Universal concept for the implementation of a single cleavable unit at tunable position in functional poly(ethylene glycol)s

Article abstract of DOI:10.1021/bm3016797

Poly(ethylene glycol) (PEG) with acid-sensitive moieties gained attention particularly for various biomedical applications, such as the covalent attachment of PEG (PEGylation) to protein therapeutics, the synthesis of stealth liposomes, and polymeric carriers for low-molecular-weight drugs. Cleavable PEGs are favored over their inert analogues because of superior pharmacodynamic and/or pharmacokinetic properties of their formulations. However, synthetic routes to acetal-containing PEGs published up to date either require enormous efforts or result in ill-defined materials with a lack of control over the molecular weight. Herein, we describe a novel methodology to implement a single acetaldehyde acetal in well-defined (hetero)functional poly(ethylene glycol)s with total control over its position. To underline its general applicability, a diverse set of initiators for the anionic polymerization of ethylene oxide (cholesterol, dibenzylamino ethanol, and poly(ethylene glycol) monomethyl ether (mPEG)) was modified and used to synthesize the analogous labile PEGs. The polyether bearing the cleavable lipid had a degree of polymerization of 46, was amphiphilic and exhibited a critical micelle concentration of 4.20 mg·L^-1. From dibenzylamino ethanol, three heterofunctional PEGs with different molecular weights and labile amino termini were generated. The transformation of the amino functionality into the corresponding squaric acid ester amide demonstrated the accessibility of the cleavable functional group and activated the PEG for protein PEGylation, which was exemplarily shown by the attachment to bovine serum albumin (BSA). Furthermore, turning mPEG into a macroinitiator with a cleavable hydroxyl group granted access to a well-defined poly(ethylene glycol) derivative bearing a single cleavable moiety within its backbone. All the acetal-containing PEGs and PEG/protein conjugates were proven to degrade upon acidic treatment.

Full text of DOI:10.1021/bm3016797

Article
pubs.acs.org/Biomac
Universal Concept for the Implementation of a Single Cleavable Unit
at Tunable Position in Functional Poly(ethylene glycol)s
Carsten Dingels,† Sophie S. Muller,†,‡ Tobias Steinbach,†,‡ Christine Tonhauser,†,‡ and Holger Frey*,†
̈
†Department of Organic Chemistry, Johannes Gutenberg-Universitat Mainz, Duesbergweg 10-14, D-55099 Mainz, Germany
̈
‡Graduate School Materials Science in Mainz, Staudingerweg 9, D-55128 Mainz, Germany
S
* Supporting Information
ABSTRACT: Poly(ethylene glycol) (PEG) with acid-sensitive
moieties gained attention particularly for various biomedical
applications, such as the covalent attachment of PEG
(PEGylation) to protein therapeutics, the synthesis of stealth
liposomes, and polymeric carriers for low-molecular-weight
drugs. Cleavable PEGs are favored over their inert analogues
because of superior pharmacodynamic and/or pharmacokinetic
properties of their formulations. However, synthetic routes to
acetal-containing PEGs published up to date either require
enormous efforts or result in ill-defined materials with a lack of
control over the molecular weight. Herein, we describe a novel methodology to implement a single acetaldehyde acetal in well-
defined (hetero)functional poly(ethylene glycol)s with total control over its position. To underline its general applicability, a
diverse set of initiators for the anionic polymerization of ethylene oxide (cholesterol, dibenzylamino ethanol, and poly(ethylene
glycol) monomethyl ether (mPEG)) was modified and used to synthesize the analogous labile PEGs. The polyether bearing the
cleavable lipid had a degree of polymerization of 46, was amphiphilic and exhibited a critical micelle concentration of 4.20
mg·L−1. From dibenzylamino ethanol, three heterofunctional PEGs with different molecular weights and labile amino termini
were generated. The transformation of the amino functionality into the corresponding squaric acid ester amide demonstrated the
accessibility of the cleavable functional group and activated the PEG for protein PEGylation, which was exemplarily shown by the
attachment to bovine serum albumin (BSA). Furthermore, turning mPEG into a macroinitiator with a cleavable hydroxyl group
granted access to a well-defined poly(ethylene glycol) derivative bearing a single cleavable moiety within its backbone. All the
acetal-containing PEGs and PEG/protein conjugates were proven to degrade upon acidic treatment.
reversible PEGylation of the desired substrate is favorable,
such as the shielding of polyplexes and liposomes.
INTRODUCTION
■
Since the pioneering work of Davis and co-workers in the
1970s,1,2 PEGylation, that is, the covalent attachment of
poly(ethylene glycol) (PEG) to a substrate, has become one of
the most important strategies to overcome the inherent
disadvantages of protein therapeutics.3−7 Pharmaceutically
interesting proteins undergo proteolytic degradation as well
as renal clearance and often are immunogenic or antigenic.
Hence, they usually exhibit short body-residence times and a
fast decrease from therapeutically effective concentrations to
ineffective doses. The attachment of a water-soluble, synthetic
polymer such as PEG to the protein leads to decreased renal
filtration, decreased proteolytic degradation, and reduced
immunogenicity of the conjugate in comparison to the
unmodified protein. These effects result in prolonged body-
residence times of the PEGylated proteins.
Besides a reduction in the PEG conjugates’ bioactivity in
most cases, PEGylation suffers from another disadvantage
regardless of the substrate: Its application is limited to
polyethers with average molecular weights below 40−60 kDa,
because PEG is not biodegradable and otherwise will not be
excreted, but accumulate in the liver.22 However, the circulation
times of PEGylated proteins improve by increasing the average
molecular weight of the synthetic polymer.23 In consequence,
biodegradable poly(ethylene glycol)s carrying cleavable moi-
eties in the backbone are of great interest. This statement is also
true for pharmaceutical applications other than the PEGylation
of proteins, polyplexes, or liposomes: Biocompatible and
biodegradable polymers are well suited to carry low molecular
weight drugs into tumor tissue making use of Ringsdorf’s drug
delivery concept24 and the enhanced permeability and retention
(EPR) effect.25,26
However, often decreased bioactivity of protein pharmaceut-
icals is observed when PEG chains are attached to their
surface.8 The attachment of PEG via a cleavable linker can be
advantageous for some protein therapeutics.9,10 PEGs with
cleavable coupling units11 or lipids12−21 have also been
investigated for other pharmaceutical applications where
Received: October 31, 2012
Revised: December 20, 2012
Published: December 20, 2012
© 2012 American Chemical Society
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Scheme 1. Synthetic Route to AROP Initiators Containing a Single Acetal Moiety
a
Scheme 2. Synthesis of Scissile PEGs with Different Termini
a
Reagents and conditions: (i) 1. CsOH·H2O, C6H6, 90 °C, vacuum. 2. Tetrahydrofuran (THF), nEO, 60 °C. 3. MeOH. (ii) 1. CsOH·H2O, C6H6, 90
°C, vacuum. 2. THF/dimethylsulfoxide (DMSO), nEO. 3. MeOH. (iii) H2, Pd(OH)2/C, dioxane/water 1:1, 80 bar, 40 °C. (iv) 1. C6H6, 90 °C,
vacuum. 2. KC10H8, THF/DMSO, nEO. 3. MeOH.
Various strategies have been employed to incorporate
cleavable moieties into the backbone of PEG, such as cis-
aconityl linkages27 and acetals,28−31 as well as esters and
disulfides.32 Unfortunately, neither of the aforementioned
materials is well-defined because all of them were synthesized
from telechelic PEGs via step-growth mechanisms. Never-
theless, promising results in terms of drug delivery and in vivo
degradation were, for example, obtained by Tomlinson et al.
who synthesized PEG-based polyacetals loaded with doxo-
rubicin, a potent anticancer drug,28 and demonstrated that
acetaldehyde, which is formed by the degradation of
acetaldehyde acetal-containing PEGs, is not cytotoxic.29
Elisseeff and co-workers partially oxidized the ether bonds of
PEG with Fenton’s reagent to generate hemiacetals at the
polyether backbone and demonstrated their degradability.33
However, no degradation kinetics data was provided. Very
recently, Lundberg et al. published a promising strategy to
synthesize more defined degradable PEG derivatives via
monomer-activated copolymerization of ethylene oxide and
epichlorohydrin and subsequent elimination of hydrogen
chloride.34 The obtained vinyl ether-containing polymers
exhibited polydispersity indices (PDI, Mw/Mn) below 1.4, but
no concept for the attachment of therapeutics was presented.
Due to their stability in extremely basic media,35 acetals and
ketals tolerate the harsh conditions of the anionic ring-opening
polymerization (AROP) of oxiranes and have been established
as protecting groups for aldehyde and hydroxyl groups in
AROP initiators36−40 and monomers.41,42 Further, these groups
have been employed to generate degradable, water-soluble
polyethers by epoxide AROP. Based on a ketal-containing
branching unit Feng et al. demonstrated the synthesis of
degradable dendrimer-like PEGs up to the seventh generation
with a molecular weight of almost half a million g·mol−1.43
Although these polyethers are well-defined, the synthetic effort
necessary for their production will probably hinder widespread
applications. Hyperbranched poly(ethylene glycol)s with acetal
groups at each branching point that can be synthesized by the
copolymerization of ethylene oxide (EO) with the inimer
glycidyloxyethyl ethylene glycol ether (GEGE) in a one-pot
reaction were recently presented by our group.44 In this
context, the recent work of the group of Kizhakkedathu has to
be mentioned, who used ketal-containing inimers for the
oxyanionic polymerization45 analogous to GEGE and multi-
functional, ketal-containing initiators for the ring-opening
multibranching polymerization of glycidol46 to produce
cleavable hyperbranched polyether polyols.
Acetal-functionalized PEGs are not solely interesting for
biomedical applications. For instance, poly(ethylene glycol)
bearing an acetaldehyde chloroethyl acetal terminus has been
used to generate scissile PEG polystyrene block copolymers,
which were investigated as precursors for porous films.47
Depending on the desired application of degradable PEG the
cleavable moieties have to be located either in the backbone or
at (one of) the functional terminus (termini). Herein, we
present the implementation of a single, cleavable acetal moiety
into conventional initiators for the anionic ring-opening
polymerization of epoxides using a straightforward two-step
protocol (Scheme 1), which gives access to both types of PEG,
either cleavable in the backbone or at one of the terminal sites
(Scheme 2). First, the original initiators, alcohols 1a−c, are
added to 2-acetoxyethyl vinyl ether48 (AcVE, 2) under acidic
conditions to form the asymmetric acetaldehyde acetals 3a−c.
Subsequently, the products are saponified to release the desired
acetal-containing initiators 4a−c.
The developed methodology is in principle applicable to all
acid stable AROP initiators and was proven for a diverse set of
alcohols to underline its general usefulness: Cholesterol (1a) as
an apolar initiator, dibenzylamino ethanol (1b), which carries
an additional, orthogonally protected functional group, and
poly(ethylene glycol) monomethyl ether (mPEG, 1c) as a
macroinitiator. The scissile macroinitiator granted access to
PEGs carrying a single acetal moiety in the backbone of the
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1 N aqueous sodium hydroxide solution. After drying over sodium
sulfate, the organic phase was evaporated to a small volume. Pure
product (4.80 g, 9.29 mmol, 72%) was obtained by column
chromatography (eluent: petrol ether/ethyl acetate 8:1) over silica.
1H NMR (400 MHz, CDCl3): δ [ppm] 5.32 (s, 1H, H-6), 4.85 (q, 1H,
JAB = 5.3 Hz, H3CCHO2), 4.26−4.12 (m, 2H, AcO-CH2), 3.78−3.58
(m, 2H, AcOCH2-CH2), 3.65−3.36 (m, 1H, H-3α), 2.38−2.12 (m,
2H, H-4), 2.05 (s, 3H, H3C−CO−), 2.03−1.94 (m, 1H, H-12α),
1.99−1.89 (m, 1H, H-7β), 1.87−1.79 (m, 2H, H-1β and H-2α), 1.87−
1.74 (m, 1H, H-16β), 1.63−0.81 (m, 22H), 1.30 (d, 3H, JAB = 5.3 Hz,
H3C-CHO2), 0.99 (s, 3H, H3-19), 0.90 (d, 3H, JAB = 6.5 Hz, H3-21),
0.84 (d, 3H, JAB = 6.6 Hz, H3-26), 0.83 (d, 3H, JAB = 6.6 Hz, H3-27),
0.65 (s, 3H, H3-18). 13C NMR (100.6 MHz, CDCl3): δ [ppm] 170.1
(1C, MeCOO), 140.9 and 140.8 (1C, C-5), 121.9 and 121.8 (1C, C-
6), 98.0 and 97.9 (1C, Me-CHO2), 75.9 and 75.8 (1C, C-3), 63.9 (1C,
AcO-CH2), 61.6 (1C, AcOCH2-CH2-), 50.3 (1C, C-9), 42.4 (1C, C-
13), 40.1 and 39.5 (1C, C-4), 39.8 (1C, C-12), 39.6 (1C, C-24), 37.5
and 37.3 (1C, C-1), 36.8 (1C, C-10), 36.3 (1C, C-22), 35.9 (1C, C-
20), 32.0 (2C, C-7 and G-8), 29.5 and 28.7 (1C, C-2), 28.3 (1C, C-
16), 28.1 (1C, C-25), 24.4 (1C, C-15), 23.9 (1C, C-23), 22.9 (1C, C-
27), 22.7 (1C, C-26), 21.1 (2C, C-11 and CH3-COO), 20.6 (1C, CH3-
CHO2), 19.5 (1C, C-19), 18.8 (1C, C-21), 12.0 (1C, C-18). MS (ESI-
MS, MeOH): m/z = 539.42 [M + Na]+, 1055.86 [2M + Na]+.
Acetoxyethyl 1-(2-Dibenzylamino ethoxy)ethyl Ether (3b).
Dibenzylamino ethanol (5.0 g, 21 mmol) and 2-acetoxyethyl vinyl
ether (5.0 g, 38 mmol) were stirred for 30 min in dichloromethane
with trifluoroacetic acid (TFA, 7.1 g, 62 mmol). The solution was
treated with triethylamine (1.5 mL) and washed with 1 N aqueous
sodium hydroxide solution. After drying over sodium sulfate, the
organic phase was evaporated to small volume. Pure product was
obtained by column chromatography (eluent: petrol ether/ethyl
polymer. The specific position of this acid labile group can be
controlled by the molecular weight of the macroinitiator and
the number of ethylene oxide (EO) units added.
EXPERIMENTAL SECTION
■
Materials. All reagents and solvents were purchased from Acros
Organics, Fluka, or Sigma-Aldrich and were used without further
purification unless stated otherwise. Ethylene glycol monovinyl ether
was purchased from TCI Europe. Deuterated solvents were purchased
from Deutero GmbH and stored over molecular sieves (except for
deuterium oxide). 2-Acetoxyethyl vinyl ether48 (2) and dibenzylamino
ethanol49 (1b) were prepared according to known protocols. Dry THF
used for the anionic ring-opening polymerization of ethylene oxide was
dried and stored over sodium. Care must be taken when handling the
toxic, flammable, and gaseous ethylene oxide.
Methods. 1H NMR spectra (400 MHz) were recorded on a Bruker
ARX 400 with a 5 mm BBO probe. 2D and 13C NMR spectra were
recorded on a Bruker Avance-II 400 (400 MHz, 5 mm BBO probe,
and B-ACS 60 auto sampler) if not stated otherwise. All spectra were
recorded with 32 scans at 294 K using a relaxation delay of 1 s, unless
stated otherwise and processed with MestReNova v6.1.1 software.
Size-exclusion chromatography (SEC) measurements in DMF
containing 0.25 g·L−1 of lithium bromide as additive were performed
on an Agilent 1100 Series as an integrated instrument using PSS
(Polymer Standards Service) HEMA column (106/105/104 g/mol),
RI detector, and UV detector operating at 275 nm. Calibration was
executed using poly(ethylene oxide) (PEO) standards from PSS.
Matrix-assisted laser desorption/ionization time-of-flight mass spec-
trometry (MALDI-ToF MS) measurements of all polymers were
recorded on a Shimadzu Axima CFR MALDI-ToF MS mass
spectrometer, equipped with a nitrogen laser delivering 3 ns laser
pulses at 337 nm. α-Cyano hydroxyl cinnamic acid (CHCA) or
dithranol was used as a matrix and potassium triflouroacetate (KTFA)
was added for ion formation. The analytes were dissolved in methanol
at a concentration of 10 g·L−1. An aliquot (10 μL) was added to 10 μL
of a solution (10 g·L−1) of the matrix and 1 μL of a solution of the
cationization agent. A total of 1 μL of the mixture was applied on a
multistage target and methanol evaporated, and a thin matrix/analyte
film was formed.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) was carried out with Tris-HCl gels (Biorad, 1.0 mm, 10 well,
8% resolving gel, 4% stacking gel). IR spectra were recorded on a
Thermo Scientific Nicolet iS10 FT-IR spectrometer, equipped with a
diamond ATR unit and were processed with OMNIC 8.1.210
software. Mass spectra were either measured on a Finnigan MAT 95
(field desorption, FD-MS) or a Waters/Micromass QToF Ultima 3
(electrospray ionization, ESI-MS). Surface tension measurements to
determine the critical micelle concentrations (CMC) were performed
on a Dataphysics DCAT 11 EC tensiometer equipped with a TV 70
temperature control unit, a LDU 1/1 liquid dosing and refill unit, as
1
acetate 6:1) over silica. Yield: 85%. H NMR (400 MHz, CDCl3): δ
[ppm] 7.42 (d, 4H, JAB = 7.2 Hz, 4 meta CHAr), 7.34 (t, 4H, JAB = 7.4
Hz, 4 ortho CHAr), 7.26 (d, 2H, JAB = 7.4 Hz, 2 para CHAr), 4.75 (q,
1H, JAB = 5.4 Hz, H3C-CHO2), 4.21 (t, 2H, JAB = 4.9 Hz, AcO-CH2),
3.77−3.67 (m, 1H, NCH2-CHa), 3.76−3.67 (m, 1H, AcOCH2-CHa),
3.69 (s, 4H, 2 Ph-CH2), 3.65−3.57 (m, 1H, AcOCH2-CHb), 3.61−3.52
(m, 1H, NCH2-CHb), 2.72 (t, 2H, JAB = 6.3 Hz, N-CH2-CH2), 2.07 (s,
3H, H3C−CO), 1.31 (d, 3H, JAB = 5.4 Hz, H3C-CHO2). 13C NMR
(100.6 MHz, CDCl3): δ [ppm] 171.0 (1C, Me-CO-O), 139.7 (2C, 2
quaternary CAr), 128.8 (4C, ortho CHAr), 128.2 (4C, meta CHAr),
126.9 (2C, para CHAr), 99.7 (1C, H3C-CHO2), 64.0 (1C, NCH2-
CH2), 63.8 (1C, AcO-CH2), 62.2 (1C, AcOCH2-CH2), 59.0 (2C, 2 Ph-
CH2), 52.9 (1C, N-CH2-CH2), 21.0 (1C, H3C-CO), 19.5 (1C, H3C-
CHO2).
1-(2-Acetoxyethoxy)ethoxy mPEG (3c). Poly(ethylene glycol)
monomethyl ether (1c, 4.0 g, 2.0 mmol), 2-acetoxyethyl vinyl ether
(1.3 g, 10 mmol), and pTSA (3.8 mg 0.2 mmol) were placed in a
round-bottom flask, dissolved in dichloromethane (DCM, 20 mL),
and stirred for 30 min. The reaction was quenched with triethylamine
(0.2 mL) and washed with 1 N aqueous sodium hydroxide solution.
The organic phase was dried over sodium sulfate and subsequently
concentrated to small volume. Pure product was obtained by
well as a RG 11 Du Nouy ring. Surface tension data was processed
̈
with SCAT v3.3.2.93 software. The CMC presented is a mean value of
three experiments. All solutions for surface tension measurements
were stirred for 120 s at a stir rate of 50%. After a relaxation period of
400 s, three surface tension values were measured. The mean values of
the three measurements were plotted against the logarithm of the
concentration. The slopes of the traces at high concentrations as well
as in the low concentration range were determined by linear
regression. The concentration at the fits’ intersection was the CMC.
1
precipitation in cold diethyl ether. Yield: 87%. H NMR (400 MHz,
CDCl3): δ [ppm] 4.77 (q, 1H, JAB = 5.4 Hz, H3C-CHO2), 4.16 (t, 2H,
JAB = 4.8 Hz, AcO-CH2), 3.86−3.36 (m, 180H, CH2O), 3.33 (s, 3H,
OCH3), 2.03 (s, 3H, H3C−CO), 1.28 (d, 3H, JAB = 5.4 Hz, H3C-
CHO2).
Glycol 1-(Cholesteryloxy)ethyl Ether (4a). Potassium hydroxide
(1.00 g, 17.8 mmol) and acetoxyethyl 1-(cholesteryloxy)ethyl ether
(3a, 1.00 g, 1.93 mmol) were stirred under reflux in a solution of
ethanol (12 mL) and water (0.5 mL) for 3 h. After cooling, brine was
added and the solution was extracted with DCM three times. After
drying over sodium sulfate the organic phase was evaporated to small
volume. Pure product (536 mg, 1.13 mmol, 59%) was obtained by
column chromatography (eluent: petrol ether/ethyl acetate 2:1) over
The Du Nouy ring was rinsed thoroughly with water and annealed in a
̈
butane flame. Turbidimetry was performed in a Jasco V-630 UV−vis
spectrometer at a wavelength of λ = 528 nm and the data was
processed with the Time Course Measurement program of Spectra
Manager v2.08 software.
Synthesis Procedures. Acetoxyethyl 1-(Cholesteryloxy)ethyl
Ether (3a). Cholesterol (5.0 g, 13 mmol) and 2-acetoxyethyl vinyl
ether (2.53 g, 19.4 mmol) were stirred for 45 min in dichloromethane
with p-toluene sulfonic acid monohydrate (pTSA, 25 mg, 0.13 mmol).
The solution was treated with triethylamine (250 μL) and washed with
1
silica. H NMR (400 MHz, CDCl3): δ [ppm] 5.32 (s, 1H, H-6), 4.81
(q, 1H, JAB = 5.3 Hz, H3C-CHO2), 3.75−3.64 (m, 2H, HO-CH2),
3.71−3.53 (m, 2H, HOCH2-CH2), 3.94−3.34 (m, 1H, H-3α), 2.53 (t,
1H, JAB = 5.8 Hz, OH), 2.35−2.12 (m, 2H, H-4), 2.04−1.94 (m, 1H,
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H-12α), 1.99−1.90 (m, 1H, H-7β), 1.90−1.78 (m, 2H, H-1β and H-
2α), 1.88−1.73 (m, 1H, H-16β), 1.63−0.80 (m, 22H), 1.32 (d, 3H, JAB
= 5.3 Hz, H3C-CHO2), 0.98 (s, 3H, H3-19), 0.89 (d, 3H, JAB = 6.5 Hz,
H3-21), 0.84 (d, 3H, JAB = 6.6 Hz, H3-26), 0.84 (d, 3H, JAB = 6.6 Hz,
H3-27), 0.65 (s, 3H, H3-18). 13C NMR (100.6 MHz, CDCl3): δ [ppm]
170.1 (1C, Me-CO−O), 140.9 and 140.8 (1C, C-5), 121.9 and 121.8
(1C, C-6), 98.0 and 97.9 (1C, Me-CHO2), 75.9 and 75.8 (1C, C-3),
63.9 (1C, AcO-CH2), 61.6 (1C, AcOCH2-CH2-), 50.3 (1C, C-9), 42.4
(1C, C-13), 40.1 and 39.5 (1C, C-4), 39.8 (1C, C-12), 39.6 (1C, C-
24), 37.5 and 37.3 (1C, C-1), 36.8 (1C, C-10), 36.3 (1C, C-22), 35.9
(1C, C-20), 32.0 (2C, C-7 and G-8), 29.5 and 28.7 (1C, C-2), 28.3
(1C , C-16), 28.1 (1C, C-25), 24.4 (1C, C-15), 23.9 (1C, C-23), 22.9
(1C, C-27), 22.7 (1C, C-26), 21.1 (2C, C-11 and CH3CO), 20.6 (1C,
CH3-CHO2), 19.5 (1C, C-19), 18.8 (1C, C-21), 12.0 (1C, C-18). MS
(ESI-MS, MeOH): m/z = 497.42 [M + Na]+, 971.81 [2M + Na]+.
Glycol 1-(2-Dibenzylamino ethoxy)ethyl Ether (4b). Potassium
hydroxide (3.2 g, 57 mmol) and acetoxyethyl 1-(2-dibenzylamino
ethoxy)ethyl ether (3b, 6.0 g, 16 mmol) were stirred under reflux in a
solution of ethanol (8.4 mL) and water (4.2 mL) for 3 h. After cooling,
brine was added and the solution was extracted with DCM three times.
After drying over sodium sulfate, the organic phase was evaporated to
a small volume. Pure product was obtained by column chromatog-
raphy (eluent: petrol ether/ethyl acetate 2:1) over silica. Yield: 58%.
1H NMR (400 MHz, DMSO-d6): δ [ppm] 7.41−7.27 (m, 8H, 4 meta-
CHAr and 4 ortho-CHAr), 7.22 (t, 2H, JAB = 7.1 Hz, 2 para-CHAr), 4.63
(q, 1H, JAB = 5.2 Hz, H3C-CHO2), 4.61 (t, 1H, JAB = 4.7 Hz, HO-
CH2), 3.68−3.57 (m, 1H, NCH2-CHa), 3.60 (s, 4H, 2 Ph-CH2), 3.53−
3.43 (m, 1H, NCH2-CHb), 3.52−3.43 (m, 1H, HOCH2-CHa), 3.51
(m, 2H, HO-CH2), 3.42−3.32 (m, 1H, HOCH2-CHb), 2.56 (t, 2H, JAB
= 6.2 Hz, N-CH2-CH2), 1.17 (d, 3H, JAB = 5.3 Hz, H3C-CHO2). 13C
NMR (100.6 MHz, DMSO-d6): δ [ppm] 139.5 (2C, 2 quaternary
CAr), 128.5 (4C, ortho-CHAr), 128.2 (4C, meta-CHAr), 126.8 (2C,
para-CHAr), 99.3 (1C, H3C-CHO2), 66.7 (1C, HOCH2-CH2), 63.1
(1C, NCH2-CH2), 60.4 (1C, HO-CH2), 58.0 (2C, 2 Ph-CH2), 52.4
(1C, N-CH2-CH2), 19.8 (1C, H3C-CHO2). MS (FD-MS, MeOH): m/
z = 329.4 [M]+, 569.6 [2M − Bn + 2H]+, 659.7 [2M + H]+, 691.7 [2M
+ MeOH + H]+.
describes the example of 6 with a degree of polymerization (Pn) of 50
(650). Cesium hydroxide monohydrate (150 mg, 893 μmol) was added
to a solution of 4b (322.7 mg, 0.9796 mmol) dissolved in benzene (7
mL) in a dry Schlenk flask. The mixture was stirred for 30 min at 60
°C under slightly reduced pressure with closed stopcock. Moisture was
removed by azeotropic distillation of benzene and subsequent drying
at 80 °C under high vacuum for 3.5 h. After cooling to room
temperature, dry THF (7 mL) was cryo-transferred into the Schlenk
flask and dry DMSO (2 mL) was added via syringe. Subsequently,
ethylene oxide (1.95 g, 44.3 mmol) was cryo-transferred via a
graduated ampule to the initiator solution and the flask was closed
tightly. The reaction mixture was stirred overnight at 40 °C and finally
quenched by the addition of methanol (2 mL). The polymer was
precipitated from methanol in cold diethyl ether twice and
1
subsequently dried under reduced pressure. Yield: 2.07 g (91%). H
NMR (400 MHz, CDCl3): δ [ppm] 7.32 (d, 4H, JAB = 7.3 Hz, 4 ortho
CHAr), 7.52 (t, 2H, JAB = 7.4 Hz, 2 meta CHAr), 7.17 (t, 1H, JAB = 7.2
Hz, para CHAr), 4.65 (q, 1H, JAB = 5.3 Hz, H3C-CHO2), 3.97 (m,
208H, CH2O), 2.62 (t, 2H, JAB = 6.3 Hz, N-CH2-CH2), 1.23 (d, 3H,
JAB = 5.3 Hz, H3C-CHO2).
α-(1-(2-Amino ethoxy)ethoxy) ω-Hydro PEG (7). Hydrogenation
of α-(1-(2-dibenzylamino ethoxy)ethoxy) ω-hydro PEG (6) was
carried out similar to the protocol described for α-dibenzylamino ω-
hydroxy-PEG.51 The following protocol describes the synthesis of 7
with a degree of polymerization of 50 (750). Compound 650 (700 mg,
0.313 mmol) was dissolved in a water/dioxane 1:1 mixture and stirred
with palladium(II)-hydroxide on activated charcoal (150 mg) under
hydrogen (80 bar) for 3 days in a stainless steel reactor. After the
solution had been filtered through Celite the filter cake was washed
with methanol (2 L). The transparent solution was reduced to small
volume and precipitated in cold diethyl ether. A second precipitation
from DCM in cold diethyl ether yielded 457 mg (0.222 mmol, 71%) of
750. 1H NMR (400 MHz, DMSO-d6): δ [ppm] 4.68 (q, 1H, JAB = 5.2
Hz, H3C-CHO2), 4.59 (s, 1H, OH), 3.80−3.37 (m, 210H, CH2O),
2.65 (t, 2H, JAB = 5.8 Hz, H2N-CH2), 1.20 (d, 3H, JAB = 5.2 Hz, H3C-
CHO2).
α-(1-mPEG ethoxy) ω-Hydro PEG (8). Compound 4c (1.00 g, 0.476
mmol) was dissolved in benzene (10 mL) in a dry Schlenk flask. The
solution was stirred for 30 min at 90 °C under slightly reduced
pressure with closed stopcock. Moisture was removed by azeotropic
distillation of benzene and subsequent drying at 90 °C under high
vacuum overnight. After cooling to room temperature dry THF (20
mL) was cryo-transferred into the Schlenk flask and potassium
naphthalenide in THF (1 mL, c = 0.5 mol·L−1, prepared under argon
from potassium (235 mg, 6.0 mmol), naphthalene (770 mg, 6.0
mmol), and dry THF (12 mL) in a glovebox) was added via syringe.
Subsequently, the generated hydrogen was evaporated including half
the amount of THF and ethylene oxide (3.0 g, 68 mmol) was cryo-
transferred via a graduated ampule to the macroinitiator solution. The
reaction mixture was stirred for 3 h at 60 °C first and overnight at 40
°C. After the polymerization had been quenched by the addition of
methanol (1.3 mL) the polymer was precipitated from methanol in
cold diethyl ether twice and subsequently dried under reduced
pressure. Yield: 3.64 g (91%). 1H NMR (400 MHz, CDCl3): δ [ppm]
4.75 (q, 1H, JAB = 5.3 Hz, H3C-CHO2), 3.99−3.37 (m, 714H, CH2O),
3.33 (s, 3H, OCH3), 1.27 (d, 3H, JAB = 5.4 Hz, H3C-CHO2).
α-(1-(2-(Squaric acid ethyl ester amido)ethoxy)ethoxy) ω-Hydro
PEG (10). Diethyl squarate (9, 60.7 mg, 357 μmol), 775 (100 mg, 29.2
μmol), and triethylamine (43 μL, 310 μmol) were stirred in a 1:1
water/ethanol solution (2 mL) for 4 h. After the ethanol had been
removed by distillation, the solution was extracted with DCM four
times. Subsequently, the organic phase was evaporated to small
volume and precipitated in cold diethyl ether. The resulting polymer
was precipitated several times from methanol in cold diethyl ether
until no more diethyl squarate was detected by thin layer
chromatography (TLC). 1H NMR (400 MHz, CDCl3): δ [ppm]
4.82−4.68 (m, 3H, H3C-CH2-O and H3C-CHO2), 3.99−3.37 (m,
311H, CH2O and CH2N), 1.50−1.38 (m, 3H, H3C-CH2O), 1.35−1.22
(m, 3H, H3C-CHO2).
1-(2-Hydroxyethoxy)ethoxy mPEG (4c). Potassium hydroxide (1.2
g, 21 mmol) and 3c (3.7 g, 1.9 mmol) were stirred under reflux in a
solution of ethanol (3.0 mL) and water (1.5 mL) for 3 h. After cooling
the solution was extracted with DCM three times and the combined
organic phases were dried over sodium sulfate. DCM was evaporated
and pure product was obtained by precipitation in cold diethyl ether.
Yield: 67%. 1H NMR (400 MHz, CDCl3): δ [ppm] 4.77 (q, 1H, JAB
=
5.5 Hz, H3C-CHO2), 3.84−3.385 (m, 180H, (CH2CH2O)n), 3.32 (s,
3H, OCH3), 1.28 (d, 3H, JAB = 5.2 Hz, H3C-CHO2).
α-(1-(Cholesteryloxy)ethoxy) ω-Hydro PEG (5). Compound 4a
(427 mg, 0.900 mmol), cesium hydroxide monohydrate (134 mg,
0.798 mmol), and benzene were placed in a Schlenk flask. The mixture
was stirred at RT for about 30 min to generate the cesium alkoxide
(degree of deprotonation 89%). The salt was dried under vacuum at
90 °C for 24 h, dry THF was added via cryo-transfer, and ethylene
oxide (2 mL, 51 mmol) was cryo-transferred first to a graduated
ampule and then to the Schlenk flask containing the initiator solution.
The mixture was allowed to warm up to room temperature, heated to
60 °C, and the polymerization was performed for 12 h at this
temperature under vacuum. The reaction was quenched with
methanol, the solvent was evaporated and the crude product was
1
precipitated into cold diethyl ether. H NMR (400 MHz, CDCl3): δ
[ppm] 5.31 (s, 1H, H-6), 4.83 (q, 1H, JAB = 5.3 Hz, H3C-CHO2),
4.10−3.21 (m, 190H, CH2O and H-3α), 2.35−2.12 (m, 2H, H-4),
2.04−1.89 (m, 2H, H-12α and H-7β), 1.88−1.73 (m, 3H, H-1β, H-2α,
and H-16β), 1.63−0.80 (m, 22H), 1.30 (d, 3H, JAB = 5.3 Hz, H3C-
CHO2), 0.98 (s, 3H, H3-19), 0.89 (d, 3H, JAB = 6.6 Hz, H3-21), 0.85
(d, 3H, JAB = 6.6 Hz, H3-26), 0.84 (d, 3H, JAB = 6.6 Hz, H3-27), 0.65
(s, 3H, H3-18).
α-(1-(2-Dibenzylamino ethoxy)ethoxy) ω-Hydro PEG (6). Com-
pound 6 was synthesized similar to protocols for N,N-dibenzylamino
ethoxide-initiated anionic ring-opening polymerization of ethylene
oxide known in the literature.50,51 The following protocol is typical and
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RESULTS AND DISCUSSION
■
A.1. Addition of Conventional AROP Initiators to
AcVE. The key step in the implementation of an acetaldehyde
moiety between the hydroxyl group and the residue of
established AROP initiators is the acid catalyzed addition of
the alcohol to AcVE (Scheme 1). This vinyl ether was chosen
for three reasons: First, upon the addition reaction the acetate
can be saponified with little effort under basic conditions that
every possible initiator for the AROP and the generated acetal
will tolerate. Second, upon saponification a new hydroxyl group
is generated, which is essential for the initiation of the anionic
epoxide polymerization. And, finally, upon deprotonation the
generated alkoxide is structurally related to the growing chain
end, i.e., an alkyloxyethoxide, which is favorable for the
initiation kinetics. The amount of acid necessary to promote
the addition of the alcohol across the vinyl ether double bond
varied depending on the nature of the alcohol (vide infra).
Hence, the applied conventional initiators have to be more or
less stable under acidic conditions. In contrast to the
aforementioned and innovative, multifunctional, ketal-contain-
ing initiators used by Kizhakkedathu and co-workers,46 our
methodology yields asymmetric acetals from established
initiators in a rapid two-step synthesis.
1
Figure 1. H NMR spectrum (400 MHz) of 3c in CDCl3 at T = 294
K.
3a were compared to those of steroids known in the
literature.52,53 In the H NMR spectra the conversion was
1
determined from the ratio of the peaks assigned to the acetal
methine protons (3a, 4.85 ppm; 3b, 4.75 ppm; 3c, 4.77 ppm)
and protons of the former alcohol residues. These were the
cholesteryl carbon−carbon double bond (H-6, 5.32 ppm), the
aromatic protons (7.50−7.20 ppm), and the methoxy group of
mPEG (3.33 ppm), respectively. Successful addition of the
alcohols across the double bond of AcVE was further indicated
by the carbonyl stretch vibrations of the acetates at 1741.2 cm−1
(3a), 1738.4 cm−1 (3b), and 1739.0 cm−1 (3c) in the IR spectra
of the acetals (SI, Figures S32−S34). Full conversion of mPEG
to 1-(2-acetoxyethoxy)ethoxy mPEG (3c) was also confirmed
by MALDI-ToF MS. The mass spectrum (Figure 2A) revealed
a single distribution of the desired polymeric species cationized
either with sodium or potassium. Within the limits of the error
Cholesterol (1a) was chosen as a model compound to
demonstrate the applicability of our acetal insertion protocol to
apolar, monofunctional initiators for the oxyanionic ROP of
epoxides. The addition of the steroid to AcVE was catalyzed by
1 mol % of p-toluene sulfonic acid. The conditions of the vinyl
ether addition had to be adjusted for dibenzylamino ethanol
(1b), because catalytic amounts of acid solely protonated the
basic tertiary amine, but did not activate the vinyl ether double
bond. The excess of trifluoroacetic acid which was necessary to
provide a satisfactory reaction rate was determined by following
1
the reaction kinetics via H NMR spectroscopy. Figure S46
displays the reaction kinetics of the addition reaction in
deuterated chloroform when 1.8 equiv of AcVE and 3 equiv of
TFA were batched (for spectra see SI, Figure S47). An 85%
conversion was achieved after 44 min, whereas under identical
conditions except for less acid fed to the reaction mixture (i.e.,
2 equiv of TFA batched with respect to 1b), 90% conversion
was reached after 18 h (data not shown).
According to our protocol, a PEG macroinitiator opens up
the synthesis of well-defined poly(ethylene glycol)s carrying a
single acetal moiety in the backbone (Scheme 2). mPEG was
chosen as the simplest precursor for this system, because it is
chemically inert, except for the mandatory hydroxyl group. The
necessary amount of acid was higher than in the analogous
reaction with 1a, reflecting the lower reactivity of the polymeric
alcohol, but significantly lower than in the reaction with 1b,
because no basic residue was competing for protons with the
double bond.
+
+
the mass peaks satisfied the following equation with MC = MNa
= 23.0 g·mol−1 for sodium cationized molecules and MC = MK
+
+
= 39.1 g·mol−1 for those cationized with potassium:
+
M3c(n) = MCH O + MCH CO + (2 + n)·MEO + MC
3
3 g
mol
g
mol
+
= 74.1
+ (2 + n)·44.05
+ MC
The SEC elugram of 3c (Figure 3) shows a monomodal
distribution and a slight increase in both the number-averaged
molecular weight (Mn = 1700 g·mol−1 to Mn = 1900 g·mol−1)
and the polydispersity index (1.06−1.07) in comparison to the
batched mPEG (1c). Data derived from SEC analysis can be
regarded as trend only, because all samples were referenced to a
PEG standard. The most important factor for the synthesis of
3c is the reaction time, since slow transacetalization may take
place. Upon longer exposure of the produced acetal to the
acidic reaction conditions, the symmetric polymeric acetal,
which is shown in Scheme 3, is formed. This side reaction can
be followed by SEC as a second mode evolving at twice the
molecular weight of the product (find elugrams in the
Supporting Information, Figure S53). Therefore, quenching
the reaction with triethylamine in time is important to yield
quantitative conversion and avoid transacetalization reactions.
A.2. Saponification of Acetal Acetates. The acetal
acetates 3a−c were saponified to yield the scissile initiators
4a−c, respectively, and the removal of the acetate was
confirmed by NMR and IR spectroscopy. For the assignment
Complete conversion of the alcohols 1a−c to the
corresponding acetal acetates 3a−c was confirmed by NMR
spectra recorded in deuterated chloroform (Figure 1 and SI,
1
Figures S1−S12). To assign all peaks in the 13C and H NMR
spectra of the purified acetal acetates 3a and 3b 2D NMR
experiments were carried out, that is, COSY (correlated
spectroscopy), HSQC (heteronuclear single quantum coher-
ence), and HMBC (heteronuclear multiple bond correlation)
experiments (find 2D NMR spectra in the Supporting
Information, Figures S3−S7 (3a) and S10−S12 (3b)).
Additionally, the chemical shifts of the cholesteryl derivative
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Figure 2. MALDI-ToF mass spectra of derivatized mPEGs. (A) Mass spectrum of 3c. (B) Mass spectrum of 4c. (C) Detail of overlay of mass spectra
of 3c (black) and 4c (red). Masses given for averaged signals, mass differences calculated from monoisotopic peaks. All spectra were recorded in
reflectron mode.
stretches at around 1740 cm−1 in the IR spectra (SI, Figures
S35−S37). Instead, all IR spectra exhibited broad hydroxyl
bands at wavenumbers of about 3400 cm−1.
The mPEG derivative 4c was further characterized by
MALDI-ToF MS and SEC. In the mass spectrum of 4c (Figure
2B) the observed mass-averaged peaks satisfied the following
equation.
+
M4c(n) = MCH O + MH + (2 + n)·MEO + MC
3 g
g
+
= 32.0
+ (2 + n)·44.05
+ MC
mol
mol
The difference of the molecular masses of M4c(n + 1) and its
precursor M3c(n) was calculated and found to be just 2 g·mol−1
(Figure 2C). Fortunately, the isotopic resolution obtained in
the investigated measuring range allowed to clearly distinguish
between the two series of polyether masses. The SEC trace of
1-(2-hydroxyethoxy)ethoxy mPEG was monomodal and shifted
to a smaller hydrodynamic volume in comparison to the acetate
derivative 3c (Figure 3). Also, the PDI increased slightly to 1.08
upon saponification.
Figure 3. SEC elugrams of 3c (black), 4c (red), and the corresponding
mPEG precursor (blue).
B.1. Use of Cleavable Initiators for EO Polymerization:
PEG with Cleavable Cholesterol Initiator. Cholesteryl
PEGs were subject to various studies for very different
purposes: They are amphiphilic, can be utilized in the synthesis
of stealth liposomes,54−56 and exhibit liquid-crystalline
behavior, if the degree of polymerization (Pn) is low.57,58
Insertion of a scissile unit between the polymer and the
initiating unit will lead to pH-responsive materials, which lose
their amphiphilic or mesogenic properties upon acidic treat-
ment. PEGs attached to cholesterol via an acid labile linker are
known already (vide supra), but the synthetic pathways
presented are laborious or the overall yield limited.13,14,19
According to our protocol, PEGs with a scissile terminal
1
of all peaks in both H and 13C NMR spectra recorded of 4a
and 4b additional 2D NMR experiments (COSY, HSQC, and
HMBC) were carried out (Supporting Information). In the 1H
NMR spectra of all cleavable initiators (SI, Figures S13, S20,
and S25) the characteristic acetyl peak at around 2.0 ppm of the
corresponding precursors had disappeared. Further, the
resonance of the methylene protons adjacent to the hydroxyl
group exhibited a clear high-field shift compared to the
corresponding signals of their analogous acetates. In the 13C
NMR spectra (SI, Figures S14 and S21), the carbonyl peaks
had vanished completely as well as the bands of the carbonyl
Scheme 3. Possible Transacetalization During Synthesis of 3c
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cholesterol unit can be synthesized rapidly in three steps. The
resulting α-(1-(cholesteryloxy)ethoxy) ω-hydro PEG (5) was
characterized by NMR spectroscopy, SEC, and MALDI-ToF
MS. The mass spectrum of 5 (SI, Figure S39) displayed the
distribution of the desired polydisperse compound cationized
with potassium.
The degradability of 5 was demonstrated by acidic hydrolysis
of the acetal, followed by turbidimetry at pH 1. In Figure 5A,
the relative transmission is plotted against the reaction time for
two different temperatures (T = 25 and 37 °C). After an initial
period, the solutions started to turn turbid, as the released
cholesterol precipitated and the transmission began to decrease.
Finally, so much cholesterol had precipitated that almost all
light was scattered. As expected, cholesterol was released faster
at higher reaction temperature as indicated by the shorter initial
phase and the more negative slope of the trace.
Successful initiation of the oxyanionic polymerization of EO
1
with 4a was also confirmed by the H NMR spectrum of 5
(Figure 4) in which, besides the acetal peaks, the H-3 and H-6
Complete removal of the steroid was confirmed by 1H NMR
spectra recorded of both the precipitate as well as the residue of
the aqueous phase of a similar experiment (Photographs of this
experiment are shown in Figure 2B−D, find protocol in the
SI).The former shows a clean cholesterol spectrum, despite of
some water (SI, Figure S29), whereas the latter just exhibits
pure PEG diol (SI, Figure S30).
B.2. Scissile Heterofunctional PEGs. Poly(ethylene
glycol)s with cleavable terminal functionalities can be used to
covalently bind PEG to different substrates such as other
synthetic polymers, proteins, or low molecular weight drug
molecules and subsequently release the substrate upon a
suitable trigger. As mentioned before, such materials have been
described in the past, but most of these approaches are based
on the extensive modification of monofunctional mPEGs and
do not allow any further modification of the polyether, such as
the addition of a labeling or targeting moiety.
Figure 4. 1H NMR spectrum (400 MHz) of 5 in CDCl3 at T = 294 K.
Peak assignment of cholesteryl moiety shown solely for characteristic
protons for clarity.
Three cleavable heterofunctional polymers 6P were synthe-
n
sized with varying degrees of polymerization and characterized
by standard methods. The proton NMR spectrum (Figure 6)
was referenced to the methyl group of the acetal and exhibits
full incorporation of the initiator. The degree of polymerization
and the number-averaged molecular weights were calculated
from the integral values using the following equation, where
IPEG is the integral of the backbone peak.
resonances as well as the characteristic pattern of the
cholesteryl methyl groups were clearly identified. The degree
of polymerization and the number-averaged molecular weight
were calculated from the area under the backbone peak
(reduced by five protons arising from the initiator) related to
the integral of a cholesteryl methyl resonance (H3-18).
IPEG − 10
3
4
P =
·
n
IMe
IPEG − 5
3
4
P =
·
= 46
g
mol
g
mol
n
IMe
Mn = P ·MEO + M4b = P ·44
+ 329
n
n
Mn = P ·MEO + M4a
n
All molecular weights found were in good agreement with
the calculated theoretical values as well as those determined by
SEC (elugrams shown in Figure 7) and are summarized in
Table 1. The SEC elugrams of all samples (Figure 7) revealed
monomodal traces of well-defined polymers with PDIs lower
than 1.08. Full incorporation of the initiator was further
confirmed by the MALDI-ToF MS (Figure 8). All mass-
averaged peaks satisfied the following equation.
g
g
mol
= 46.44
+ 475
mol
g
mol
≈ 2500
These values are in very good agreement with the
theoretically expected ones (Pn = 45, Mn = 2450 g·mol−1),
whereas the Mn derived from SEC analysis (see SEC elugram in
the SI, Figure S44) was smaller (1690 g·mol−1). The
underestimation of Mn found by SEC results from the rather
large apolar initiating moiety, which leads to a contraction of
the polyether coil to reduce initiator/solvent interactions.
Nevertheless, the SEC elugram corresponded to a well-defined
polymer with a polydispersity index of 1.08. Because cholesteryl
PEGs are known to be amphiphilic and form micelles in
aqueous solutions, the critical micelle concentration of 5 was
determined by measuring the surface tension of aqueous
solutions of the scissile cholesteryl PEG with a ring
+
M6(n) = M4b + n·MEO + MK
g
g
mol
g
mol
= 329.4
+ n·44.05
+ 39.1
mol
In conclusion, we successfully applied 4b as an initiator for
the oxyanionic polymerization of ethylene oxide and confirmed
its full incorporation into well-defined poly(ethylene glycol)s
with adjustable molecular weights.
To obtain heterofunctional polyethers, all samples of 6 were
hydrolyzed to the corresponding α-amino -ω-hydro PEGs 750,
775, and 7140. Complete removal of the benzyl groups was
tensiometer. Compound 5 has a CMC of 4.20
0.39
mg·L−1, which is in the order of CMCs expected for
verified by the absence of aromatic signals in the H NMR
1
amphiphilic polyethers of similar molecular weights.54,59
spectrum (exemplary spectrum of 750 shown in the Supporting
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Figure 5. Acidic cleavage of scissile cholesteryl PEG 5. (A) Cleavage followed by turbidimetry (λ = 528 nm) at pH 1 for two different temperatures
(25 and 37 °C). (B−D) Photographs of 5’s cleavage in comparison to regular cholesteryl PEG (cholPEG). (B) Aqueous polymer solutions. (C)
Onset of cholesterol precipitation after addition of hydrochloric acid. (D) Precipitated cholesterol in case of scissile cholesteryl PEG 5.
1
Figure 7. SEC elugrams of 6 with various degrees of polymerization;
RI detector signal.
Figure 6. H NMR spectrum (400 MHz) of 650 recorded in CDCl3.
IPEG − 6
3
4
Information, Figure S26). The residual initiator proton signals
(quadruplet of methine at 4.68 ppm, triplet of methylene
adjacent to the amino group at 2.65 ppm, and doublet of
methyl group at 1.20 ppm) were still observed. The degrees of
polymerization, determined by the following equation, were in
good agreement with those of the corresponding precursors
(Table 1). Hence, no cleavage of the acetal occurred during the
hydrogenation step.
P =
·
n
IMe
The MALDI-ToF mass spectra of 7 (SI, Figures S40−S42)
supported this result, as each of them revealed the distribution
of mass peaks of the desired species.
As mentioned in a previous study, PEGs carrying a terminal
amino group exhibit an apparent broadening of the molecular
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Table 1. Characteristic Data of Scissile Heterofunctional
PEGs 6, 7, and 10
a
b
c
c
Mn
polymer (g·mol−1
Mn
Mn
Mw
b
c
)
(g·mol−1
)
(g·mol−1
)
(g·mol−1
)
Pn
PDI
650
675
6140
750
775
7140
10
2320
3330
5730
2330
3450
6310
2510
3630
6490
2400
3470
6500
3610
2140
3040
2320
3260
50
1.08
1.07
75
5590
d
5900
d
140
51
1.06
d
d
d
d
d
d
d
75
144
76
3570
3160
3360
1.06
a
b
c
Calculated. Determined by 1H NMR. Determined by SEC,
d
referenced to PEG standards. Not determined, due to apparent
broadening of molecular weight distribution in SEC analysis.
Figure 9. SEC elugrams of 675 (red, RI detector), 775 (dotted black, RI
detector) and 10 (blue, UV detector). Note that amino PEGs often
revealed a broadening of the mass distribution in the SEC analysis (on
our system) leading to a apparent increase of the PDI.
cold diethyl ether during the workup. Full conversion of the
amino terminus to the squaric acid ester amide and complete
retention of the acetal was verified by MALDI-ToF MS. The
mass spectrum (SI, Figure S43) shows the expected distribution
of potassium cationized polymer peaks. In the 1H NMR
spectrum of 10 (SI, Figure S28), the methyl protons showed an
isolated signal around 1.28 ppm, whereas the corresponding
methylene signal was superimposed by the acetal methine
resonance at 4.76 ppm. The Pn determined from the NMR
spectrum is in very good agreement with the values calculated
for the amino PEG precursor. However, a small amount (<5
mol %) of acetaldehyde was detected in the proton NMR
spectrum. Since the ethyl ester’s methyl group integrated to the
expected value, the MALDI-ToF MS detected a single
distribution, and TLC of 10 showed a single UV-active
compound, the detected partial acetal cleavage had occurred in
the NMR tube.
A closer look was taken on the acidic cleavage of the acetals
by following the hydrolysis of the α-(1-(2-dibenzylamino
ethoxy)ethoxy) ω-hydro PEG 675 at 37 °C in acidic D2O
solutions with various pD values (pD 2.4, 4.4, 4.9, and 5.4) with
1H NMR spectroscopy. All integrals were referenced to the
aromatic resonances of the benzyl groups and the integral of
the acetal methyl signal was monitored to determine the
residual acetal. The acidic acetal cleavage was also investigated
for 10 at pD 4.9 in an experiment analogous to those described
before. In Figure 10, the normalized integral values of the
residual acetals of 675 and 10 are plotted against time (find
corresponding NMR spectra in the SI, Figures S48−S52).
Because the solvent served as a reagent in the degradation and
the samples were diluted (c6 = 5.74 mM, c10 = 2.38 mM), it was
assumed that acetal hydrolysis followed pseudo-first-order
kinetics and, therefore, the experimental data was fitted with
an exponential decay function:
Figure 8. MALDI-ToF mass spectrum of 650 and detail. Masses given
for averaged signals, mass difference calculated from monoisotopic
peaks. Spectrum was recorded in reflectron mode.
weight distributions in SEC analysis with our system.51 This
effect was also observed for all samples of 7 (exemplary SEC
elugram of 750 shown in SI, Figure S45), which is why no
reasonable molecular weight average and thus no PDI could be
determined by SEC. However, upon derivatization of the amino
group SEC revealed well-defined PEG with narrow molecular
weight distribution (vide infra).
To confirm the accessibility of 7’s cleavable terminal amino
group for further derivatization reactions and activate the
polyether for the bioconjugation with proteins, the recently
established conversion of α-amino ω-hydroxyl PEGs into the
corresponding squaric acid ester amides51 was carried out.
Compound 775 was reacted with diethyl squarate to yield 10
(Scheme 4).
In Figure 9 the superimposed SEC traces of 10 and its
precursors 675 and 775 are shown. The SEC elugrams of 10 and
675 exhibited monomodal traces of well-defined polymers with
corresponding molecular weight distributions. The PDI of 10
(1.06) was slightly lower than the PDI found for 675 (1.07)
which was attributed to a loss of a small low-molecular weight
fraction of the polymer upon repetitive precipitation of 10 in
Scheme 4. Synthetic Route to Scissile Squaric Acid Ester Amido PEGs
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+
hydrolysis of (simple) noncyclic acetals in water is around kD /
kH = 2.6−2.7.60
+
B.3. PEG with Scissile Backbone. Well-defined functional
PEGs with a cleavable group in the backbone can be
synthesized rapidly with our methodology using an AROP
macroinitiator such as 1-(2-hydroxyethoxy)ethoxy mPEG (4c).
The amount of EO batched in the polymerization was
calculated to add a PEG block with a number-averaged
molecular weight of 6.0 kg·mol−1, allowing separation and
distinction of this block from the macroinitiator precursor (2.0
kg·mol−1) via SEC upon acidic cleavage of the acetal. For the
interpretation of the 1H NMR spectrum of the scissile mPEG 8
(SI, Figure S27) the peak integrals were again referenced to the
signal of the methoxy group. Except for the larger backbone
signal the spectrum was almost identical to that of the
precursor. In agreement with the expected theoretical value the
1
Mn was calculated from the H NMR spectrum to be 7.9
kg·mol−1 using the following equation:
Figure 10. Acidic cleavage of scissile PEGs 675 (circles) and 10
(squares) in deuterium oxide followed by H NMR spectroscopy at
various pD values at T = 37 °C: crossed circles, pD 2.4; dotted circles,
pD 4.4; full circles/squares, pD 4.9; open circles, pD 5.4; solid lines,
exponential fits.
1
I
g
g
3
Mn = P ·MEO + MCH OH
=
·
PEG ·44
+ 32
n
3
4 ICH
mol
mol
3
This value also corresponds well to the Mn determined from
the SEC trace (7.5 kg·mol−1, elugram shown in Figure 11).
Ot
I6 (pD, t) = e−k ·I6 (pD, t = 0)
D
2
75
75
The cleavage rate constants in deuterium oxide kD O and
2
corresponding half-lives t1/2 of all hydrolyses were calculated
from the exponential fits and are listed in Table 2. Similar to the
Table 2. Acetal Cleavage Rate Constants and Half-Lives of
Scissile PEGs in Acidic Deuterium Oxide
polymer
pD
kD O (s−1
)
t1/2 (h)
2
675
675
675
675
10
2.4
4.4
4.9
5.4
4.9
1180 100
25.4 0.7
17.4 0.3
2.60 0.23
40.2 1.9
2.12 0.18
98.2 2.9
143.1 2.9
961.4 86.2
62.1 3.0
acidic degradation of recently presented poly(glycidyloxyethyl
ethylene glycol ether) (PGEGE) copolymers,44 a strong pH-
dependence of the degradation kinetics was observed. At pD
2.4, half the amount of 675 had been hydrolyzed after 2 h,
whereas at pD 5.4 the polymer exhibited a half-life of over a
month (t1/2 = 40 4 d). Most interestingly, 10 degrades much
faster than 675 under the same conditions. The basic tertiary
amino group adjacent to the acetal of 675 is protonated and
carries a positive charge in acidic media. Hence, the pre-
equilibrium protonation of one of the acetal oxygens (due to
the Coulomb forces most probably the one at the PEG side),
which occurs prior to the cleavage of the acetal carbon−oxygen
bond,60 is hindered, and therefore, the hydrolysis rate of 675 is
comparably low. The corresponding nitrogen of 10 will not be
protonated, because its electron lone pair is delocalized in the
squaric acid amide bond, resulting in a faster acetal cleavage.
Nevertheless, squaric acid amides are known to be protophilic61
and 10 will be protonated at its squaric acid moiety to some
extent. This explains why under comparable conditions the
half-life of 10 is longer than that of Bn2NTrisP(G-co-GEGE),
whose focal amino group is spatially separated from most of its
acetaldehyde acetals.44 Unfortunately, these values cannot be
transferred directly to protic systems, as it has to be taken into
account that the kinetic deuterium isotope effect for the
Figure 11. SEC elugrams of mPEG 1c (blue) and scissile mPEG 8
(black) before and after acetal cleavage; RI detector channel.
Note, that the trace was referenced to a PEG standard.
Compared to the macroinitiator, the molecular weight
distribution became broader and the polydispersity index
increased to 1.09, still indicating a well-defined polyether.
The cleavability of the in-chain acetal in acidic media was
demonstrated by stirring the scissile mPEG 8 in aqueous 0.11
M p-toluene sulfonic acid for 3 h at room temperature. In
Figure 11, the normalized SEC elugrams of 8 before and after
degradation as well as its mPEG precursor 1c are shown. Note
that the degraded sample exhibits a bimodal trace and the mode
corresponding to a smaller hydrodynamic radius fits well to that
of 1c. The second mode is significantly shifted to a lower
molecular weight in comparison to the SEC trace of 8 and was
clearly assigned to the PEG block polymerized onto 4c. Both
modes of the degraded sample corresponded to narrowly
distributed PEGs with number-averaged molecular weights of
about 1.8 and 5.7 kg·mol−1, respectively, which is in very good
agreement with the expected values. Hence, we successfully
457
dx.doi.org/10.1021/bm3016797 | Biomacromolecules 2013, 14, 448−459

Biomacromolecules
Article
demonstrated the incorporation of a single acetal into the
backbone of well-defined poly(ethylene glycol) monomethyl
ether with full control over its position in the chain and the final
cleavability of that moiety under acidic conditions.
C. Exploratory Results on Bioconjugation to BSA. A
total of 13 equiv of 10 were coupled to BSA using a protocol
for the squaric acid mediated PEGylation recently published by
our group.51 Successful covalent attachment of the acetal-
containing squaric acid amido PEGs was evidenced by SDS-
PAGE. In Figure 12 the Coomassie Blue-stained gel obtained
molecular weight initiators, polyethers with cleavable initiator
moieties, but completely different properties were generated.
PEG carrying the cleavable cholesterol unit is an amphiphile
with a CMC of 4.20 mg·L−1, whereas the cleavable heterofunc-
tional PEGs obtained from dibenzylamino ethanol could be
subsequently derivatized and activated for the recently
described squaric acid mediated PEGylation. The latter was
proven in a first exploratory model reaction to BSA. In the case
of the scissile macroinitiator, PEG carrying a single acetal
moiety in the backbone of the polymer was synthesized. All
PEGs were characterized by NMR spectroscopy, MALDI ToF
mass spectrometry, and size-exclusion chromatography (SEC).
The incorporation of a single acetal unit at the desired position
was verified for each of the well-defined polyethers (PDIs ≤
1.09). All of the obtained polymers and the PEGylated BSA
were proven to be cleaved at the acetal moieties in acidic media.
In conclusion, we established a rapid methodology to
incorporate a single acid labile moiety at a desired position in
well-defined functional poly(ethylene glycol)s. These materials
are highly interesting for the development of new pharmaceut-
icals as well as for materials science. Possible applications of the
acid sensitive cholesteryl PEGs, for example, for the reversible
stabilization of liposomes or acid sensors are subjects to
ongoing studies. Also, the PEGylation of other proteins than
BSA with the scissile polyethers, the pharmaceutical properties
of the conjugates, especially the in vivo bioactivities, as well as
their toxicities are under current investigation.
Figure 12. SDS-PAGE of PEGylated BSA: lane 1, MW marker; lane 2,
BSA; lane 6, BSA PEGylated with 10; lane 7, PEGylated BSA after
acidic treatment.
ASSOCIATED CONTENT
* Supporting Information
■
S
from the electrophoresis is shown. Compared to BSA (lane 2),
the BSA-PEG conjugate (lane 6) exhibits a clear shift toward
higher molecular masses. Lane 2 further reveals the presence of
a fraction of dimerized BSA, which also was PEGylated
completely (high-molecular weight band in lane 6). The
polydispersity of both the molecular weight of the synthetic
polyether and the number of polymer chains attached to the
protein result in the clear broadening of the protein−polymer
conjugate band.
Further synthetic protocols, NMR spectra, IR spectra, SEC
elugrams, and MALDI-ToF mass spectra, as well as procedures
and spectra of reaction kinetics, followed by NMR spectros-
copy. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: hfrey@uni-mainz.de.
To demonstrate the cleavability of the acetals and the
resulting release of BSA, the synthesized BSA-PEG conjugate
was hydrolyzed in 1 N hydrochloric acid. SDS-PAGE of the
product (Figure 12, lane 7) exhibits complete release of the
protein, which migrated the same distance through the gel as
the unmodified BSA. This is also true for the dimer. The slight
broadening of the bands was attributed to the polydisperse
number of squaramide linkers still attached to the protein. In
conclusion, we successfully demonstrated that the incorpo-
ration of an acetal moiety into squaric acid amido PEGs does
not decrease their applicability as PEGylation agents, but gives
access to acid-sensitive protein−PEG conjugates. The detach-
ment of the polyether from the conjugate under physiologically
more relevant conditions is under current investigation.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors are thankful to Jacky Thill, Eric Hoffmann, Alina
Mohr, and Margarete Deptolla for technical support. C.D. is
grateful to the Max Planck Graduate Center with the Johannes
Gutenberg-Universitat Mainz (MPGC) for a fellowship and
̈
financial support. S.S.M. and T.S. are recipients of a fellowship
through funding of the Excellence Initiative (DFG/GSC 266)
in the context of the graduate school of excellence “MAINZ”
(Materials Science in Mainz).
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