Silver Oxide Mediated Monotosylation of Poly(ethylene glycol) (PEG): Heterobifunctional PEG via Polymer Desymmetrization

Article abstract of DOI:10.1021/acs.macromol.7b01787

Heterobifunctional poly(ethylene glycol)s (PEGs) are key structures for bioconjugation in the context of the PEGylation strategy to enhance blood circulation times of, for example, peptide drugs or stealth liposomes. The formation of heterobifunctional PEGs from symmetric PEG diols is challenging because of limited yields of the targeted monofunctional product and difficulties associated with separation steps. On the basis of a detailed comparison of reaction conditions, we have investigated a polymer desymmetrization strategy to maximize the yields of monofunctional PEG tosylate. The tosylation reaction in the presence of the heterogeneous catalyst silver oxide and potassium iodide in a specific stoichiometric ratio proved to be highly efficient, resulting in 71-76% yield of monofunctional PEG depending on molecular weight, exceeding the expected value of 50% in a statistical reaction without addition of a catalyst. For characterization as well as for the preparative separation of monotosylated PEG, we developed a HPLC method, using an evaporative light scattering detector, enabling both analytical and semipreparative separation of monotosylated PEGs on gram scale up to 20 000 g mol^-1. To demonstrate the efficiency of the procedure, an α-azido-ω-methacryloyl-PEG was prepared as a building block suitable for azide-alkyne click-type reactions that can be incorporated into polymer networks via radical polymerization. We click-functionalized α-azido-ω-methacryloyl-PEG with a mannose-functionalized alkyne to enable functionalization of nanogels for enhanced cellular uptake via the mannose receptor. The synthesis strategy is suitable for a broad range of applications in the field of PEGylation and for hydrogel and nanogel functionalization.

Full text of DOI:10.1021/acs.macromol.7b01787

Article
Cite This: Macromolecules XXXX, XXX, XXX−XXX
Silver Oxide Mediated Monotosylation of Poly(ethylene glycol) (PEG):
Heterobifunctional PEG via Polymer Desymmetrization
Hannah Pohlit,†,‡,§ Matthias Worm,‡ Jens Langhanki,‡ Elena Berger-Nicoletti,‡ Till Opatz,‡
and Holger Frey*,‡
†Department of Dermatology, University Medical Center Mainz, Langenbeckstr. 1, 55131 Mainz, Germany
‡Institute of Organic Chemistry, Johannes Gutenberg University Mainz, Duesbergweg 10-14, 55128 Mainz, Germany
§Graduate School Materials Science in Mainz, Staudinger Weg 9, 55128 Mainz, Germany
S
* Supporting Information
ABSTRACT: Heterobifunctional poly(ethylene glycol)s
(PEGs) are key structures for bioconjugation in the context of
the “PEGylation” strategy to enhance blood circulation times of,
for example, peptide drugs or “stealth” liposomes. The formation
of heterobifunctional PEGs from symmetric PEG diols is
challenging because of limited yields of the targeted monofunc-
tional product and difficulties associated with separation steps.
On the basis of a detailed comparison of reaction conditions, we
have investigated a “polymer desymmetrization” strategy to
maximize the yields of monofunctional PEG tosylate. The
tosylation reaction in the presence of the heterogeneous catalyst
silver oxide and potassium iodide in a specific stoichiometric ratio
proved to be highly efficient, resulting in 71−76% yield of
monofunctional PEG depending on molecular weight, exceeding the expected value of 50% in a statistical reaction without
addition of a catalyst. For characterization as well as for the preparative separation of monotosylated PEG, we developed a HPLC
method, using an evaporative light scattering detector, enabling both analytical and semipreparative separation of monotosylated
PEGs on gram scale up to 20 000 g mol−1. To demonstrate the efficiency of the procedure, an α-azido-ω-methacryloyl-PEG was
prepared as a building block suitable for azide−alkyne click-type reactions that can be incorporated into polymer networks via
radical polymerization. We click-functionalized α-azido-ω-methacryloyl-PEG with a mannose-functionalized alkyne to enable
functionalization of nanogels for enhanced cellular uptake via the mannose receptor. The synthesis strategy is suitable for a broad
range of applications in the field of PEGylation and for hydrogel and nanogel functionalization.
(“alkyne−azide click”, AAC), or thioether formation (“thiol−
ene click”).
INTRODUCTION AND MOTIVATION
■
Poly(ethylene glycol) (PEG) shows highly desirable character-
istics for biomedical applications. It is soluble both in water and
in a variety of organic solvents. PEG is nontoxic, chemically
inert, and mostly nonimmunogenic. These advantages render
PEG the current gold standard polymer for antifouling
materials, drug-delivery systems, or attachment to therapeutic
molecules to enhance water solubility, blood circulation time,
and bioavailability.1,2 To achieve targeted drug delivery, the
attachment of targeting molecules to nanocarrier systems is
often required.3,4 For conjugation of targeting molecules to the
nanocarrier surface, heterobifunctional PEGs (PEGs bearing
two different end groups) are particularly interesting. Depend-
ing on the desired end groups of the polymer chain, there are
different conjugation strategies to attach targeting molecules
(e.g., antibodies or sugar-based substrates).5 Popular con-
jugation chemistries target functional amino acid residues
present in proteins and peptides (e.g., reaction of mPEG-N-
hydroxysuccinimide with lysine residues), click chemistry
At present, various heterobifunctional PEGs are commer-
cially available. Three different strategies have been described to
prepare heterobifunctional oligo(ethylene glycol)s (OEGs) and
PEGs: (i) The first is ring-opening polymerization of ethylene
oxide (EO) using an initiator and a termination reagent with
different functional moieties. This method limits the availability
of functional groups, as they have to tolerate the strongly basic
conditions of the oxyanionic polymerization. Furthermore, the
synthesis needs to be carried out under inert atmosphere to
exclude polymer chain growth initiated by traces of water and
requires handling of the toxic gas EO.6−24 (ii) The second
method involves iterative coupling reactions of heterobifunc-
tional oligomers to build up OEGs and PEGs. This method
requires considerable synthetic effort and purification in
Received: August 17, 2017
Revised: November 10, 2017
© XXXX American Chemical Society
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between the individual coupling steps.25,26 (iii) Another
strategy consists of a monofunctionalization of readily available
OEG/PEG-diol with a functional group and subsequent
(chromatographic) isolation of the desired heterobifunctional
PEG molecule. Symmetrical PEG-diols as inexpensive starting
materials are available in large quantities and with a broad range
of molecular weights. For statistical reasons, this functionaliza-
tion always results in a mixture of non-, mono-, and
disubstituted PEGs, affording moderate yields of the desired
monofunctional product. Furthermore, difficulties have been
reported for the purification of higher molecular weight PEGs
(with Mn exceeding 1000 g mol−1), as the chemical difference
(none, one, or two end groups) between the reaction products
decreases with molecular weight as a result of the decreasing
influence of the end group.27−37 For this reason, the third
approach has been mostly used for low molecular weight
oligomers or heterobifunctional PEGs that possess ionizable
end groups that facilitate separation by ion exchange
chromatography.38 In the current work, the third method,
“polymer desymmetrization” of PEG, is studied and developed
for the synthesis of monotosylated PEG (Mn = 1500−20 000 g
mol−1), which is a valuable intermediate that can be converted
into a broad range of heterobifunctional PEGs.
In recent years, several reports have described an enhanced
selectivity during tosylation of oligo(ethylene glycol)s (OEG)
to monosubstituted product based on the addition of silver
oxide and potassium iodide. Bouzide et al. first reported the
benefits of the use of silver oxide in combination with
potassium iodide for the tosylation reaction of monofunctional
OEGs.39 This method mediates sulfonation reactions of
alcohols in high yields under neutral reaction conditions
without the release of chloride as a byproduct. Potassium iodide
is required because it converts tosyl chloride to the much more
reactive tosyl iodide in situ and consequently accelerates the
reaction rate.39 In a subsequent publication, Bouzide et al.
investigated monotosylation reactions and observed high yields
of monotosylated OEGs (yield 61−92% monotosylated
product for OEG with molecular weights of 62−282 g
mol−1). It is obvious from simple statistical considerations
that for a 1:1 reaction of PEG with tosyl chloride one would
expect a product ratio of 25% PEG-diol (1), 50% PEG-
monotosylate (2), and 25% PEG-ditosylate (3) (see Scheme
1).
Scheme 2. Coordination of Oligo(ethylene glycol) with
Silver Cations As Proposed by Bouzide et al. (Left) versus
Surface Controlled Asymmetric Activation of PEG at the
Heterogeneous Silver Oxide Catalyst Particles (Right)
that intramolecular hydrogen bonding is responsible for
deprotonation of one hydroxyl group by silver oxide, suggesting
that the other hydroxyl group becomes less acidic.41,42
However, it is unlikely that the preferred monosubstitution of
poly(ethylene glycol) depends on the coordination of all
oxygen atoms to the silver cation, as it was proposed for
oligo(ethylene glycol). Furthermore, due to the low solubility
of silver oxide in toluene, free silver cations would rather
interact with iodide than with the ether oxygens. The general
concept can be transferred to monoacylation, monobenzylation,
or monophosphorylation of e.g. carbohydrates43−45 and has
also been used for other oligo(ethylene glycol)s46 or poly-
(ethylene glycol)s.28,41,42,47,48 Obviously, the silver oxide-based
strategy bears high promise although it currently lacks a
conclusive, mechanistic picture. Unfortunately, none of the
existing publications suggest a mechanism for the preferential
monotosylation reaction of poly(ethylene glycol), and
analytical data are limited or missing.
Based on our previous experience, characterization of
1
monosubstitution products by H NMR and size exclusion
chromatography (SEC) is not adequate, since the global
information obtained from both methods cannot conclusively
distinguish between a monotosylated PEG and a mixture of
non- and ditosylated PEG. On the other hand, using only
MALDI-ToF (matrix-assisted laser desorption ionization time-
of-flight) mass spectrometry is not suitable to quantify the
amount of the different PEG structures in a reaction mixture
with non-, mono-, and ditosylated PEG. To the best of our
knowledge, no systematic comparison of tosylation reactions of
dihydroxy-functional (= nonfunctionalized) PEG has been
conducted using silver oxide and potassium iodide as additives
versus conventional, i.e., random tosylation reactions without
any catalyst added. In the first part of this work, the effect of
silver oxide and potassium iodide on the yield of monotosylated
PEG is investigated. Subsequently, an example for the synthetic
value of monotosylated PEG, i.e., modification of PEG chains
with an azide functionality as well as a methacryloyl unit in ω-
position is presented, permitting both azide−alkyne click
reactions and covalent incorporation of the heterobifunctional
polymers into chemical networks via radical polymerization.
The authors tentatively explained the enhanced yields of
monotosylated OEG by coordination of the silver cation with
the oxygen atoms in the OEG backbone (see Scheme 2).40 Hsu
et al. as well as Mahou et al. attribute the selectivity toward
monofunctionalized PEG with intramolecular hydrogen bond-
ing, which is favored by the chelate formation. They propose
Scheme 1. Tosylation Reaction of OEG or PEG with Silver
Oxide and Potassium Iodide in Toluene at 40 °C with
Product Mixture Due to Reaction Statistics
RESULTS AND DISCUSSION
■
Synthesis and Purification of Monotosylated PEG. The
monotosylation step is crucial as it represents the “desymmet-
rization” of the PEG diol. Subsequently, recovery of the pure
monofunctionalized product is essential for the following
transformations to obtain pure products. For statistical reasons,
in a stoichiometric ratio of PEG and tosyl chloride, i.e., using a
1:1 ratio of PEG and tosyl chloride, one would expect a mixture
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of the possible reaction products PEG-diol (1), PEG-
monotosylate (2), and PEG-ditosylate (3) in a ratio of 1:2:1.
Improving the yield of monotosylated PEG is highly desirable.
An enhanced selectivity toward monotosylation of PEG has
been proposed for reactions in the presence of silver oxide and
potassium iodide in toluene.41,42 To the best of our knowledge,
the effects of silver oxide and potassium iodide on the
tosylation reaction have not been studied for polymer diols,
such as PEG in comparison to statistical tosylation reactions so
far. Therefore, we decided to vary and compare different
reaction conditions with regard to the amount of monotosy-
lated PEG produced.
Table 1. Comparison of the Influence of Different Reaction
Conditions on the Amount of Monotosylated PEG
a
reaction conditions (1.0
equiv PEG, 1.0 equiv TsCl,
40 °C, 16 h)
PEG
(Mn/g
yield
yield
yield
condition
mol−1
)
1 (%) 2 (%) 3 (%)
C1
1.5 equiv Ag2O, 0.2 equiv KI,
12.5 mL toluene
1500
17.9
76.1
90.1
14.4
15.8
99.5
22.8
29.6
99.4
74.6
99.5
87.9
77.2
42.9
42.3
73.3
23.5
9.9
8.8
0.4
0.0
14.4
7.9
0.0
7.5
0.0
0.0
2.1
0.0
0.0
0.3
1.9
1.8
C2
1.5 equiv NEt3, 12.5 mL
1500
1500
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
b
DCM
C3
1.5 equiv NEt3, 25 mL
b
DCM
C4
1.5 equiv Ag2O, 0.2 equiv KI,
12.5 mL toluene
71.2
76.2
0.5
1
To date, merely H NMR and MALDI-ToF MS have been
C5
1.5 equiv Ag2O, 0.2 equiv KI,
25 mL toluene
employed to confirm formation of the pure monotosylated
1
C6
1.5 equiv Ag2O, 0.2 equiv KI,
125 mL toluene
product in the Ag2O-catalyzed tosylation. However, H NMR
spectroscopy is inappropriate to exclude the presence of diol-
PEG and ditosylated PEG. The integrals of the hydroxyl group
and the tosylate group may be in good agreement for
C7
15 equiv Ag2O, 2 equiv KI,
12.5 mL toluene
69.7
70.4
0.6
C8
30 equiv Ag2O, 4 equiv KI,
12.5 mL toluene
1
monotosylated PEG product in H NMR (as an example see
C9
0.15 equiv Ag2O, 0.02 equiv
KI, 12.5 mL toluene
Figure S1); however, detailed investigation of the sample by
MALDI-ToF mass spectrometry shows distributions that can
be unambiguously assigned to the monotosylated product (2),
diol-PEG (1), or ditosylated PEG (3) (see Figure S3). On the
other hand, it is not possible to quantify the amount of non-,
mono-, and ditosylated PEG using MALDI-ToF MS, as we
were able to confirm by mixing the purified components in
different ratios, subsequently analyzing the integrals appearing
in the MALDI-ToF MS (see Figures S46−Figure S48). The
signals cannot clearly be assigned to the non-, mono-, and
ditosylated compounds 1, 2, and 3, since combination with
different cations may result in the same m/z value (see Figure
S46). Furthermore, the fraction of polymer coordinating to a
certain type of cation is not predictable and not easily
reproducible. These facts lead to a visible trend upon mixing
of different PEG species, but it is not sufficient to obtain a
calibration curve (see Figure S49). The observation that
quantification of different polymer species possessing different
end groups in a polymer mixture is not possible by MALDI-
ToF-MS alone is relevant for numerous other works. For
example, α-(2-propenyl)-ω-(2-propenyloxy)poly(oxyethylene),
α-(2-propenyl)-ω-carboxymethylthiopoly(oxyethylene), and α-
carboxymethylthioethyl-ω-carboxymethylthio-oligo(ethylene
C10
C11
C12
C13
C14
C15
1.5 eqiv AgNO3, 0.2 equiv
KI, 12.5 mL toluene
23.2
0.5
1.5 equiv Ag2O, 12.5 mL
toluene
1.5 equiv Ag2O, 12.5 mL
toluene, 96 h
12.1
22.5
55.2
55.9
1.5 equiv NEt3, 12.5 mL
toluene
1.5 equiv NEt3, 12.5 mL
b
DCM
1.5 equiv NEt3, 12.5 mL
b
DCM, TsCl dissolved in
DCM added dropwise
C16
1.5 equiv NEt3, 25 mL
2000
81.2
18.8
0.0
b
DCM
C17
C18
12.5 mL pyridine
2000
2000
96.9
38.7
3.1
0.0
6.0
1.5 equiv NEt3, 12.5 mL
DCM, subsequent addition
of Ag2O
55.3
C19
C20
C21
C22
C23
C24
C25
C26
C27
C28
C29
1.5 equiv Ag2O, 0.2 equiv KI,
12.5 mL toluene
4000
4000
4000
8000
8000
8000
8000
8000
8000
10000
20000
57.3
26.6
74.0
88.3
65.2
90.6
73.4
55.2
87.4
61.3
77.4
41.8
60.2
25.2
11.4
32.8
9.4
0.9
13.2
0.8
0.3
2.0
0.0
1.0
2.7
0.0
3.1
0.8
3 equiv Ag2O, 0.4 equiv KI,
12.5 mL toluene
1.5 equiv NEt3, 12.5 mL
b
DCM
glycol) were described by Volcker et al.29
1.5 equiv Ag2O, 0.2 equiv KI,
12.5 mL toluene
̈
To investigate how silver oxide and potassium iodide
enhance the yield of monotosylated product, statistical
tosylation of PEGs with different molecular weights was
performed in a variety of solvents in order to compare it to
the catalysed reaction. Both the stoichiometric ratio (PEG/
solvent/silver oxide) and the mode of addition of tosyl chloride
(in one dose or via slow addition) was varied and analyzed. To
exclude adsorption of one component of the reaction mixture
to the silver oxide surface, silver oxide was also added
subsequent to a tosylation reaction in an experiment under
statistical conditions (see Table 1, sample C18). Silver oxide is
insoluble in the solvents employed, which results in a
heterogeneous character of the reaction mixture and influences
its interplay with PEG and tosyl chloride. Reverse phase HPLC
was applied to quantify the amounts of the three possible
reaction products and was optimized to separate the
monotosylated PEG (Mn = 1500−20 000 g mol−1) on a
semipreparative scale for further reactions. A range of molecular
weights of PEG was chosen (see Table 1) that represents the
most commonly used molecular weights for PEGylation and
3 equiv Ag2O, 0.4 equiv KI,
12.5 mL toluene
1.5 equiv NEt3, 12.5 mL
b
DCM
1.5 equiv Ag2O, 0.2 equiv KI,
12.5 mL toluene, 48 h
25.6
42.0
12.6
35.5
21.7
3 equiv Ag2O, 0.4 equiv KI,
12.5 mL toluene, 48 h
1.5 equiv NEt3, 12.5 mL
b
DCM, 48 h
6 equiv Ag2O, 0.8 equiv KI,
12.5 mL toluene, 48 h
6 equiv Ag2O, 0.8 equiv KI,
12.5 mL toluene, 48 h
a
Amounts of reaction products were calculated from peak areas in
analytical HPLC measurements. Nonitalic fields: uncatalyzed reaction
b
conditions. Italic fields: Ag2O-catalyzed reaction conditions. DCM =
dichloromethane.
functionalization of nanoparticles or liposomes.5 Using a 20
min HPLC gradient (40%−100% acetonitrile in water), it was
C
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possible to separate all three possible reaction products. Elution
of the reaction products was monitored by means of an
evaporative light scattering detector. Subsequently, the purity of
the separated PEG products, proven by absence of signals for
the other reaction products, was confirmed by MALDI-ToF-
MS. This method is of general interest to distinguish between
nonfunctionalized and functionalized polymers. It is highly
relevant for determination of the content of undesired diols in
methoxy-PEG.
15.8%−77.4%. A systematic comparison of the influence of
different reaction conditions on the yield of monotosylated
product is summarized in Table 1.
To investigate (and eventually to confirm or dispute) the
influence of silver oxide and potassium iodide, statistical
tosylation (noncatalyzed tosylation reaction) of PEG in
dichloromethane with triethylamine and tosyl chloride was
performed. Under these conditions, regardless of slow addition
of tosyl chloride to the solution or addition in one portion, the
tosylation afforded 55.2% and 55.9% of monotosylated PEG
2000 (2), respectively (see Table 1, samples C14 and C15).
These results are in line with simple probability considerations
and are also in good agreement with literature regarding the
tosylation of PEG 2000, giving values of 34% PEG, 49% PEG-
monotosylate, and 12% PEG-ditosylate.27 The reaction with
silver oxide constantly produced higher yields of monotosylated
PEG (2) for PEG with Mn in the range of 1500−8000 g mol−1
compared to the statistical reaction conditions (see Table 1,
samples C1, C4, C19, and C22).
Figure 1 shows the typical separation and characterization
elugram, employing a mixture of PEG-1500-diol 1, PEG-1500-
To elucidate to which extent this effect depends on the
stoichiometric ratio of PEG, silver oxide, and solvent, as well as
reaction time and temperature, another set of experiments was
conducted. 15 or 30 equiv of silver oxide instead of 1.5 equiv
under otherwise the same reaction conditions resulted in
comparable amounts of monotosylation product (see Table 1,
samples C7 and C8). In contrast, dilution (only 0.15 equiv of
silver oxide or 10-fold the amount of solvent) resulted in
drastically decreased yields of monotosylated product 2 (see
Table 1, samples C6 and C9). The catalyst-free statistical
reaction with triethylamine in toluene or dichloromethane or in
pyridine resulted in a lower yield of monotosylated PEG 2
compared to catalyzed reactions, independent of molecular
weight (see Table 1, samples C2, C3, C10, C13−18, C21, C24,
and C27). To exclude an adsorption effect of one subset of
products, the reaction was performed under statistical
conditions in DCM. After 16 h of reaction time, Ag2O was
added and removed by filtration after 30 min of stirring.
However, the amount of monotosylated product and side
products remained constant, in full analogy to the reactions in
which no silver oxide was added (see Table 1, sample C18).
Mahou et al. proposed that the selectivity toward
monofunctionalized PEG depends on coordination of hydroxyl
groups to silver cations and activation through intramolecular
hydrogen bonding. However, if silver nitrate is used as a catalyst
instead of silver oxide, no selectivity toward monofunctional-
ized PEG was observed at all (see Table 1, sample C10). It is
noteworthy that even silver nitrate is insoluble in toluene and
reacts with potassium iodide to precipitated silver iodide.
The role of KI was also investigated by Bouzide et al.39
Potassium iodide was reported to enhance tosylation reaction
rates, as it converts the tosyl chloride to the much more reactive
tosyl iodide in situ.39 In accordance with this report, we
discovered almost no monotosylated PEG after 18 h of reaction
time and merely 12.1% of PEG-monotosylate after 96 h of
reaction time under otherwise the same reaction conditions,
when no potassium iodide was added (see Table 1, samples
C11 and C12).
Figure 1. Typical separation of a crude mixture of PEG-1500-diol 1,
PEG-1500-monotosylate 2, and PEG-1500-ditosylate 3 with reverse
phase HPLC. The purity of the separated PEG-derived structures was
confirmed by MALDI-ToF-MS after separation.
monotosylate 2, and PEG-1500-ditosylate 3, obtained in a
reaction using silver oxide and potassium iodide (1.5 equiv of
Ag2O, 0.2 equiv of KI, 12.5 mL of toluene) that was analyzed
with the HPLC method. For detailed mass spectrometry data of
the distinct tosylation products, see Figures S5−S7. Remark-
ably, HPLC separation of unreacted diol 1, the targeted
monotosylated PEG 2, and ditosylated PEG 3 was applicable
even for PEG chains with higher molecular weights (up to
20 000 g mol−1) with only little variation of the gradient
program (see Figure S19). Integration of the peak area allows
to determine the relative amounts of the reaction products
obtained from the crude reaction mixture.
The tosylation reaction catalyzed with silver oxide and
potassium iodide yields between 76.2% and 21.7% of
monotosylated PEG 2, depending on molecular weight (see
Table 1). The amount of the targeted monotosylated product 2
decreased with increasing molecular weight of PEG. The
difunctional side product ditosylate-PEG 3 was produced in
surprisingly low yields in the range of 14.4%−0.8%, and
unreacted PEG 1 was recovered with yields in the range of
In general, with increasing molecular weight of PEG, the
conversion and the yields of monotosylated PEG under the
same reaction conditions decreased. Therefore, we altered the
reaction conditions to 3 equiv of Ag2O and 48 h reaction time
for PEG-4000 and PEG-8000 and 6 equiv of silver oxide and
48 h of reaction time for PEG-10000 and PEG-20000,
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respectively (see Table 1, samples C19−C29). With 3 equiv of
Ag2O, PEG-4000 could be converted to PEG-monotosylate
with yields of 60.2% (2, see Table 1, sample C20) which
compares favorably to 25.2% of PEG-monotosylate (2, see
Table 1, sample C21) obtained under uncatalyzed reaction
conditions. For PEG-8000, prolongation of reaction times from
16 to 48 h resulted in 32.8% and 42.0% of the targeted
monotosylated product compared to 9.4% and 12.6% from
uncatalyzed, but otherwise similar, reaction conditions (see
Table 1, samples C23, C24, C26, and C27). Tosylation of PEG-
20000 still yields 21.7% of monotosylated product, which is
remarkable for such high molecular weights.
To sum up, the series of experiments regarding variation of
all reaction parameters showed that Ag2O in combination with
KI reproducibly provides an enhanced selectivity toward
monotosylation reactions of poly(ethylene glycol)s (42−76%
monotosylated product depending on molecular weight for
PEG-1500 to PEG-8000). In contrast, the uncatalyzed
tosylation reaction resulted in a systematically lower yield of
monotosylated product ranging from 12 to 55%, depending on
molecular weight in a range expected for the statistical
transformation. For PEG-2000 or lower molecular weights,
1.5 equiv of Ag2O was sufficient to obtain high yields of
monotosylated product (see Table 1, samples C1 and C5). For
PEG with a molecular weight exceeding 2000 g mol−1, the
amount of catalyst had to be increased to 3 or 6 equiv, and the
reaction time had to be prolonged to 48 h to obtain high
monotosylation yields (see Table 1, samples C20, C26, C28,
and C29). Slower reaction kinetics for larger PEGs are not
surprising, as larger PEGs are sterically disfavored for reaction
at both hydroxyl groups of the polymer coils.
the basis of the different end groups (see Table 1, sample C29,
and Figure S19). 20 000 g mol−1 is the largest molecular weight
used for bioconjugation to enable polymer elimination from the
body through renal excretion. The results assessed in the
analytical HPLC measurements were reproduced on a
semipreparative scale (up to 200 mg of crude product per
injection), again with baseline separation of the three relevant
PEG species. Thus, this polymer desymmetrization method
permits to obtain pure monotosylated product on a gram scale
after several runs of semipreparative HPLC. The purified
monotosylated product could be converted to heterobifunc-
tional PEGs with a variety of functional groups. As an example,
the synthesis of α-4-(α-D-mannopyranosyloxymethylene)-
1,2,3,triazol-1-yl-ω-methacryloyl-PEG, a polymer which is
suited for hydrogel and nanogel functionalization, is described
below.
Transformation of Heterobifunctional PEG to α-4-(α-
D-Mannopyranosyloxymethylene)-1,2,3-triazol-1-yl-ω-
methacryloyl-PEG. This section shows an example for the
preparative value of the monotosylated PEGs. Targeting of the
mannose receptor on CD11c positive cells (e.g., dendritic cells)
is important to enhance efficient cell-specific uptake of
nanoparticles.51−54 For the conjugation of mannose to the
polymer chain, a copper(I)-catalyzed alkyne−azide cyclo-
addition (CuAAC)55,56 click reaction was used. In a four-step
sequence, the commercial PEG-diol was transformed into α-4-
(α-D-mannopyranosyloxymethylene)-1,2,3,triazol-1-yl-ω-meth-
acryloyl-PEG, a building block that is highly valuable to
incorporate a functionality into nanoparticles synthesized by
radical polymerization (see Scheme 3). Although mannose has
Scheme 3. Reaction Scheme of the Transformation of PEG−
Monotosylate (2) to α-4-(α-D-
Mannopyranosyloxymethylene)-1,2,3-triazol-1-yl-ω-
methacryloyl-PEG (10) in a Three-Step Reaction
From our observations we derive the following hypothesis:
the underlying key effect for enhanced monotosylation adding
silver oxide compared to the statistical reaction is the
insolubility of silver oxide in organic solvents and its low
solubility in water (solubility product Ksp(Ag2O) in water at
20 °C = 1.69 × 10−6 mol3 L−3).49 Silver(I) oxide shows a rather
inhomogeneous particle size distribution in the size range of a
few nanometers up to 5 μm, which supports a heterogeneous
catalysis process at the surface of the silver oxide particles (see
Figures S50 and S51). The hydroxyl end groups of PEG can
dynamically interact with free coordination sites at the silver
oxide surface, forming weak interactions with the silver(I) oxide
particles, thereby promoting polarization and tosylation of
hydroxyl groups (see Scheme 2). Backfolding, i.e., loop
formation, of polymer chains is entropically disfavored;
therefore, coordination of both hydroxyl groups of PEG to
Ag2O is considerably less likely. Furthermore, Svedhem et al.
proposed that silver oxide acts as a halide acceptor in SN2
reactions of alkyl halides, which we consider quite possible.50
On the basis of our results (Table 1), we suggest a surface-
controlled activation of PEG and tosyl chloride at the
heterogeneous surface of the silver oxide particles. However,
the significantly enhanced amount of monotosylated product
must be due to a complex interplay between surface activation
and exchange dynamics at the surface.
been grafted onto polymers or attached via click chemistry to
(co)polymers before,57−60 to the best of our knowledge, this is
the first report regarding a facile method to synthesize a
mannose−methacrylate−PEG building block. This building
block can be used in (nano)hydrogel synthesis due to the
presence of methacrylate (enabling radical polymerization),
mannose (as targeting unit), and PEG (for miscibility and
aqueous solubility of the building block).
Efficient removal of unsubstituted PEG 1 and disubstituted
product 3 is a key requirement for the preparation of pure
monotosylated product 2. For all molecular weights inves-
tigated, excellent separation via analytical as well as semi-
preparative HPLC was achieved. Remarkably, even the reaction
products of PEG with 20000 g mol−1 can be separated with the
HPLC method described with baseline resolution, merely on
In the first step, we substituted the tosylate moiety by an
azide to obtain α-azido-ω-hydroxyl-PEG. Already in the two-
step reaction of PEG-diol to α-azido-ω-hydroxyl-PEG, the
preparative value of the synthesized heterobifunctional product
increases dramatically. The chemical characterization is shown
1
in Figures S21−S23. All resonances in the H NMR spectrum
E
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Scheme 4. Preparation of Propargyl-α-D-mannopyranoside (9)
Reaction temperatures referred to the temperature of the particular
cooling/heating bath.
can be assigned, and there are no remaining signals indicating
unreacted tosylate moieties. SEC and MALDI-ToF-MS
measurements also show a monomodal distribution and the
absence of residual monotosylate−PEG (confirmed by
MALDI-ToF-MS). The free hydroxyl group at the ω-position
can be further functionalized to enable cross-linking by radical
polymerization. This was achieved using the coupling agent
N,N'-Diisopropyl-carbodimide (DIPC) at room temperature.
Complete functionalization with methacrylate was confirmed
Chromatography. Thin-layer chromatography (TLC) was carried
out on silica gel 60 F254 plates (Merck) or RP silica gel RP-18 F254s
plates (Merck). Compounds were visualized using UV light and/or by
immersion in a solution of m-methoxyphenol (0.1 mL) in ethanol (95
mL) and sulfuric acid (2 mL) followed by heating.
Chromatography was performed using flash chromatography of the
indicated solvent system on 35−70 μm silica gel (Acros Organics).
1
Characterization Methods. H NMR spectra (400 MHz) were
1
recorded using a Bruker AMX400 spectrometer and referenced
internally to residual proton signals of the deuterated solvent. 1H
NMR spectra were analyzed with MestReNova v10.0.1.
by H NMR, SEC, and MALDI-ToF-MS analysis (see Figures
S24−S26).
Propargyl α-D-mannopyranoside was synthesized in a three-
step sequence as shown in Scheme 4 and Figures S31−S37.
The CuAAC reaction was tested with phenyl acetylene as a
For size exclusion chromatography (SEC) measurements, an
Agilent 1100 series integrated instrument was used, including a PSS
HEMA column (106/104/102 Å porosity) and a refractive index (RI)
and ultraviolet (UV) detector. Samples were measured in
dimethylformamide (DMF, containing 0.25 g/L of lithium bromide
as an additive), and calibration was carried out using poly(ethylene
oxide) standards. The standards and the software used for analysis
(PSS WinSEC Unity v7) were provided by Polymer Standards Service
GmbH. Data were plotted with MATLAB.
MALDI-ToF mass spectrometry was performed on a Shimadzu
Axima CFR MALDI-ToF mass spectrometer equipped with a nitrogen
laser delivering 3 ns laser pulses at 337 nm. Dithranol was used as
matrix, and potassium trifluoroacetate was added to facilitate
ionization of polymer samples. The MS spectra were analyzed with
Kompact Version 2.4.1 from Kratos Analytical Ltd.
The exact mass measurements (Figure S45) was performed on a
SYNAPT G2-Si mass spectrometer (Waters Corp., Manchester, U.K.)
equipped with a matrix-assisted laser desorption/ionization (MALDI)
source. The mass resolution was set to 15 000, and the instrument was
calibrated by clusters of red phosphorus. For sample preparation 1,8,9-
trihydroxyanthracene (dithranol) was used as a MALDI matrix.
Analyte and matrix were dissolved in tetrahydrofuran in a mixing ratio
of 1:500 by weight.
1
model reaction. According to H NMR and MALDI-ToF-MS,
the click reaction was quantitative, and no residual α-azido-ω-
methacryloyl-PEG could be detected (see Figures S27−S29).
The whole chain of reactions from PEG-diol to α-1,2,3-triazole-
4-phenyl-ω-methacryloyl-PEG was additionally monitored by
FTIR (see Figure S30).
For the application in nanogel synthesis, the covalent
attachment of target molecules like mannose to the polymer
is a crucial requirement. Therefore, we used copper(I)-
catalyzed alkyne−azide cycloaddition to attach a mannose-
substituted alkyne to the α-azido-ω-methacryloyl-PEG.
All resonances could be assigned in the NMR spectra. The
integral of H-5 of the triazole ring at 8.11 ppm, the
methacrylate methylene group at 6.12 and 5.65 ppm, and the
peaks of the mannose are well in accord with expected values
(see Figures S38−S43). A shift toward higher molecular
weights (2240 g mol−1 and Đ = 1.06) is visible in SEC
measurements compared to α-azido-ω-methacryloyl-PEG (see
Figure S44). The successful click reaction was confirmed by
MALDI-ToF-MS (see Figure S45).
The freeze-drier used was a Christ Alpha 2-4 LD Plus.
The gradient HPLC system for analytical and semipreparative setup
consisted of an Agilent Technologies 1260 Infinity system with a 1260
Quat pump, a 1260 ALS autosampler, a 1260 VWD UV−vis variable-
wavelength spectrophotometric detector, and a Softa 1300 evaporative
light scattering detector. The UV detector was operated at a
wavelength of 254 nm. For analysis, a Reprosil 100 (C18, 5 μm
particle size, 4.6 × 250 mm i.d.) column from Maisch GmbH was used.
All data were analyzed with PSS WinSEC Unity v7, provided by
Polymer Standards Service GmbH. For separation, a Reprosil 100
(C18, 5 μm particle size, 20 × 250 mm i.d. protected with 20 × 30 mm
i.d. column guard) column from Maisch GmbH was used. The mobile
phases consisted of acetonitrile (inlet A) and water (inlet B).
Fourier transform infrared spectroscopy (FTIR) was measured
using a Nicolet iS10 spectrometer from Thermo Fisher Scientific Inc.
The software for spectra analysis was Omnic Software v8.1.210.
Synthetic Procedures. Synthesis of α-Tosyl-ω-hydroxyl-PEG (1,
2, and 3). The synthesis of α-tosyl-ω-hydroxyl-PEG was modified
from Mahou et al.42 Exemplarily, the synthesis of α-tosyl-ω-hydroxyl-
PEG-1500 is described below. PEG (1.00 g, M = 1500 g mol−1, n =
0.667 mmol) was dissolved in benzene (5 mL) in a Schlenk flask and
dried under high vacuum after azeotropic distillation of benzene to
remove water. For the standard experiment, PEG was dissolved in dry
toluene (12.5 mL), and the reaction solution was heated to 40 °C.
Under vigorous stirring, Ag2O (261 mg, 1.5 equiv, n = 1.00 mmol) and
potassium iodide (25 mg, 0.2 equiv, n = 0.15 mmol) were added under
EXPERIMENTAL PART
■
Materials. Dimethylformamide, potassium trifluoroacetate, tol-
uene, dichloromethane, benzene, methanol, acetonitrile, methacrylic
acid, N,N′-diisopropylcarbodiimide (DIPC), 4-(dimethylamino)-
pyridine (DMAP), silver(I) oxide, silver nitrate, potassium iodide,
Cu(I)Br, and diethyl ether were purchased from Sigma-Aldrich. LiBr,
magnesium sulfate, phenylacetylene, tosyl chloride, N,N,N′,N″,N″-
pentamethyldiethylenetriamine (PMDETA), sodium azide, and hydro-
quinone were purchased from Acros. Dithranol was purchased from
Fluka. Deuterated DMSO and methanol were obtained from Deutero
GmbH. PEG standards were acquired from Polymer Standards Service
GmbH. Lewatit Mono Plus S100CHC4124 was a gift from Lanxess.
Dimethylformamide DMF, extra dry, AcroSeal) and pyridine were
purchased from Acros. All chemicals were used without further
purification, unless otherwise stated. Acetonitrile and dichloromethane
were distilled from calcium hydride. The eluents for column
chromatography (cyclohexane and ethyl acetate) were distilled prior
to use. Deuterochloroform was stored over alumina (Brockmann
activity I).
All reactions involving air or moisture sensitive reagents or
intermediates were performed under an inert atmosphere of argon
in glassware that was oven-dried using standard Schlenk techniques.
F
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inert conditions. After 15 min of stirring, tosyl chloride (150 mg, 1.05
equiv, n = 0.788 mmol) was added under argon counterflow, and the
reaction mixture was rigorously stirred overnight at 40 °C. The
amount of solvent, silver oxide, reaction temperature, and reaction
time were modified according to Table 1. The solution was filtered
through a filtration paper at 40 °C followed by solvent removal by
rotary evaporation. The crude product was purified by 2-fold
precipitation from methanol into ice-cold diethyl ether. The pure
product was dried under high vacuum for 24 h. The product mixture
(1, 2, and 3) was recovered as colorless powder (891 mg, yield = 80%,
M = 1670 g mol−1). 1H NMR (400 MHz, DMSO-d6) δ [ppm] = 7.78
(d, 2H, CaromH−C−SO3), 7.48 (d, 2H, CaromH−C−CH3), 4.58 (t, 1H,
−OH), 4.11 (m, 2H, CH2−SO3), 3.40−3.56 (m, 119H, (CH2−CH2−
O)n), 2.42 (s, 3H, −CH3) (see Figure S1).
For the noncatalyzed tosylation reaction, a procedure reported by Li
et al. was used and slightly modified.27 In short, PEG (1.0 g, M = 2000
g mol−1, n = 0.50 mmol) was dried with azeotropic distillation of
benzene. PEG was dissolved in DCM (12.5 mL), followed by the
addition of tosyl chloride (95 mg, 1 equiv, n = 0.50 mmol) and TEA
(138 μL, 2 equiv, n = 9.96 mmol), and stirred under an argon
atmosphere at 40 °C for 16 h. The solvent was removed by rotary
evaporation. The crude product was purified by 2-fold precipitation
from methanol into ice-cold diethyl ether. The pure product was dried
under high vacuum for 24 h. The pure product mixture (1, 2, and 3)
was recovered as colorless powder (873 mg, yield = 78%, M = 2170 g
mol−1).
mol−1, n = 0.299 mmol) and sodium azide (97 mg, 5 equiv, n = 1.5
mmol) were dissolved in dry dimethylformamide (DMF, 10 mL) and
stirred overnight at 90 °C under an argon atmosphere. After cooling to
room temperature, DMF was removed under reduced pressure. The
crude product was dissolved in dichloromethane (DCM, 5 mL) and
extracted twice with brine (5 mL) each and twice with deionized water
(5 mL) each. The organic phase was dried with magnesium sulfate.
After removal of DCM the crude product was precipitated twice from
DCM into ice-cold diethyl ether. The pure product was dried under
high vacuum for 24 h. The pure product was recovered as colorless
1
powder (266 mg, yield = 58%, M = 1541 g mol−1). H NMR (400
MHz, DMSO-d6) δ [ppm] = 4.58 (t, 1H, −OH), 3.46−3.56 (m, 132H,
(CH2−CH2−O)n), 3.41 (m, 4H, −(CH2)2−N3) (see Figure S21).
Synthesis of α-Azido-ω-methacryloyl-PEG (5). Exemplarily, the
synthesis of α-azido-ω-methacryloyl-PEG-2000 is described below. α-
Azido-ω-hydroxyl-PEG (4, 100 mg, M = 2041 g mol−1, n = 0.0499
mmol) was dissolved in dichloromethane (1 mL). Methacrylic acid
(4.21 μL, M = 86.06 g mol−1, n = 0.0499 mmol), N,N′-
diisopropylcarbodiimide (DIPC, 1.3 equiv, 10.2 μL, M = 126.2 g
mol−1, n = 0.0649 mmol), and 4-(dimethylamino)pyridine (DMAP,
0.5 equiv, 2 mg, M = 122.17 g mol−1, n = 0.0250 mol) were added, and
the reaction was allowed to proceed for 16 h at room temperature.
Hydroquinone (approximately 2 mg) was added prior to purification.
The crude product was purified by 3-fold precipitation from
dichloromethane into ice-cold diethyl ether. The pure product was
dried under high vacuum for 24 h. The pure product was recovered as
colorless powder (202 mg, yield = 73%, M = 1609 g mol−1). 1H NMR
(400 MHz, DMSO-d6) δ [ppm] = 6.03 (m, 1H, CH2−C−CH3), 5.69
(m, 1H, CH2−C−CH3), 4.20 (m, 2H, CH2−O−CO), 3.65 (m, 2H,
CH2−CH2−O−CO), 3.43−3.56 (m, 119H, (CH2−CH2−O)n), 3.39
(m, 2H, CH2−N3), 1.88 (s, 3H, −CH3) (see Figure S24).
Synthesis of α-4-Phenyl-1,2,3-triazol-1-yl-ω-methacryloyl-PEG
(6). Exemplarily, the synthesis of α-4-phenyl-1,2,3,triazol-1-yl-ω-
methacryloyl-PEG-1500 is described below. α-Azido-ω-methacryloyl-
PEG (5, 150 mg, M = 1609 g mol−1, n = 0.0932 mmol),
phenylacetylene (15 μL, 1.5 equiv, n = 0.14 mmol, m = 14 mg),
N,N,N′,N″,N″-pentamethyldiethylentriamine (PMDETA, 5.9 μL, 0.3
equiv, n = 0.028 mmol, m = 4.9 mg), and methanol (2 mL) were
mixed in a Schlenk flask. Three cycles of freeze pump−thaw were
performed for degassing the solvent. Addition of CuBr (4 mg, 0.3
equiv, n = 0.03 mmol) under argon counterflow. Stirring at room
temperature overnight, protected from light by tin foil. Addition of
three tips of a spatula acidic ion-exchange resin Lewatit Mono Plus,
stirring for approximately 1 h until discoloration of the solution. The
solution was filtered through a filtration paper followed by solvent
removal by rotary evaporation. The crude product was purified by 2-
fold precipitation from methanol into ice-cold diethyl ether. The pure
product was dried under high vacuum for 24 h. The pure product was
recovered as colorless powder (139 mg, yield = 87%, M = 1711 g
mol−1). 1H NMR (400 MHz, DMSO-d6) δ [ppm] = 8.53 (s, 1H, CH−
N), 7.84 (m, 2H, CaromH(ortho)), 7.44 (m, 2H, CaromH(meta)), 7.34
(m, 2H, CaromH(para)), 6.03 (m, 1H, CH2−C−CH3), 5.69 (m, 1H,
CH2−C−CH3), 4.57 (m, 2H, CH2−N), 4.20 (m, 2H, CH2−O−CO),
3.87 (m, 2H, CH2−CH2−N), 3.65 (m, 2H, CH2−CH2−O−CO),
3.43−3.56 (m, 158H, (CH2−CH2−O)n), 1.88 (s, 3H, −CH3) (see
Figure S27).
For the different reaction conditions, reactants and solvent
conditions were modified, as stated in Table 1.
Analysis and Purification of α-Tosyl-ω-hydroxyl-PEG (1, 2, and 3)
via HPLC. Analytical. The mixture of α-tosyl-ω-hydroxyl-PEG (2),
diol-PEG (1), and α,ω-ditosyl-PEG (3) (1 mg) was dissolved in
acetonitrile (ACN):water (40:60, 1 mL), filtered through a Rotilabo
syringe filter (PTFE, pore size 0.45 μm), and analyzed by RP-HPLC
with an acetonitrile/water gradient. Gradient conditions: linear
gradient from 40% ACN to 70% ACN over 7.5 min, then a linear
gradient to 100% ACN over 1.5 min, and then isocratic at 100% ACN
for 3 min. Subsequently the starting gradient was restored in 1 min
and a 8 min reconditioning time of 40% ACN:60% water was allowed
before further gradient analyses. Total run time was 20 min at a flow
rate of 1 mL/min. For PEG 4000−10 000 g mol−1, a linear gradient
from 40% ACN to 50% ACN over 7.5 min, then a linear gradient to
100% ACN for 1.5 min, and then isocratic at 100% ACN for 3 min
were used. Then the starting gradient was restored in 1 min, and an 8
min reconditioning time of 40% ACN:60% water was allowed before
further gradient analyses. For PEG-20000, an isocratic solvent
composition of 44.5% ACN and 55.5.% water was kept for 4 min,
and then the composition was changed to 46% ACN within 1 min,
keeping this composition for 7 min. Within 1 min, the composition
was changed to 60% ACN and kept for 5 min. Subsequently, the
starting gradient was restored in 1 min, and an 8 min reconditioning
time was allowed before further sample injection.
Semipreparative Scale. The mixture of α-tosyl-ω-hydroxyl-PEG
(2), diol-PEG (1), and α-tosyl-ω-tosyl-PEG (3) (100 mg) was
dissolved in acetonitrile (ACN):water (40:60), filtered through a
Rotilabo syringe filter (PTFE, pore size 0.45 μm), and separated by
RP-HPLC with an acetonitrile/water gradient. Gradient conditions:
linear gradient from 40% ACN to 70% ACN over 7.5 min, then a
linear gradient to 100% ACN over 1.5 min, and then isocratic at 100%
ACN for 3 min. Subsequently, the starting gradient was restored in 1
min, and an 8 min reconditioning time of 40% ACN:60% water was
allowed before further sample injection. Total run time was 20 min at a
flow rate of 10 mL/min. The sample fraction of monotosylated PEG
(2) between 7 and 8 min (PEG-1500) was collected, dried in a freeze-
drier, and analyzed by MALDI-ToF-MS. For PEG 4000−10000 and
PEG 20000 the gradient program was used as described for analytical
measurements.
Synthesis of 1,2,3,4,6-Penta-O-acetyl-α,β-D-mannopyranose (7).
The synthesis of peracetylated mannose was modified from Kartha et
al.61 Iodine (560 mg, 2.2 mmol, 0,04 equiv) and acetic anhydride (50
mL) were mixed under an Ar atmosphere. D-Mannose (10.0 g, 55.5
mmol, 1 equiv) was added portion by portion at 0 °C. After stirring for
30 min at 0 °C and additionally for 18 h at room temperature TLC
(cyclohexane/toluene/ethyl acetate 3:3:1) showed complete con-
sumption of the starting material. The reaction mixture was diluted
with dichloromethane (50 mL) and was washed twice with cold
saturated aqueous Na2SO3 solution (2 × 80 mL) and then with a
saturated aqueous solution of NaHCO3 (4 × 50 mL). The separated
organic layer was dried over anhydrous MgSO4. The solvent was
removed in vacuo to afford the desired peracetylated D-mannose (21.5
g, 55.1 mmol, 99%, mixture of both anomers α/β 1:4.75) as a
Synthesis of α-Azido-ω-Hydroxyl-PEG (4). The synthesis of α-
azido-ω-hydroxyl-PEG was developed based on Mahou et al.42
Exemplarily, the synthesis of α-azido-ω-hydroxyl-PEG-1500 is
described below. α-Tosyl-ω-hydroxyl-PEG (2, 500 mg, M = 1670 g
G
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yellowish high viscous oil. Rf = 0.30 (silica gel, cyclohexane/toluene/
2.4 mg), hydroquinone (1 tip of a spatula), and methanol (1 mL) were
mixed in a Schlenk flask. Three cycles of freeze−pump−thaw were
performed for degassing the solvent. Addition of CuBr (2 mg, 0.3
equiv, n = 0.01 mmol) under argon counterflow. Stirring at room
temperature overnight, protected from light by tin foil. Addition of two
tips of a spatula acidic ion-exchange resin Lewatit Mono Plus, stirring
for approximately 1 h until discoloration of the solution. The solution
was filtered through a filtration paper followed by solvent removal by
rotary evaporation. The crude product was purified by 2-fold
precipitation from methanol into ice-cold diethyl ether. The pure
product was dried under high vacuum for 24 h and thereafter
recovered as slightly brownish powder (103 mg, yield = 96%, M =
ethyl acetate, 3:3:1).
1
Signals Assignable to β-Anomer. H NMR, COSY (400 MHz,
CDCl3) δ (ppm) = 6.09 (d, 3J = 1.9 Hz, 1H, H-1), 5.34−5.36 (m, 2H,
2
3
H-3, H-4), 5.25−5.27 (m, 1H, H-2), 4.28 (dd, J = 12.4 Hz, J = 4.9
Hz, 1H, H-6a), 4.10 (dd, 2J = 12.4 Hz, 3J = 2.5 Hz, 1H, H-6b), 4.03−
4.07 (m, 1H, H-5), 2.18, 2.17, 2.10, 2.05, 2.01 (5 × s, 15H, COCH3).
13C NMR, HSQC, HMBC (100.6 MHz, CDCl3) δ (ppm) = 170.8,
170.2, 169.9, 169.7, 168.2 (5 × COCH3), 90.7 (C-1), 70.7 (C-5), 68.8
(C-3), 68.4 (C-2), 65.6 (C-4), 62.2 (C-6), 21.0, 20.9, 20.9, 20.8, 20.8
(5 × COCH3) (see Figures S32−S33).
The spectral data are in accordance with the literature.62
1
2324.2 g mol−1). H NMR, COSY (400 MHz, CD3OD) δ [ppm] =
Synthesis of Propargyl-2,3,4,6-tetra-O-acetyl-α-D-mannopyrano-
side (8). The synthesis of propargyl-2,3,4,6-tetra-O-acetyl-α-D-manno-
pyranoside was modified from Daly et al.63 1,2,3,4,6-Penta-O-acetyl-
α,β-D-mannopyranose (7, 10.0 g, 25.6 mmol, 1 equiv) and propargyl
alcohol (7.18 g, 7.48 mL, 128 mmol, 5 equiv) were dissolved in
dichloromethane (100 mL). After stirring for 20 min at room
temperature, the reaction mixture was cooled to 0 °C, and BF3·OEt2
(16.22 mL, 128 mmol, 5 equiv) was added dropwise. The mixture was
stirred for 15 min at this temperature and then at room temperature
for 24 h. The solution was treated with saturated NaHCO3 solution
(25 mL). Subsequently the aqueous layer was extracted with
dichloromethane (2 × 50 mL), and the combined organic layers
were dried over anhydrous MgSO4. The solvent was removed in vacuo,
and the residue was purified by flash column chromatography
(cyclohexane/ethyl acetate, 1:1) to give the title compound (7.23 g,
18.7 mmol, 73%) as a colorless viscous oil. Rf = 0.43 (silica gel,
cyclohexane/ethyl acetate, 1:1).
8.09 (s, 1H, CH−N), 6.13 (m, 1H, CH2−C−CH3), 5.66 (m, 1H,
CH2−C−CH3), 5.65 (m, 1H, CH2−C−CH3), 4.87 (1H, H-1,
overlapped by solvent signal), 4.64−4.80 (dd, 2H, O−CH2−CR
CR), 4.59 (m, 2H, N−CH2−CH2), 4.28 (m, 2H, CH2−O−CO), 3.91
(m, 2H, N−CH2−CH2), 3.87 (m, 1H, H-6a), 3.79 (m, 1H, H-3), 3.75
(m, 2H, CO−O−CH2−CH2), 3.72 (m, 1H, H-6b), 3.54−3.68 (m,
259H, (CH2−CH2−O)n, 1H, H-4, 1H, H-2), 3.46 (m, 1H, H-5), 1.95
(s, 3H, −CH3) (see Figure S38). 13C NMR, HSQC, HMBC (100.6
MHz, CD3OD) δ (ppm) = 169.0 (O−CO−C), 145.5 (−CCH−
N−), 138.5 (CH2C−CH3), 126.4 (CH2C−CH3), 126.3 (−C
CH−N−), 100.8 (C-1), 75.0 (C-5), 72.5 (C-3), 72.0 (C-2), 71.6
((−CH2−CH2−O−)n), 70.4 (N−CH2−CH2−O), 70.1 (CO−O−
CH2−CH2), 68.6 (C-4), 65.1 (CO−O−CH2−CH2), 63.0 (C-6),
60.7 (−CCH−N−), 51.5 (N−CH2−CH2−O), 18.5 (CH2C−
CH3) (see Figure S39).
1H NMR, COSY (400 MHz, CDCl3) δ (ppm) = 5.36 (m, 3H, H-2,
CONCLUSION
3
■
H-3, H-4), 5.02 (d, 1H, J = 1.7 Hz, H-1), 4.31−4.25 (m, 3H, H-6a,
2
3
CH2−CC), 4.10 (dd, J = 12.2 Hz, J = 2.5 Hz, 1H, H-6b), 4.01
(ddd, 3J = 9.3 Hz, 3J = 5.2 Hz, 3J = 2.5 Hz, 1H, H-5), 2.47 (t, 4J = 2.4
Hz, 1H, CH), 2.15, 2.09, 2.03, 1.98 (4 × s, 12H, COCH3). 13C NMR,
HSQC, HMBC (100.6 MHz, CDCl3) δ (ppm) = 170.8, 170.1, 170.0,
169.8 (4 × COCH3), 96.4 (C-1), 86.0 (CCH), 75.7 (CCH), 69.5
(C-2), 69.1 (C-5), 69.1 (C-3), 66.1 (C-4), 62.4 (C-6), 55.1 (CH2),
21.0, 20.9, 20.8, 20.8 (4 × COCH3). IR (ATR) λmax/cm−1 1756, 1738,
1431, 1256, 1232, 1186, 1056, 1013, 979, 795, 691. [α]2D2 + 53.4° (c =
1.00, CHCl3) (see Figures S34−S35). HRMS (ESI): calculated for
[C17H22O10 + Na]+: 409.1111, found: 409.1116.
Heterobifunctional, i.e., α,ω-functionalized, PEGs are highly
valuable polymers that are widely used for bioconjugation
(“PEGylation”) of e.g. proteins, nanoparticles, hydrogels,
nanogels, and liposomes.64 The preparation of heterobifunc-
tional PEGs from the readily available symmetrical PEG diols is
desirable, but the “polymer desymmetrization” step is
challenging due to low yields and demanding separation of
the reaction products, especially for PEG samples with
molecular weight exceeding 1000 g mol−1. The monotosylation
of PEG via an Ag2O/KI catalyzed strategy was compared to the
noncatalyzed tosylation reaction with respect to enhancing the
amount of the valuable monotosylated reaction product. The
addition of silver oxide and potassium iodide resulted in higher
yields of monofunctionalized PEG (71−76%, Mn 2000 g
mol−1) compared to the uncatalyzed, statistical tosylation
reaction (below 56%) with 1 equiv of tosyl chloride. The yield
for monotosylated product decreased under the same reaction
conditions with increasing molecular weight of the polymer due
to inferior accessibility of the chain ends of the coiled polymer
chains (11.4% for Ag2O-catalyzed compared to 9.4%
uncatalyzed for PEG-8000). For this reason, reaction time
and silver(I) oxide equivalents were adjusted to yield 42.0% of
monotosylated PEG-8000 compared to 12.6% for the
uncatalyzed reaction under comparable reaction conditions.
An analytical HPLC method was developed for rapid
characterization of the crude reaction mixture. Monotosylated
PEG was separated from the crude product by an adapted
analytical or semipreparative HPLC method with baseline
resolution. It is remarkable that even tosylated PEG samples
with 20 000 g/mol, which translates to 450 monomer units,
could be separated according to the different end groups on a
semipreparative scale by HPLC.
Synthesis of Propargyl-α-D-mannopyranoside (9). The synthesis
of propargyl-α-D-mannopyranoside was modified from Daly et al.63
Propargyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (8, 7.00 g, 17.9
mmol) was dissolved in methanol (70 mL), and sodium methoxide
was added until pH 9−10 (approximately 60 mg). The reaction
mixture was stirred at room temperature for 16 h. Subsequently, the
solution was neutralized with Amberlite 120 H+ resin until pH 7. The
mixture was filtered over Celite, which was washed thoroughly with
methanol. The solvent was removed in vacuo to afford the desired 1-
propargyl-α-D-mannopyranoside (3.83 g, 17.6 mmol, 98%) as a highly
viscous syrup which solidified soon to an amorphous solid. Rf = 0.85
1
(RP-silica gel, acetonitrile/water, 1:9). H NMR, COSY (400 MHz,
CD3OD) δ (ppm) = 4.96 (d, 3J = 1.7 Hz, 1H, H-1), 4.27 (d, 1H, 4J =
2.4 Hz, CH2), 3.84 (dd, 2J = 11.8 Hz, 3J = 2.3 Hz, 1H, H-6a), 3.79 (dd,
3
3
1H, J = 3.1 Hz, J = 1.7 Hz, H-2), 3.74−3.58 (m, 3H, H-3, H-4, H-
6b), 3.54−3.47 (m, 1H, H-5), 2.86 (t, J = 2.4 Hz, CH). 13C NMR,
4
HSQC, HMBC (100.6 MHz, CD3OD) δ (ppm) = 99.8 (C-1), 80.0
(CCH), 76.0 (CCH), 75.1 (C-5), 72.5 (C-3), 72.0 (C-2), 68.5
(C-4), 62.8 (C-6), 54.8 (CH2−CCH) (see Figures S36−S37). IR
(ATR) λmax/cm−1 3403, 3276, 2934, 2909, 1586, 1343, 1252, 1134,
1061, 963, 916, 814, 663. [α]2D2 + 116.7° (c = 1.00, MeOH). HRMS
(ESI): calculated for [C9H14O6 + Na]+: 241.0688, found: 214.0692.
Synthesis of α-4-(α-D-Mannopyranosyloxymethylene)-1,2,3-tria-
zol-1-yl-ω-methacryloyl-PEG (10). Exemplarily, the synthesis of α-4-
(α-D-mannopyranosyloxymethylene)-1,2,3-triazol-1-yl-ω-methacrylo-
yl-PEG-2000 is described below. α-Azido-ω-methacryloyl-PEG (6, 96
mg, M = 2106 g mol−1, n = 0.046 mmol), 1-propargyl-α-D-
mannopyranoside (9, 1.24 equiv, M = 219.30 g mol−1, n = 0.057
mmol, 12.5 mg), PMDETA (2.9 μL, 0.3 equiv, n = 0.014 mmol, m =
As an example of the further use of the monotosylated PEG-
synthon, using a three-step route, the α-tosyl-ω-hydroxyl-PEG
could be converted into the complex, heterobifunctional α-4-
H
DOI: 10.1021/acs.macromol.7b01787
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Article
(α-D-mannopyranosyloxymethylene)-1,2,3,triazol-1-yl-ω-meth-
acryloyl-PEG, which can be applied for functionalization of
hydrogels and nanogels. To the best of our knowledge, this is
the first report on the combination of a PEG spacer, mannose,
and methacrylate to obtain α-4-(α-D-mannopyranosyl-
oxymethylene)-1,2,3,triazol-1-yl-ω-methacryloyl-PEG. Since
click reactions are highly selective even in the presence of
numerous other functional groups, this synthesis strategy is
suitable for broad application in the field of hydrogel and
nanogel functionalization. In brief, the desymmetrization
strategy for PEG has been proven to represent a versatile
method to provide pure α-tosyl-ω-hydroxyl-PEG in a molecular
weight range of 1500−20 000 g mol−1. This compound is a key
intermediate for a wide range of α,ω-heterobifunctional PEGs,
rendering this approach highly valuable for a broad range of
new PEGylation reagents.
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.macro-
mol.7b01787.
Characterization of compounds (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*(H.F.) E-mail hfrey@uni-mainz.de.
ORCID
Till Opatz: 0000-0002-3266-4050
Holger Frey: 0000-0002-9916-3103
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors gratefully acknowledge the support of this research
by Tobias Bauer, Jan Blankenburg, Steffen Hildebrand, Ulrike
Kemmer-Jonas, Monika Schmelzer, and Patrick Verkoyen. The
authors thank Mirko Montigny and Romina Schroder for TEM
̈
measurements and Dr. Hans-Joachim Rader for MALDI-ToF
̈
measurement of Figure S45. This work was supported by the
Max Planck Graduate Center with the Johannes Gutenberg
University Mainz (MPGC) as well as by funding of the
Excellence Initiative (DFG/GSC 266) in the context of the
graduate school of excellence “MAINZ” (Materials Science in
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ABBREVIATIONS
■
ACN, acetonitrile; CD11c, cluster of differentiation 11c;
CuAAC, copper(I)-catalyzed alkyne−azide cycloaddition;
DCM, dichloromethane; DMF, dimethylformamide; DMSO,
dimethyl sulfoxide; ELSD, evaporative light-scattering detector;
EO, ethylene oxide; FTIR, Fourier transform infrared spec-
troscopy; HPLC, high-performance liquid chromatography;
MALDI-ToF-MS, matrix-assisted laser desorption ionization
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resonance spectroscopy; OEG, oligo(ethylene glycol); PEG,
poly(ethylene glycol); PMDETA, N,N,N′,N″,N″-pentamethyl-
diethylenetriamine; RI, refractive index; SEC, size exclusion
chromatography; TFA, trifluoroacetic acid; THF, tetrahydro-
furan; TsCl, tosyl chloride; UV, ultraviolet.
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