Synthesis of Chemical Linkers for
Bionanoconstructs
Trevor B. Parker, Liang Chen, Michael B. Bostwick, Nishi
Begum, Richard D. Gandour
Department of Chemistry, Virginia Tech, Blacksburg. VA,
24061
tparker4@vt.edu
INTRODUCTION
Sterol surfactants, a class of poly(ethylene glycol) (PEG)
lipids, find many uses in topical formulations (e.g. cosmetics
and skin creams) and in drug delivery (e.g. micelles and
protective coatings for “stealth” liposomes)1. The amphiphilic
nature and low toxicity of PEG lipids make them prime
candidates for these purposes. Much recent research has
focused on the drug encapsulation by micelles utilizing PEG
lipids. Drugs with low water solubility can aggregate within
the lipid core of micelles, and thus be dissolved in solution for
easier drug delivery2.
PEG lipids play increasingly large roles in liposomal and
nanoparticle drug delivery3,4,5. Conventional liposomes can be
beneficial when targeting the immune system (e.g. the
mononuclear phagocyte system, which includes nonspecific
defense). When targeting other systems, conventional
liposomes face major disadvantages such as short blood
circulation time and low target selectivity. Stealth liposomes
(or PEGylated liposomes) incorporate PEG lipids in the
liposomal phospholipid membrane such that PEG chains
occupy the exterior of the liposome. These PEG chains hinder
binding between blood serum and liposomes. This increases
blood circulation time and liposomal stability, while
decreasing both uptake by the phagocyte system and drug
delivery to untargeted cells3, 4.
Other research has investigated the attaching of a
headgroup to the end of the polymer chain on PEG lipids6.
These headgroups are often dendritic, and can serve to modify
physical properties or adhere to solid surfaces. The capability
for adhesion gives rise to many possible applications in
bionanoconstructs.
Our interest resides in using modified cholestanol – PEGs
as building blocks for novel bionanoconstructs. Attachment of
a dendritic headgroup to the end of the PEG chain creates
novel amphiphilic polymers. A tricarboxyl headgroup presents
interesting possibilities in its potential to adhere to metal oxide
nanoparticles. These novel polymers may enable the creation
of cutting-edge bionanoconstructs for drug delivery.
EXPERIMENTAL
Materials. 3β,5α-Cholestanol, recrystallized from EtOH,
was heated at 70 °C under high vacuum for 24 h.
Tetrahydrofuran (THF), dried with sodium in the presence of
benzophenone, was distilled after the solution turned dark
blue. Potassium naphthalenide was prepared every 2 weeks in
dry THF from naphthalene (99%, Acros) and potassium (in
mineral oil, Aldrich) with a concentration of ~0.9 mol/L and
stored under argon in an aluminum foil wrapped flask.
Ethylene oxide (EO, 99.5+%, Aldrich) was transferred to a
flame-dried-under-argon vial that was sealed with a septum,
secured with copper wire. Before transferring EO, the vial,
which was marked with a specific volume, was placed in a dry
ice/acetone bath. EO was condensed in the vial until the
specified volume was reached; the weight of EO was
estimated by presuming a density (0.95 g/cm3 at –40 °C).3
Then, EO was transferred to the reaction flask via a cannula
under argon pressure. Weisocyanate™, (98%) recrystallized in
hexane. Pentamethyldiethyltriamine, (Acros Organics)
purified by distillation. TFA, (99% Sigma Aldrich).
Polymerization. Cholestanol (0.1–0.5 g) was dissolved in
dry THF (20–40 mL) in a 100-mL flask. Potassium
naphthalenide in THF(~0.9 M, 0.9 eq) was added dropwise by
syringe into the stirred flask. The flask was placed into a dry
ice/acetone bath and cooled for at least 10 min. Then,
condensed EO (10–200 eq) was added to the flask. After
addition, the bath was removed, and the flask was allowed to
reach room temperature. After 24 h, the reaction was
quenched with concentrated HCl (1 or 2 drops). The reaction
mixture was poured into hexane (100 mL). Work-up depended
on the molecular weight of the polymer.
Isolation. For polymers with a MW ≥ 2K, a white solid
precipitated and was isolated by vacuum filtration. The solid
was dried under high vacuum overnight. For polymers with a
MW ≤ 2 K, the solution was concentrated by rotary
evaporation to give an oil or wax, which was dried under high
vacuum.
PEG Lipid Chromatography. Crude polymerization
products (~2g) were purified and fractionated by using a
13.5×4.5-cm silica gel column. Varying ratios of
CH2Cl2:MeOH (20:1 to 5:1) served as the gradient eluent. A
large fraction (150 mL) followed by 40–60 fractions (25 mL)
were collected. Typically, 10–20 fractions were pooled and
concentrated by rotary evaporation and dried overnight under
high vacuum.
Headgroup Attachment. Fractionated polymerization
products with low PDIs were chosen to be used in reactions
with Weisocyanate. PEG lipid (0.055 mmol) was reacted
with a 3:1 molar excess of Weisocyante (0.164 mmol). PEG
lipid and Weisocyanate were placed in a 50-mL round
bottom flask with a stir bar, and covered with a septum. An oil
bath was used to bring the temperature to 70 C. Two drops of
pentamethyldiethylenetriamine were then added to the
reaction flask. After 24 h of stirring, argon was flushed
through the flask by syringe for 2 h, exiting through a bubbler.
This was carried out in attempt to remove the
pentamethyldiethylenetriamine. The flask contents were then
dissolved in CH2Cl2 (20 mL), followed by concentration by
rotary evaporation. The gel was then dried under high vacuum
for 24 h.
Triester Chromatography. Crude products (~1g) were
purified and fractionated by using a 13.5×4.5-cm silica gel
column. Varying ratios of CH2Cl2:MeOH (20:1 to 5:1) served
as the gradient eluent. A large fraction (200 mL) followed by
75-150 fractions (25 mL) were collected. Typically, 10-25
fractions were pooled, concentrated by rotary evaporation, and
dried overnight under high vacuum.
Deprotection. Purified triester (~0.1g) was placed in an
RB flask, and sealed with a septum. TFA (~0.5 mL) was
added by syringe. The solution was stirred for 5 h at RT. After
5 h, CH2CL2 (~20 mL) was added to the flask. The CH2Cl2
was then removed by rotary evaporation. This solvent chase
was repeated 7 times in an attempt to remove the TFA. The
resulting oils were dried on high vacuum for 24 h.
Linker Purification. Crude triacids were treated with
activated carbon to remove any fluorinated compounds.
Triacids were dissolved in CH2Cl2 (~15 mL), and activated
carbon (~2g) was added and stirred. After 5 min, solution was
vacuum filtered over MgSO4. Vacuum filtration was repeated
4 times to remove all the carbon. Clear solutions were
concentrated by rotary evaporation to yield clear oils.
Characterization. All products, before and after
purification, were characterized with 1H NMR in DMSO-d6 on
a Varian Inova 400 MHz. Integrations were normalized to the
C19 methyl group (δ 0.72, 3H). SEC was used to determine
PDI and molecular weight of PEG lipids and triesters,
employing chloroform as a solvent.
RESULTS AND DISCUSSION
The living polymerization of ethylene oxide on the C-3
alcohol group of cholestanol with K naphthalenide as an
initiator yielded crude PEG lipids with varying molecular
weights. Varying molecular weights were desired because the
optimum polymer length is not yet known. Reactions were
terminated by adding a drop of concentrated HCl before
pouring into hexane (Table 1). The polymers were
chromatographed and several showed excellent PDIs (>1.05)
and agreement between the MW determined by NMR and that
determined by SEC. NMR MWs were calculated with
integrations of the PEG peak at δ 3.5 ppm.
Polymer
B1
B2
B3
B4
B5
B6
B7
B8
B9
B10
B11
B12
B13
B14
B15
B16
B17
B18
B19
B20
B21
B22
Table 1. PEG Lipids After Chromatography
MW (NMR)
MW (SEC)
PDI (SEC)
762
531
1.03
828
548
1.09
1048
670
1.14
1202
1334
1.05
1400
1410
1.09
1851
2266
1.05
1994
1973
1.12
2269
2104
1.09
2280
2819
1.03
2478
2868
1.05
2731
2708
1.10
3028
3809
1.02
3512
3131
1.11
3688
3849
1.20
3908
2931
1.12
4000
4416
1.03
4271
5591
1.02
4524
4122
1.30
4876
5595
1.02
5888
6828
1.03
6500
6994
1.14
9012
8342
1.07
Figure 1. PEG Lipid 1H NMR
Polymers with low polydispersities and agreeing
molecular weights by NMR and SEC were chosen for
reactions with Weisocyanate™.
Scheme 1. Anionic Polymerization of EO on Cholestanol
Scheme 2. Headgroup Attachment on Terminal End of
PEG Chain
CONCLUSIONS
The techniques discussed in this report show a successful
method for the synthesis and purification of linker molecules
to be used in bionanoconstructs. Firstly, PEG lipids of
varying molecular weights were synthesized. Secondly, PEG
lipids were reacted with Weisocyanate™ to form triester—
PEG—Cholestanol polymers. Thirdly, these protected linker
molecules were deprotected to form triacid—PEG—
Cholestanol polymers. These linker molecules will serve
interesting purposes in coated nanoparticles and other
bionanoconstructs.
Figure 3. Triester 1H NMR.
Triesters were successfully synthesized and purified by
the methods discussed in this report, confirmed by NMR
(Figure 2.) The appearance of the peak at δ 6.9 (N–H) and δ
1.4 (tert-butyl groups) are good indicators of headgroup
attachment.
Scheme 3. Deprotection of Triester to Yield Triacid
Figure 3. Triacid 1H NMR
Triacids were successfully synthesized and purified by the
methods discussed in this report, confirmed by NMR (Figure
3). Appearance of the broad peak around δ 12 (carboxylic
acids) and disappearance of the peak at δ 1.4 (tert-butyl
groups) are good indicators of reaction completion.
ACKOWLEDGEMENTS
The National Science Foundation is acknowledged for
funding through grant DMR-0851662. TP thanks Dr. Maggie
Bump ad Dr. Judy Riffle for their guidance and support during
the MII SURP program, and also Teresa Dickerson and Cindy
Graham for their work with the program. Virginia Tech is
acknowledged for its exceptional laboratory facilities.
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