Trehalose glycopolymers for stabilization of protein conjugates to environmental stressors

Article abstract of DOI:10.1021/ja2120234

Herein, we report the synthesis of trehalose side chain polymers for stabilization of protein conjugates to environmental stressors. The glycomonomer 4,6-O-(4-vinylbenzylidene)-α,α-trehalose was synthesized in 40% yield over two steps without the use of protecting group chemistry. Polymers containing the trehalose pendent groups were prepared via reversible addition-fragmentation chain transfer (RAFT) polymerization using two different thiol-reactive chain transfer agents (CTAs) for subsequent conjugation to proteins through disulfide linkages. The resulting glycopolymers were well-defined, and a range of molecular weights from 4200 to 49 500 Da was obtained. The polymers were conjugated to thiolated hen egg white lysozyme and purified. The glycopolymers when added or covalently attached to protein significantly increased stability toward lyophilization and heat relative to wild-type protein. Up to 100% retention of activity was observed when lysozyme was stressed ten times with lyophilization and 81% activity when the protein was heated at 90 °C for 1 h; this is in contrast to 16% and 18% retention of activity, respectively, for the wild-type protein alone. The glycopolymers were compared to equivalent concentrations of trehalose and poly(ethylene glycol) (PEG) and found to be superior at stabilizing the protein to lyophilization and heat. In addition, the protein-glycopolymer conjugates exhibited significant increases in lyophilization stability when compared to adding the same concentration of unconjugated polymer to the protein.

Full text of DOI:10.1021/ja2120234

Article
pubs.acs.org/JACS
Trehalose Glycopolymers for Stabilization of Protein Conjugates to
Environmental Stressors
Rock J. Mancini, Juneyoung Lee, and Heather D. Maynard*
Department of Chemistry and Biochemistry and California NanoSystems Institute, University of California, Los Angeles, 607 Charles
E. Young Drive East, Los Angeles, California 90095-1569, United States
S
* Supporting Information
ABSTRACT: Herein, we report the synthesis of trehalose side chain
polymers for stabilization of protein conjugates to environmental stressors.
The glycomonomer 4,6-O-(4-vinylbenzylidene)-α,α-trehalose was synthe-
sized in 40% yield over two steps without the use of protecting group
chemistry. Polymers containing the trehalose pendent groups were prepared
via reversible addition−fragmentation chain transfer (RAFT) polymerization
using two different thiol-reactive chain transfer agents (CTAs) for
subsequent conjugation to proteins through disulfide linkages. The resulting
glycopolymers were well-defined, and a range of molecular weights from
4200 to 49 500 Da was obtained. The polymers were conjugated to thiolated
hen egg white lysozyme and purified. The glycopolymers when added or
covalently attached to protein significantly increased stability toward
lyophilization and heat relative to wild-type protein. Up to 100% retention
of activity was observed when lysozyme was stressed ten times with lyophilization and 81% activity when the protein was heated
at 90 °C for 1 h; this is in contrast to 16% and 18% retention of activity, respectively, for the wild-type protein alone. The
glycopolymers were compared to equivalent concentrations of trehalose and poly(ethylene glycol) (PEG) and found to be
superior at stabilizing the protein to lyophilization and heat. In addition, the protein−glycopolymer conjugates exhibited
significant increases in lyophilization stability when compared to adding the same concentration of unconjugated polymer to the
protein.
there is a growing demand for novel stabilizing excipients.23
Specifically, various carbohydrates have been used to mitigate
INTRODUCTION
There is considerable interest in proteins as therapeutics and as
■
biochemical and chemical reagents.1−3 However, proteins are
stressors typically experienced by proteins including thermal
fluctuations and lyophilization.24−26 We report herein polymers
based on a natural disaccharide trehalose as potentially new
stabilizers for proteins.
inherently unstable to thermal fluctuations.4 Furthermore, most
therapeutics are known to degrade upon storage, transport, and
use, necessitating regulated temperatures, controlled solvation,
and addition of stabilizing excipients.5−10 Protein activity also
diminishes upon exposure to physical or chemical insults such
as desiccation,11 heat,12 light,13 and pH change,14 further
complicating application of many biomolecules. As such,
attachment of poly(ethylene glycol) (PEG) to proteins has
been widely used to increase in vitro and in vivo stability for
therapeutic proteins;7−9 however, PEGylation alone provides
limited stability to temperature, desiccation, or storage.
To overcome this problem, lyophilization (freeze-drying) has
been widely adopted.11,15 However, during lyophilization
stresses are present on the protein that are tangential to a
freeze−thaw process.16 Therefore, engineering research must
be employed to find the optimal buffer formulation for
lyophilization of each individual protein type.17,18 Formation
of ice crystals, change in solute concentration, and variation of
pH are common stresses present during lyophilization that can
contribute to protein denaturation.19 As a result, high
concentrations of excipients are commonly added,20 particularly
for protein therapeutics and biochemical reagents,21,22 and
In nature, many plants and animals endure complete
dehydration stress by accumulating large amounts of
trehalose.27,28 This alpha-linked glucose disaccharide has been
known to impart unusual stability to organisms tolerating
anhydrobiosis (desiccation) and cryobiosis (low temperature)
by protecting cells and proteins.24,29−33 For instance, Westh
and Ramløv reported in 1991 that Tardigrades, commonly
known as water bears, accumulate trehalose from 0.1% to 2.3%
dry weight within 5−7 h under dehydration conditions.30 In
addition, Crowe and co-workers showed Aphelenchus avenae
(nematodes) can increase the concentration of trehalose to as
much as 15% dry body weight in response to desiccation.31
Other groups have reported that brine shrimp eggs, Artemia,
survive up to 15 years under anhydrous conditions when
trehalose is present.32 There is much debate over the
mechanism of this dramatic stabilization of organisms by
Received: December 23, 2011
Published: April 20, 2012
© 2012 American Chemical Society
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trehalose, and there are three main hypotheses that are not
mutually exclusive: water replacement,34,35 water entrapment,36
and vitrification.37 Proponents of the water replacement
hypothesis claim that hydroxy groups in the trehalose molecule
interact with proteins via hydrogen bonding and protect them
from desiccation. In the water entrapment hypothesis, a layer of
water is intimately isolated between the biomolecule and
sugars. This surrounding water then protects the protein by
maintaining hydration. In the third hypothesis, vitrification,
glassy sugar matrices protect protein tertiary and quaternary
structure from unfolding. To date, the precise mechanism of
how trehalose can stabilize dehydrated organisms has not been
proven, and it may be that the mechanism is dependent on the
nature of the dehydration method.20 Most importantly,
trehalose acts as a protectant of proteins against environmental
stresses. We hypothesized that polymers with pendent trehalose
carbohydrates would likewise stabilize proteins to environ-
mental stressors (Figure 1).
selectivity to form linear polymer versus the branched one was
unclear at that time.42 Polymerization of diamino-type trehalose
was explored to overcome the issue of branching, but the
overall process was more complicated.43 Acetalization,44
enzymes,45 Diels−Alder reactions,46 and click chemistry47,48
have been exploited to synthesize trehalose-based linear
polymers, with the latter study being extended to biological
systems. However, most incorporate trehalose into the polymer
backbone rather than as a side chain. To date, a well-defined
trehalose polymer synthesized by controlled radical polymer-
ization has not been reported, and trehalose-based protein−
glycopolymer conjugates have yet to be realized. Conjugating a
trehalose polymer directly to the protein has the advantage of
increasing the effective concentration of polymer relative to
protein, thereby enhancing protective effects of the polymer. In
addition, for therapeutics, a trehalose-based protein−polymer
conjugate could combine the advantages of environmental
stabilization due to the sugar with improved pharmacokinetics
resulting from the polymer. Herein, we describe the synthesis of
a trehalose side chain polymer and stabilization of protein
conjugates. Trehalose was attached to a styrene monomer via a
4,6-acetal linkage. Subsequent RAFT polymerization generated
well-defined, cysteine-reactive glycopolymers. These were
covalently attached to thiolated hen egg white lysozyme, a
protein known to lose activity due to heat49 or lyophilization.50
The resulting lysozyme−glycopolymer conjugates were ex-
posed to these stresses, and significant increases in retention of
activity relative to wild-type protein were observed. Inves-
tigation of the stabilization of wild-type lysozyme in the
presence of unconjugated polymer is also presented.
RESULTS AND DISCUSSION
■
Synthesis of a Trehalose-Based Glycomonomer. One
typical protection scheme for trehalose involves treatment with
dimethoxy toluene to selectively afford acetal protection as
4,6,4′,6′-O-dibenzylidene trehalose in high (>90%) yield.51
Therefore, we envisioned that reaction with 4-vinylbenzalde-
hyde diethyl acetal (1)52 would exhibit similar regioselectivity
to install a polymerizable group. Terephthaldehyde mono-
diethyl acetal was employed to form 1 via a Wittig reaction in
97% yield. This in turn was used to effect a transacetal reaction
with trehalose exclusively at the 4,6 position under acidic
conditions affording 4,6-O-(4-vinylbenzylidene)-α,α-trehalose
in 41% yield (2, Scheme 1).
Figure 1. (a) Side chain trehalose polymer with a protein-reactive
group at one end. (b) Trehalose polymer conjugates protect proteins
from lyophilization and heat.
A unique disaccharide, trehalose, is a food additive that is
generally regarded as safe (GRAS).38 Although endogenous
production has not been exhibited by mammals, metabolism
occurs in humans through fragmentation about the anomeric
center by trehalase enzymes found in the intestinal villi to
produce glucose.39 This stabilization ability combined with the
safety of a known metabolic fate has contributed to the
proliferation of the use of trehalose as an excipient in protein-
based therapeutics, with at least four retail drugs currently
having it in their formulation.27 In this report, trehalose side
chain polymers to add to proteins and to conjugate to proteins
as stabilization agents were synthesized.
Previously, trehalose-based materials have been produced as
cross-linked polymer networks22 such as polysubstituted
trehalose vinylbenzyl ether thermo-set resins.40 Achieving
trehalose linear polymers has been challenging as the anomeric
centers are relatively unreactive due to the 1,1-glycosidic
linkage.41 Therefore, typical synthetic routes to produce
trehalose-based monomers contain several protecting and
deprotecting steps, use of bifunctional monomers targeting
the 6,6′ positions, or produce mixtures of regio-isomers that are
not well-defined. For example, a simple strategy for synthesiz-
ing a trehalose linear polymer was first reported in 1979, but
Reactions necessitating anhydrous conditions along with
statistical monosubstituion of unmodified trehalose are
generally difficult to achieve, partially due to the dramatic
decrease in solubility of anhydrous trehalose in organic solvents
relative to the dihydrate form.53 Although a significant amount
of bis-functionalized byproduct was formed, production of the
desired product 2 occurred in 41% yield after HPLC
purification; no other isomers were observed indicating that
preference for the 4,6 position was retained for the substituted
benzaldehyde diethyl acetal. In addition to 1H NMR, 13C
NMR, HMQC, and COSY NMR (see Supporting Information,
Figure S1),54 heteronuclear single quantum coherence total
correlation spectroscopy (HSQC-TOCSY) experiments were
performed to verify both the selectivity of the reaction as well as
the integrity of the product ring systems (see Supporting
Information, Figure S2). Furthermore, NOESY NMR was
undertaken to demonstrate retention of the unique con-
formation of the native disaccharide whereby the two glucose
subunits are held in a rigid clamshell shape about the α,α-1,1-
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a
Scheme 1. Synthesis of 4-Vinylbenzaldehyde Diethyl Acetal
1 by Wittig Olefination and Regioselective Reactivity with
Trehalose to Yield 4,6-O-(4-vinylbenzylidene)-α,α-trehalose
2
Table 1. RAFT Polymerization of 2 with 3 or 5
entry
CTA CTA:M:AIBN target Mn conv. (%) Mn NMR PDI
Poly 1
Poly 2
Poly 3
Poly 4
Poly 5
Poly 6
Poly 7
Poly 8
3
3
3
3
5
5
5
5
1:21:0.2
1:29:0.2
1:35:0.2
1:50:0.2
1:20:0.4
1:60:0.2
1:76:0.2
1:120:0.4
7000
9600
72
77
84
85
94
40
73
81
4200
9400
1.05
1.07
1.11
1.14
1.10
1.13
1.20
1.47
13800
20000
9000
14700
19000
8000
11400
25800
44800
15400
24500
49500
a
Polymerization was conducted in DMF at 80 °C with 0.8 M
monomer concentration for all trials.
Scheme 2. Activation of the Known Starting Material 1-
Mercaptotriethylene Glycol with Aldrithiol to Yield
Activated Disulfide 4 before Subsequent Carbodiimide
Coupling to Produce the Thiol Reactive Chain Transfer
Agent 5
glycosidic linkage (see Supporting Information, Figure S3).55
This result is important as it is thought to be a critical
component in generating the unique physiochemical and
protective properties of the carbohydrate.56
Synthesis of a Thiol Reactive Glycopolymer by RAFT
Polymerization. To form polymers to conjugate to proteins
and to achieve low polydispersity indices (PDIs) and targeted
molecular weights, RAFT polymerization was used. RAFT
polymerization readily provides well-defined polymers with
protein-reactive end groups.57 The CTA 3 was prepared with a
pyridyl disulfide group because this functionality has been
frequently reported as an effective reactive moiety for reaction
with free cysteines in proteins.58,59 Synthesis of CTA 3 was
accomplished by esterification of 2-(ethyl sulfanylthiocarbonyl
sulfanyl)-propionic acid with a pyridyl disulfide alcohol in 76%
yield.
(ethyl sulfanylthiocarbonyl sulfanyl)-propionic acid afforded
the thiol reactive chain transfer agent 5 in 62% yield. RAFT
polymerization of 2 in the presence of 5 was shown to proceed
for a range of molecular weights (Table 1, Poly 5−8) with slight
deviation from narrow polydispersities obtained for the highest
molecular weight attempted (Poly 8). After purification by
dialysis (1 kDa MWCO, H2O, 3 days), these thiol reactive
glycopolymers were characterized by NMR spectroscopy to
confirm end-group retention and complete removal of
monomer (see Supporting Information, Figure S7).
Conjugation of Glycopolymer to Thiopropionyl Hen
Egg White Lysozyme. Trehalose polymers were then
conjugated to thiolated lysozyme. Thiols were added to hen
egg white lysozyme by treatment with N-succinimidyl-S-
acetylthiopropionate (SATP) in a procedure known to
covalently attach thioacetate functionality to proteins via
amide bonds.61 After deprotection with the hydroxyl amine
and removal of excess SATP, free thiols were quantified on the
thiolated lysozyme (LyzSH) by Ellman’s assay resulting in a
thiol:protein ratio of 1:1.4 (71% thiol). The LyzSH was then
conjugated to various trehalose polymers (Poly 2, 5−8, Scheme
3). After incubation with trehalose polymers, conjugates were
seen by SDS-PAGE (see Supporting Information, Figures S6
and S11). The conjugation yield was lower for Poly 2 as
compared to Poly 5−8. One possible explanation was that the
TEG linker moves the thiol reactive end group away from the
bulky and hydrated trehalose side chain of the polymer
improving access to the protein surface.
RAFT polymerization of 2 with 3 was performed at 80 °C
(Scheme 1). The ratio used for polymerization was [CTA]:
[monomer]:[AIBN] = 1:29:0.2, with a concentration of 0.8 M
monomer. After 6 h, the polymerization was stopped to obtain
77% conversion. The polymer was dialyzed against aqueous
sodium bicarbonate with a MWCO 2000 g/mol. The molecular
1
weight of the polymer was analyzed by H NMR spectroscopy
and was 9600 Da by comparing the integration of the end-
group pyridine peaks to the aromatic ring from styrene (see
Supporting Information, Figure S4). The PDI by GPC was
1.07, demonstrating that a well-defined polymer was formed.
The kinetic study of the trehalose polymer demonstrated that
PDIs were well below 1.1 throughout the polymerization (see
Supporting Information, Graph S1). The [CTA]:[monomer]:
[AIBN] ratios were then altered to obtain other molecular
weights ranging from 4200 to 19 000 g/mol (Table 1, Poly 1−
4) with narrow PDIs obtained in all cases.
In addition, a new CTA 5 was devised to contain a
hydrophilic triethylene glycol spacer between the trithiocar-
bonate and pyridyl disulfide end-group (Scheme 2). Tri-
(ethylene glycol) (TEG) was modified by tosylation followed
by refluxing with thiourea.60 Upon addition of base, the
resulting 1-mercapto-tri(ethylene glycol) was treated with
Aldrithiol to obtain activated disulfide 4. Subsequent
carbodiimide-mediated coupling to the acid moiety on 2-
Lysozyme−glycopolymer conjugates (Lyz−Poly 5−8) were
purified by fast protein liquid chromatography (FPLC) with
subsequent runs of LyzSH and glycopolymer alone used to
determine which fractions would be pure of starting materials
(see Supporting Information, Figure S10). These fractions were
collected and utilized in subsequent studies. Additionally, SDS-
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stabilization effects of trehalose are enhanced by using a
polymer where the entropic barrier to having several
carbohydrate moieties organized around the protein has already
been included in the excipient. Trehalose itself has been
proposed to stabilize proteins to desiccation via water
replacement, water entrapment, and vitrification. The mecha-
nisms by which the trehalose polymers stabilize the protein are
not yet understood, and investigative studies are underway.
Regardless of the mechanism, the results suggest that trehalose
polymers may be useful as a replacement for the disaccharide in
formulations of unmodified biomolecules, particularly in
instances where the additional materials properties imparted
by the polymer would be advantageous.
Scheme 3. RAFT Polymerization of 2 with CTA 5 to
Produce the Thiol Reactive Glycopolymers Poly 5 through
a
Poly 8
a
The glycopolymers were then conjugated to thiolated lysozyme.
To investigate the ability of the polymer to stabilize when
conjugated to the protein, Lyz−Poly 5−8 were exposed to the
same 10 lyophilization cycles. In this case, the conjugates
exhibited between 59 and 100% retention of original activity
compared to 16% retention by wild-type lysozyme (Figure 2).
The smallest molecular weight Lyz−Poly 5 had the lowest
activity, while the largest polymer conjugate Lyz−Poly 8 had
full retention of activity. However, the activities of the medium-
sized polymers did not directly correlate to molecular weight. It
is possible that this is due to differing numbers of polymers
attached to the protein in the fractions analyzed; the number of
polymers attached in the collected fractions could not be
accurately estimated by SDS-PAGE due to the low concen-
trations involved. Importantly though, the conjugates were all
significantly more stable compared to samples containing a
similar concentration of unattached polymer added (1 equiv to
protein); addition of polymer resulted in only 18−47%
retention of lysozyme activity. These results show that
conjugating the polymer to the protein is advantageous with
regard to environmental stability. Studies to determine the in
vitro and in vivo stability of protein conjugates prepared from
these polymers are underway to evaluate pharmacokinetic
properties and potential use of the polymer in therapeutic
conjugates.
PAGE was used to confirm isolation of the conjugate as
demonstrated by the absence of a lysozyme band under
nonreducing conditions. Reappearance of the band under
reducing conditions indicated that the polymer was conjugated
to the lysozyme through a reducible disulfide bond (see
Supporting Information, Figure S12). Activities of the purified
Lyz−Poly conjugate fractions were confirmed by observing
active lysis of the FITC labeled Gram-positive bacteria
Micrococcus luteus in the EnzChek lysozyme activity assay.
Lysozyme−Glycopolymer Conjugate Resistance to
Environmental Stress. The ability of the polymers to
stabilize lysozyme to lyophilization was first verified, and the
results were compared to trehalose at the same concentrations.
Poly 5−8 at 100 equivalents to protein were added (not
conjugated) to wild-type Lyz containing no free thiols. Samples
with trehalose added at equivalent concentrations relative to
the trehalose in the various polymers were also tested. The
samples were exposed to 10 lyophilization cycles, and the
protein activity was determined. Wild-type protein retained
16% activity after this treatment, while with a 100-fold excess of
polymer, full retention (100%) of activity was observed (Figure
2). The same effect was seen regardless of the molecular weight
Trehalose is also known to stabilize biomolecules to increases
in heat.24,62,63 Therefore, an identical array of lysozyme samples
were also stressed by exposure to a heat burden of 90 °C for 1 h
(Figure 3). Although trehalose was found to confer marginal
stabilization at the highest concentration tested (31% activity
retention for 100 equiv comparable to Poly 8), the trehalose
Figure 2. Activity of the lysozyme−glycopolymer conjugate, wild-type
lysozyme with glycopolymer (1 or 100 equiv relative to lysozyme), or
wild-type lysozyme with trehalose (1 or 100 equiv relative to polymer
monomer units) as excipients exposed to 10 cycles of lyophilization.
Data shown are repeated 6 times with p < 0.01 for all polymer 100×
and conjugate samples relative to wild-type.
tested (8000 to 49 500 Da). Trehalose at equivalent
concentrations to that in the 100-fold polymers only stabilized
the lysozyme between 18 and 31%. The results confirm that the
polymers are able to protect the protein during lyophilization.
Furthermore, the data show that the polymer is significantly
more effective than trehalose at the same concentration relative
to monomer units present in the polymer. This indicates that
Figure 3. Activity of the lysozyme−glycopolymer conjugate, wild-type
lysozyme with glycopolymer (1 or 100 equiv relative to lysozyme), or
wild-type lysozyme with trehalose (1 or 100 equiv relative to polymer
monomer units) as excipients exposed to a heat burden of 90 °C for 1
h. Data shown are repeated 6 times with p < 0.001 for all polymer and
conjugate samples relative to the wild-type.
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polymer effects superior protection to heat, both added and as a
conjugate. Up to 81% retention of activity was observed under
these rigorous conditions. In this particular study, the
protective effect was not concentration dependent as adding
1 equiv and 100 equiv or conjugating the polymer gave similar
retention of activity for all molecular weights tested. However,
in other studies, which were longer in duration and at a
different concentration of protein, some concentration depend-
ence was observed (data not shown). Overall, the results
demonstrate that the glycopolymers stabilize lysozyme to high
temperatures. Furthermore the data suggest that the polymers
should be investigated further as an excipient and conjugate to
mollify the rigorous storage requirements that are typical for
proteins, particularly during transport where large fluctuations
in temperature may be observed.
Additional initial studies were conducted to compare
trehalose glycopolymers to the commonly used excipient
poly(ethylene glycol) (PEG). As such, PEG (Mn = 2−20
kDa) was combined with wild-type lysozyme at 1 or 100 equiv
and stressed by lyophilization or heat in an identical fashion as
before. The lyoprotective effect of PEG (see Supporting
Information, Figures S13 and S14) was found to depend on
weight. Glycopolymer at 1 equiv performed similarly to 1 equiv
of PEG, and 100 equiv of glycopolymer was a more effective
lyoprotectant than PEG depending on the molecular weight.
Thermal stability of PEG relative to the glycopolymers was also
investigated by exposing the relevant samples to a heat burden
of 90 °C for 1 h as before. Although PEG mitigates the thermal
stress to a minimal extent, the trehalose glycopolymer
outperforms PEG based on DP or molecular weight for all
samples tested (see Supporting Information, Figures S13 and
S14). The data collectively indicate that trehalose side chain
polymers are highly effective and superior to PEG as stabilizers
of a representative protein, lysozyme, to heat stress, and similar
or better stabilizers to lyophilization stress.
AUTHOR INFORMATION
■
Corresponding Author
maynard@chem.ucla.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Science Foundation
(CHE-1112550). The ARX 500 NMR data was obtained on
equipment supported by the National Science Foundation
(CHE-1048804). R.J.M. thanks the NSF and California
NanoSystems Institute for a Materials Creation Training
Program Fellowship. J.L. is grateful for additional funding
from a Yonsei International Foundation Scholarship. The
authors thank Thi (Kathy) Nguyen for preparing the thiolated
lysozyme.
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ASSOCIATED CONTENT
* Supporting Information
■
S
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