary
amines of our PPI dendron by studying the z potential of 11 at a pH
range from 2 to 7. By studying 11 instead of 12, we avoided in-
terferences from the protonation of the terminal primary amines of
12. As shown in Fig. 3, a decrease in pH from 7 to 5 induces only
a slight increase in the z potential of 11; however, the overall charge
of 11 dramatically increases when the pH decreases from 5 to 4,
suggesting a high degree of protonation when dropping the pH
below 5. This pH of 4e5 corresponds to lysosomal pH, and conse-
quently, protonation of our PPI dendron at this pH is likely to induce
osmotic swelling and lysis of the lysosome through the proton-
sponge effect (as already observed using PEI50), and hence enable
release of the dendron into the cytosol.51 This would constitute
a significant benefit in the field of drug delivery. Further experi-
ments will be carried out in order to check this hypothesis.
Fig. 2. Top: 1H NMR of TmobS-TEG-G3NH2 (12) in CHCl3; bottom: APCI mass spectrum
of TmobS-TEG-G3NH2 (12).
purification. While protected dendron 11 is soluble in chloroform,
once the thiol group is deprotected, its solubility in CHCl3 is neg-
ligible and it becomes solely soluble in polar solvents, such as water,
methanol or acetone. 1H NMR in deuterated acetone (Fig. S11,
Supplementary data) allows for the observation of the thiol proton,
with a signal at 1.26 ppm. Also, the signal at 6 ppm, corresponding
3. Conclusion
We prepared a PPI dendron of generation 3 (displaying eight
branches) with its focal point coupled to a Tmob-protected thio-
lated TEG. This dendron presents several attractive features: (1) the
protected form (dendron 12) is soluble in a variety of solvents
Scheme 4. Deprotection of TmobS-TEG-G3CN (11).

2804
H. Pan et al. / Tetrahedron 69 (2013) 2799e2806
amino function) was heated to reflux for 24 h. After 24 h, the solution
70
60
50
40
30
20
10
0
was cooled to room temperature, and excess acrylonitrile was re-
moved under reduced pressure. The resulting oil was dissolved in
chloroform, washed with ammonium hydroxide and water, dried
over sodium sulfate, filtered, and concentrated under reduced pres-
sure. The resulting white solid was recrystallized using chloroform to
yield the product as a white solid (2.077 g, 85% yield): 1H NMR
(400 MHz, CDCl3):
d
(ppm) 7.04 (d, J¼8.2 Hz, 2H), 6.75 (d, J¼8.7 Hz,
2H), 2.88 (t, J¼6.9 Hz, 4H), 2.65e2.77 (m, 4H), 2.39 (t, J¼6.9 Hz, 4H).
13C NMR (100 MHz, CDCl3):
d (ppm) 154.4, 131.6, 130.1, 118.8, 115.6,
1
2
3
4
5
6
7
8
56.1, 50.1, 33.6, 17.4. IR nmax (neat) 2245 cmꢀ1 (nitrile). HRMS (ESI)
calcd for C14H18N3O (MþHþ) 244.1444, observed 244.1448.
pH
Fig. 3. Protonation of TmobS-TEG-G3CN (11) at different pHs:
TEG-G3CN (11) versus pH in water.
z potential of TmobS-
4.2.2. G1NH2 (2). To a solution of 1 (1.02 g, 4.19 mmol) in anhy-
drous THF (20 mL) under argon was added borane dimethyl sulfide
complex (4.5 mL, 47 mmol, 5 equiv per nitrile group) using a sy-
ringe. The reaction mixture was stirred at room temperature until
gelification was evident. The borane dimethyl sulfide complex was
added in two more additions over the course of one day. The
mixture was allowed to stir overnight. Methanol was added slowly
at 0 ꢂC until no further bubbling or reaction was observed. The
solvent was removed under reduced pressure, after which fresh
methanol (50 mL) was added to the residual oil and heated to reflux
overnight. Upon cooling to room temperature, the solvent was re-
moved under reduced pressure. The oily residue was taken up in
water (10 mL) and washed with diethyl ether (3ꢁ5 mL), hexane
(3ꢁ5 mL), and ethyl acetate (1ꢁ5 mL), and lyophilized to yield
1.005 g of the product as a clear colorless oil (95% yield). 1H NMR
ranging from chloroform to water, which will ease further conju-
gation of the dendritic branches with diverse moieties; (2) the
deprotected form (dendron 13) is stable against thiol oxidation in
air, so no specific precautions are needed during its handling or
short-term storage; (3) the thiolated spacer will assist with grafting
of the PPI dendron onto other constructs, such as other dendrons,
surfaces, nanoparticles etc.; (4) the tertiary amines of the PPI
dendron interior show potential to act as proton-sponges for the
biological purpose of endosomal escape. Overall, this novel PPI
dendron represents a suitable candidate for biomedical use, and
displays features that facilitate its use as a building-block in the
construction of diverse multifunctional systems.
4. Experimental section
4.1. Materials
(400 MHz, CD3OD)
d
(ppm) 6.91 (d, J¼8.2 Hz, 2H), 6.59 (d, J¼8.7 Hz,
2H), 2.43e2.55 (m, 12H), 1.53 (m, 4H). 13C NMR (100 MHz, CD3OD):
d
(ppm) 157.2, 132.3, 130.8, 116.5, 62.9, 52.9, 41.2, 33.2, 22.4. HRMS
(ESI) calcd for C14H26N3O (MþHþ) 252.2070, observed 252.2075.
All solvents and reagents were purchased from commercial
sources and used without purification except where specified.
Anhydrous solvents (THF, CH2Cl2, CH3CN, MeOH, and DMF) were
retrieved from the solvent purification system (mBraun MB-SPS)
and stored under a nitrogen atmosphere shortly before use.
Proton and carbon nuclear magnetic resonance spectra (1H NMR
and 13C NMR, respectively) were obtained using a JEOL ECX
400 MHz NMR spectrometer, operated at 400 and 100 MHz, re-
4.2.3. G2CN (3). Acrylonitrile (50 mL), 2 (3.076 g, 12.24 mmol) and
glacial acetic acid (2.5 mL, 20 mmol per primary amino function)
were added together at room temperature and heated to reflux for
48 h. After 48 h, the solutionwas cooled to room temperature. Excess
acrylonitrile was removed under reduced pressure. The resulting oil
was dissolved in chloroform, washed with ammonium hydroxide
and water, dried over sodium sulfate, filtered, and concentrated
under reduced pressure. The crude yellow oil was purified via col-
umn chromatography (80:10:1 CHCl3/MeOH/NH4OH) to yield 3.21 g
of the product as a cloudy viscous oil (57% yield): Rf¼0.472 (SiO2,
spectively. Chemical shifts (d) are in parts per million using an in-
ternal standard to TMS as the reference, set to 0.00 ppm.
Abbreviations used in proton data are as follows: s (singlet),
d (doublet), t (triplet), q (quartet), quin (quintet), dd (doublet of
doublets), dt (doublet of triplet), m (multiplet), br (broad). NMR
solvents were purchased from Cambridge Isotope Laboratories
(Andover, MA). Mass spectra were recorded at the UMBC Molecular
Characterization and Analysis Complex (MCAC) (Catonsville, MD).
Fourier transform infrared spectroscopy (FTIR) was performed us-
ing a Perkin Elmer Paragon 500 with a horizontal ATR accessory
and ZnSe crystal flat plate.
80:10:1 CHCl3/MeOH/NH4OH). 1H NMR (400 MHz, CDCl3)
d (ppm)
7.00 (d, J¼8.2 Hz, 2H), 6.72 (d, J¼8.2 Hz, 2H), 2.76 (t, J¼6.9 Hz, 8H),
2.63 (br s, 4H), 2.52 (t, J¼7.3 Hz, 4H), 2.46 (t, J¼6.9 Hz, 4H), 2.41 (t,
J¼6.9 Hz, 8H), 1.56 (quin, J1¼7.3 Hz, J2¼6.9 Hz, 4H). 13C NMR
(100 MHz, CDCl3): d (ppm) 154.7, 132.2, 130.0, 119.1, 115.6, 55.7, 51.6,
51.5, 49.7, 32.3, 24.9, 17.0. IR nmax (neat) 2249 cmꢀ1 (nitrile). HRMS
(ESI) calcd for C26H38N7O (MþHþ) 464.3132, observed 464.3133.
Thin layer chromatography on silica gel (SiO2) was performed
4.2.4. G2NH2 (4). To a solution of 3 (0.270 g, 0.582 mmol) in an-
hydrous THF (20 mL) under argon was added borane dimethyl
sulfide complex (1.1 mL, 11.65 mmol, 5 equiv per nitrile group)
using a syringe. The reaction mixture was stirred at room tem-
perature until gelification was evident. The borane dimethyl sulfide
complex was added in two more additions over the course of the
day. The mixture was allowed to stir for one additional day at room
temperature. Methanol was added slowly at 0 ꢂC until no further
bubbling or reaction was observed. The solvent was removed under
reduced pressure, after which fresh methanol (50 mL) was added to
the residual oil and heated to reflux overnight. Upon cooling to
room temperature, the solvent was removed under reduced pres-
sure. The oily residue was taken up in 15 mL water and washed with
hexane (2ꢁ10 mL) and diethyl ether (2ꢁ10 mL) and lyophilized
overnight to yield the product as a white fluffy powder (0.203 g,
ꢁ
using Whatman Partisil K5 150 A glass plates (250
mm) without
preadsorbent zone. Column chromatography was performed using
silica gel (63e200 m) from Uline, Inc., and eluted with the in-
m
dicated solvent system.
Reactions at ‘room temperature’ (RT) were conducted at ambi-
ent laboratory temperature (20e26 ꢂC). Distillation of solvents on
a rotary evaporator at 20e50 ꢂC and 25e15 mmHg is referred to as
‘removal of solvent under reduced pressure’; ‘in vacuo’ refers to
pressures of 1.0e0.25 mmHg.
4.2. Syntheses of dendrons
4.2.1. G1CN (1). Acrylonitrile (100 mL), tyramine (1.373 g,
10.01 mmol) and glacial acetic acid (1.25 mL, 20 mmol per primary

H. Pan et al. / Tetrahedron 69 (2013) 2799e2806
2805
73% yield). 1H NMR (400 MHz, CD3OD)
2H), 2.81e2.43 (m, 28H), 1.69e1.56 (br m, 12H). 13C NMR (100 MHz,
d
(ppm) 7.00 (d, 2H), 6.69 (d,
1.90 mmol, 46% yield) was yielded as a yellow oil through flash
column chromatography (2:1 EtOAc/Hex): Rf¼0.208 (SiO2, 2:1
CD3OD):
d
(ppm) 155.5, 131.1, 129.4, 115.0, 61.5, 55.7, 51.8, 51.3, 39.2,
EtOAc/Hex); 1H NMR (400 MHz, CDCl3)
d (ppm) 5.98 (s, 2H), 3.67 (s,
31.9, 27.3, 23.5. HRMS (ESI) calcd for C26H54N7O (MþHþ) 480.4384,
6H), 3.65 (s, 3H), 3.61 (s, 2H), 3.57 (br, 2H), 3.51e3.43 (m, 12H), 2.55
observed 480.4391.
(t, 2H). 13C NMR (100 MHz, CDCl3)
d
(ppm) 160.0, 158.5, 107.6, 90.3,
77.2, 72.3, 70.6, 70.3, 70.2, 70.1, 69.9, 61.3, 55.5, 55.1, 30.7, 23.5. HRMS
4.2.5. G3CN (5). To a solution of 4 (6.38 g, 13.31 mmol) in methanol
(100 mL) was added acrylonitrile (50 mL, 56 equiv). The solution
was heated to reflux for 48 h. The reaction was then cooled to room
temperature and the solvent was removed under reduced pressure.
The residue was taken up in deionized water and extracted with
dichloromethane. The combined organic layers were dried over
sodium sulfate, filtered and the solvent was removed under re-
duced pressure. The crude oil was washed with hexane and diethyl
ether until no further impurities were observed via 1H NMR to yield
a clear yellow oil (7.54 g, 63% yield). 1H NMR (400 MHz, CDCl3)
(ESI) calcd for C18H30O7S (MþHþ) 391.1785, observed 391.1762.
4.3.4. TmobS-TEG-OTs (9). To a solution of 21 (538 mg, 1.38 mmol)
in THF (50 mL) was added 2 mL 2 M NaOH at room temperature. The
solution was stirred for 30 min until TsCl (688 mg, 4 mmol) was
added. The reaction mixture was stirred overnight before addition of
30 mL of water. The resulting mixture was separated and the water
layer was washed with ethyl acetate (3ꢁ30 mL). The combined or-
ganic layer was washed with brine, dried over magnesium sulfate,
filtered, and concentrated under reduced pressure. The product
(638 mg, 1.17 mmol, 85% yield) was yielded as an oil through flash
column chromatography (2:1 EtOAc/Hex): Rf¼0.65 (SiO2, 2:1 EtOAc/
d
(ppm) 7.01 (d, 2H), 6.73 (d, 2H), 2.20e2.92 (m, 60H), 1.51e1.64 (br
m, 12H). 13C NMR (100 MHz, CDCl3):
d
(ppm) 155.1, 131.7, 129.9,
119.1, 115.7, 55.9, 52.2, 51.7, 51.5, 49.6, 32.4, 24.9, 24.3, 17.0. IR nmax
Hex); 1H NMR (400 MHz, CDCl3)
d
(ppm) 7.62 (d, 2H), 7.17 (d, 2H), 5.97
(s, 2H), 3.98 (t, 2H), 3.65e3.40 (m, 23H), 2.52 (t, 2H), 2.26 (s, 3H). 13C
NMR (100 MHz, CDCl3) (ppm) 160.4,158.7,144.8,132.9,129.9,127.9,
(neat) 2247 cmꢀ1 (nitrile). HRMS (ESI) calcd for C50H78N15
O
(MþHþ) 904.6508, observed 904.6526.
d
107.9, 90.5, 70.8, 70.6, 70.5, 70.5, 70.1, 69.4, 68.6, 30.9, 23.8, 21.5. HRMS
4.3. Synthesis of spacer
(ESI) calcd for C25H37O9S2 (MþNaþ) 567.1693, observed 567.1686.
4.3.1. TsO-TEG-OH (6). To
a
solution of tetraethylene glycol
4.3.5. TmobS-TEG-Br (10). To a solution of 9 (420 mg, 0.77 mmol)
in 40 mL acetone was added LiBr (0.90 g, 10.4 mmol) at room
temperature and then heated to reflux overnight. The solution was
concentrated under reduced pressure and the resulting oil was
redissolved in ethyl acetate (80 mL) and washed with water and
brine, dried over magnesium sulfate, filtered, and concentrated
under reduced pressure. The product (198 mg, 0.44 mmol, 57%
yield) was yielded as a pale yellow oil through flash column chro-
matography (2:1 Hex/EtOAc): Rf¼0.3 (SiO2, 2:1 Hex/EtOAc); 1H
(100.1 g, 515.2 mmol) in 230 mL THF was added a solution of so-
dium hydroxide (6.89 g, 172.3 mmol) dissolved in 20 mL deionized
water. The mixture was cooled to 0 ꢂC and toluene sulfonyl chloride
(9.81 g, 51.5 mmol) in 20 mL THF was added dropwise. The reaction
was allowed to stir at 0 ꢂC for 2 h. The solution was poured into
deionized water and the aqueous layers were separated and
extracted with dichloromethane. The organic layers were com-
bined and washed with water, dried over sodium sulfate, filtered
and concentrated under reduced pressure to yield 16.17 g of
product as a clear colorless oil (90% yield, based on the toluene
sulfonyl chloride): Rf¼0.634 (SiO2, EtOAc); 1H NMR (400 MHz,
NMR (400 MHz, CDCl3)
3.68 (s, 2H), 3.58e3.54 (m, 10H), 3.39 (t, 2H), 2.62 (t, 2H). 13C NMR
(100 MHz, CDCl3) 160.3, 158.8, 108.1, 90.6, 71.2, 71.0, 70.7, 70.6,
d (ppm) 6.05 (s, 2H), 3.74e3.72 (m, 11H),
d
CDCl3)
d
(ppm) 7.79 (d, 2H), 7.33 (d, 2H), 4.16 (t, 2H), 3.55e3.72 (m,
70.2, 55.8, 55.4, 31.0, 30.6, 23.9. HRMS (ESI) calcd for C18H30BrO6S
14H), 2.44 (s, 3H). Spectral data agreed with literature values.52
(MþHþ) 453.0941, observed 453.0940.
4.3.2. HS-TEG-OH (7). Absolute ethanol (65 mL) and thiourea
(0.471 g, 6.19 mmol) were added to dried 6 (2.03 g, 5.83 mmol) and
allowed to reflux under argon for 24 h. After 24 h, the reaction was
cooled to room temperature and sodium hydroxide (0.700 g,
17.51 mmol) in absolute ethanol/water (9:1 v/v, 8.5 mL) was added.
The reactionwas heated to refluxunderargonfor 3 h. Thereactionwas
then cooled to room temperature, acidified with concentrated
hydrochloric acid to pH 2, filtered to remove the salts, and concen-
trated under reduced pressure. The crude oil was purified via column
chromatography(10:1EtOAc/absoluteethanol)toobtain1.022gof the
product as a pale yellow oil (83.3% yield, 2 steps): 1H NMR (400 MHz,
4.4. Dendron-spacer
4.4.1. TmobS-TEG-G3CN (11). To
a solution of 5 (230 mg,
0.25 mmol) in 10 mL of anhydrous THF under argon was added
sodium hydride (60% dispersion in mineral oil) (23 mg, 0.58 mmol).
The mixture was stirred at room temperature for several hours
until the solution turned yellow. At this point, the yellow mixture
was transferred (using cannula) to a solution of 10 (410 mg,
0.90 mmol) in anhydrous THF and the resulting solution was stirred
at room temperature for 48 h. 1 mL of methanol was then added to
neutralize the excess of sodium hydride. The solvent was evapo-
rated under reduced pressure after being stirred for 15 more
minutes. The oily residue was washed with diethyl ether (3ꢁ5 mL).
The washed oil was dissolved in a minimum of ethyl acetate and
reprecipitated with cold diethyl ether. The residue was dried in
vacuo to yield the product as light-yellow viscous oil (236 mg,
CDCl3)
d (ppm) 3.58e3.75 (m, 14H), 2.70 (dt, 2H), 2.46 (broad s, 1H),
1.62 (t, 1H). Spectral data agreed with literature values.45,53
4.3.3. TmobS-TEG-OH (8). To a solution of 7 (870 mg, 4.14 mmol) in
40 mL of anhydrous dichloromethane under nitrogen was slowly
added trifluoroacetic acid (4 mL) at room temperature. The solution
was stirred for 3 min until 2,4,6-trimethoxybenzyl alcohol (984 mg,
4.97 mmol) in 5 mL of anhydrous dichloromethane was added.
[2,4,6-Trimethoxybenzyl alcohol was previously prepared by fol-
lowing the procedure from the literature.54] The reaction mixture
was stirred for 2 h at room temperature after which saturated
NaHCO3 was slowly added to neutralize the solution. The resulting
mixture was separated and the water layer was washed with
dichloromethane (3ꢁ50 mL). The combined organic layer was
washed with brine, dried over magnesium sulfate, filtered, and
concentrated under reduced pressure. The product (743 mg,
0.18 mmol, 74% yield). 1H NMR (400 MHz, CDCl3)
2H), 6.80 (d, 2H), 6.03 (s, 2H), 4.00 (t, 2H), 3.72e3.71 (m, 11H), 3.66
(s, 2H), 3.62e3.51 (m, 10H), 2.75e2.71 (m, 14H), 2.61e2.57 (m, 8H),
2.47e2.32 (m, 40H), 1.52e1.48 (m, 12H). 13C NMR (100 MHz, CDCl3)
d (ppm) 7.06 (d,
d
(ppm) 160.3, 158.8, 157.1, 132.9, 129.6, 119.0, 114.6, 108.0, 90.6,
70.6, 70.5, 70.4, 70.3, 69.9, 69.5, 67.2, 55.8, 55.6, 55.2, 51.9, 51.9, 51.4,
51.2, 49.3, 32.2, 30.8, 24.7, 24.3, 23.6, 16.6. HRMS (ESI) calcd for
C68H106N15O7S (MþHþ) 1276.8115, observed 1276.8170.
4.4.2. TmobS-TEG-G3NH2 (12). To
0.074 mmol) in 5 mL of anhydrous THF under argon was slowly
a solution of 11 (94 mg,

2806
H. Pan et al. / Tetrahedron 69 (2013) 2799e2806
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added borane dimethyl sulfide complex (1.2 mL, 11.84 mmol) at
room temperature. The mixture was stirred at room temperature
for 2 h until another addition of borane dimethyl sulfide complex
(1.2 mL, 11.84 mmol). The mixture was stirred for 24 h at room
temperature until 10 mL methanol was added slowly at 0 ꢂC. The
reaction mixture was evaporated under reduced pressure and dried
in vacuo for 1 h. The white residue was washed with ethyl ether
(3ꢁ10 mL) and ethyl acetate (3ꢁ5 mL). After washing, the residue
was dried in vacuo for 1 h until 20 mL of anhydrous methanol was
added. The solution was heated to reflux overnight and then cooled
to room temperature. The solution was condensed under reduced
pressure and then washed with ethyl ether (3ꢁ10 mL). The residue
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12. Majoros, I. J.; Myc, A.; Thomas, T.; Mehta, C. B.; Baker, J. R., Jr. Biomacromolecules
0.06 mmol, 82% yield). 1H NMR (400 MHz, CDCl3)
d (ppm) 7.11 (br,
2006, 7, 572e579.
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2H), 6.85 (d, 2H), 6.17 (s, 2H), 4.06 (br, 2H), 3.77 (br, 9H), 3.68e3.55
(m, 14H), 2.65e2.47 (br m, 62H), 1.70e1.62 (br, 28H). 13C NMR
(100 MHz, CD3OD)
d (ppm) 162.1, 160.3, 158.8, 134.1, 130.9, 115.9,
109.2, 91.8, 72.2, 71.9, 71.8, 71.4, 71.0, 67.0, 62.9, 56.5, 56.4, 56.0,
54.2, 53.7, 53.4, 52.9, 41.1, 41.0, 32.0, 30.7, 30.3, 26.2, 24.8, 15.6.
HRMS (APCI) calcd for C68H138N15O7S (MþHþ) 1309.0619, observed
1309.0771.
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4.4.3. HS-TEG-G3CN (13). To a 2-neck 25 mL-round-bottom-flask
containing 11 (93 mg, 0.073 mmol) and a stir bar, 9 mL anhydrous
dichloromethane was added under argon and the solution was
stirred. Triethylsilane (0.3 mL, 3% vol), followed by trifluoroacetic
acid (0.5 mL, 5% vol) were then injected to the solution of 24 under
argon. The reaction mixture was stirred under argon at room
temperature overnight. Evidence of deprotection is displayed by
the observation of orange droplets at the surface of the CH2Cl2
solution, due to the insolubility of the formed thiol in CH2Cl2. After
12 h, the mixture was evaporated in vacuo at room temperature for
several hours (to ensure TFA removal). The residue obtained was
washed with CHCl3 (3ꢁ5 mL) and hexane (2ꢁ5 mL) and dried in
vacuo to yield 26 (75 mg, 0.068 mmol, 93%) as a pale yellow oil. 1H
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NMR (CD3COCD3):
d (ppm) 7.25 (d, 2H), 6.89 (d, 2H), 4.09 (t, 2H),
3.78 (t, 2H), 3.63e3.56 (br m, 10H), 3.44 (m, 24H), 2.89 (t, 16H), 2.73
(t, 4H), 2.66 (t, 16H), 2.51 (br t, 2H), 2.03 (m, 12H), 1.26 (s, 1H); 13C
32. Das, J.; Frechet, J. M. J.; Chakraborty, A. K. J. Phys. Chem.
13768e13775.
B 2009, 113,
NMR (100 MHz, CD3COCD3) d (ppm) 157.1, 132.9, 129.6, 119.0, 114.6,
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1999, 26, 501e502.
72.7, 70.6, 70.5, 70.4, 70.0, 69.5, 67.5, 55.8, 51.9, 51.3, 50.1, 49.9, 48.9,
29.6, 29.2, 23.7, 21.4, 15.9. IR nmax (neat) 2248 cmꢀ1 (nitrile). HRMS
(ESI) calcd for C58H94N15O4S (MþHþ) 1096.73339, observed
1096.7560.
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The authors are grateful for the financial support from the AACR/
PanCAN (Career Development Award, grant no. 08-20-25-DANI)
and from the Department of Defense (Prostate Cancer Research
Program, CDRMP, grant no PC081299). They also would like to ac-
knowledge Josh Wilhide for the acquisition of the HRMS spectra.
43. Daniel, M.-C.; Grow, M. E.; Pan, H.-M.; Bednarek, M.; Ghann, W. E.; Zabetakis,
K.; Cornish, J. New J. Chem. 2011, 35, 2366e2374.
Supplementary data
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1H NMR spectra and 13C NMR spectra of G1CN (1), G1NH2 (2),
G2CN (3), G2NH2 (4), G3CN (5), TmobS-TEG-OH (8), TmobS-TEG-
OTs (9), TmobS-TEG-Br (10), TmobS-TEG-G3CN (11), TmobS-TEG-
G3NH2 (12), and HS-TEG-G3CN (13). ESI spectrum of HS-TEG-G3CN
(3). FTIR of compounds 1, 2, 3, 4, and 5. Control experiment on the
effect of the presence of trace transition metal on the thiol group of
dendron 13. Supplementary data related to this article can be found
at http://dx.doi.org/10.1016/j.tet.2013.01.070.
References and notes
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