Modification of proteins with cyclodextrins prevents aggregation and surface adsorption and increases thermal stability

Article abstract of DOI:10.1021/la203271u

This work describes a general approach for preventing protein aggregation and surface adsorption by modifying proteins with β-cyclodextrins (βCD) via an efficient water-driven ligation. As compared to native unmodified proteins, the cyclodextrin-modified

Full text of DOI:10.1021/la203271u

ARTICLE
pubs.acs.org/Langmuir
Modification of Proteins with Cyclodextrins Prevents Aggregation and
Surface Adsorption and Increases Thermal Stability
Deepali Prashar, DaWei Cui, Debjyoti Bandyopadhyay, and Yan-Yeung Luk*
Department of Chemistry and Department of Biomedical and Chemical Engineering, Syracuse University, Syracuse, New York 13244,
United States
S Supporting Information
b
ABSTRACT: This work describes a general approach for preventing protein aggrega-
tion and surface adsorption by modifying proteins with β-cyclodextrins (βCD) via an
efficient water-driven ligation. As compared to native unmodified proteins, the
cyclodextrin-modified proteins (lysozyme and RNase A) exhibit significant reduction
in aggregation, surface adsorption and increase in thermal stability. These results reveal
a new chemistry for preventing protein aggregation and surface adsorption that is likely
of different mechanisms than that by modifying proteins with poly(ethylene glycol).
’ INTRODUCTION
Carbohydrates and their derivatives are a class of molecules
known to stabilize protein folding.11 In particular, cyclodextrin in
solution has the ability to function as an artificial chaperone that
can refold protein structures, likely by stripping the detergents
that are associated with the denatured proteins.12 Cyclodextrin
dimers have also been shown to disrupt protein aggregation.13
These target the disruption of hydrophobic interactions between
proteins or between proteins and surfactants and strongly suggest
that cyclodextrin supports both protein folding and refolding. In
addition to the hydrophobic patches in proteins that can cause
protein association and aggregation,14 chaotropic additives such
as urea and guanidinium hydrochloride can also promote protein
denaturation and aggregation.15 In this work, we explore direct
covalent modification of proteins with β-cyclodextrin (βCD) to
prevent protein aggregation and surface adsorption. The con-
tribution of lysine residues to the stability of proteins depends on
the nature of counteranions,15,16 and certain lysines in proteins
may be of chaotropic nature and may destabilize protein folding.17
Thus, in addition to their abundance in proteins and chemical
reactivity, modifying the lysine residues in proteins may increase
protein stability.
Understanding the fundamental science of aggregation and
surface adsorption of proteins is critically important for developing
protein-based drugs,1 for studying aggregation-induced diseases,2
and for preventing biofouling, which impacts a wide range of
industries including biomedical research.3 For most efforts, aggre-
gation4 and surface adsorption5 of proteins are studied as two
separate problems. In spite of this difference, in their severe cases,
both aggregation and surface adsorption share a common phe-
nomenon of protein denaturation. Development of protein-based
drugs, while bearing enormous potential,6 has been impeded by
aggregate formation and surface adsorption. Protein aggregation
not only causes production loss but, more importantly, induces
toxic side effects and sometimes fatal immunogenicity.1 The sur-
face adsorption also causes loss of efficacy of proteins in syringes
for intravenous delivery. Since its development in the 1970s,7
modification of proteins with poly(ethylene glycol) (PEG) has
remained the major approach to reduce protein aggregation.7,8 As
a synthetic polymer, PEG is large, polydispersed with undefined
conformations.9 The multiple ether groups can also form unde-
sired complexes with high valent metal ions.10 Considering the
diverse structures and properties of proteins, a different and com-
petent chemistry for preventing protein aggregation and surface
adsorption is highly desired. Because the severe cases of aggrega-
tion and surface adsorption are associated with protein denatura-
tion, we explore a general working hypothesis that covalent graft-
ing of proteins with organic molecules known to promote protein
folding will also increase protein stability and reduce aggregation
and surface adsorption. Here, we describe a chemistry for modify-
ing proteins with cyclodextrins that results in reducing aggregation
and surface adsorption of the proteins and increasing the thermal
stability of the proteins.
Modifying proteins with sugar and their derivatives by chemi-
cal methods18,19 has been shown to enhance solubility and
increase stability and sometimes the activity of proteins.20 Existing
methods for conjugating monosaccharides or oligosaccharides
to proteins and peptides use a wide range covalent linkages,
including acetamidine, amine, diazo, thiourea, amide, sulfide, and
disulfide bond formation.18 Although N-hydroxysuccinimide
Received: March 18, 2011
Revised:
September 8, 2011
Published: September 08, 2011
r
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Scheme 1
(NHS) ester method is broadly used, other methods such as
diazotization appear to be more desirable for modifying proteins
with sugar derivatives. For example, Lemieux and co-workers
have extensively used carbonylazide groups to conduct chemical
glycosylation of proteins.19 A squarate-based chemistry has been
reported to couple oligosaccharide amines to proteins using
diethyl squarate.21 Some of these methods have also been
combined with site-directed mutagenesis. Bertozzi and co-work-
ers have demonstrated chemical glycosylation on cell surfaces
using Staudinger ligation.22 Copper catalyzed click reactions
between alkyne modified protein and sugar azides have also
been used for glycosylation of proteins.23
While significant advances emerged for bioconjugation in
recent years,24 modification of proteins with cyclodextrins, parti-
cularly with a well-defined bonding identity, remains chal-
lenging.25À28 One of the difficulties is likely that cyclodextrin is
capable of catalyzing the hydrolysis of ester bonds,29 which is an
intermediate in many activation methods for making amide
bonds. Recently, we reported a class of water-driven chemose-
lective reactions of squarate derivatives with cysteine or unpro-
tected peptides containing N-terminus cysteine.30 Here, we
demonstrate the use of water-driven ligations30À32 to modify the
lysine residues of proteins with βCD that results in preventing
aggregation and surface adsorption of proteins and increasing
the thermal stability of proteins.
performed using SILICYCLE, Silica-P Flash Silica Gel with 40À63 μ
1
1
mesh size. H and 13C NMR spectra were recorded at 300 MHz. H
chemical shifts were reported in ppm relative to CDCl3 δ 7.26 or D2O δ
4.80; 13C chemical shifts were reported relative to CDCl3 δ 77.23. High
resolution mass spectra (HRMS) were recorded by positive ion electro-
spray on a Bruker 12 T APEX ÀQe FTICR-MS instrument with an
Apollo II ion source, unless otherwise reported.
Synthesis of βCD-Derivatized Amino Squarate Reagents.
Scheme 1 shows the optimized synthesis of βCD squarate reagents 6À8.
Briefly, compounds 1,31 4,30 and mono-6-amino-6-deoxy-β-cyclodextrin
5
33 were synthesized using literature-reported procedures. Compounds
2 and 3 were obtained by coupling 1 with 3,5-difluorophenol or
4-fluorophenol, respectively, in the presence of triethylamine (TEA).
Compounds 6, 7, and 8 were obtained by mixing 5 with compound 2, 3,
or 4, respectively, in a mixture of solvents (water, dimethylformamide,
and acetone).
Synthesis of 2. Compound 131 (1.407 g, 9.384 mmol) was dis-
solved in benzene (10 mL), and the solution was cooled to 0 °C. 3,5-
Difluorophenol (2.440 g, 18.766 mmol) was added to the above solution
in one portion, followed by slow addition of excess TEA (2.090 g, 20.674
mmol), and the reaction mixture was stirred at room temperature over-
night. The reaction mixture was filtered, and the residue obtained was
washed with benzene to yield a white solid. The solid obtained was
purified using flash silica gel column (20% EtOAc in hexanes) to give 2
(0.80 g, 27%) as a white solid. Rf = 0.59 (20% EtOAc in hexanes). 1H
NMR (300 MHz, CDCl3): δ 6.90À6.88 (m, 4H), 6.84À6.78 (m, 2H).
13C NMR (75 MHz, CDCl3): δ 186.2, 182.4, 165.3, 165.1, 161.9, 161.7,
154.0, 153.6, 103.7, 103.5, 103.4, 103.3, 103.2, 102.9. HRMS found =
339.0280 [M + H] +, calcd for [C16H6F4O4 + H] + = 339.0275.
Synthesis of 6. Mono-6-amino-6-deoxy-β-cyclodextrin (5).33
(10.0 mg, 0.008 mmol) was dissolved in water (0.5 mL). A solution of
compound 2 (6.00 mg, 0.017 mmol) in DMF (0.1 mL) was added to the
above solution dropwise. The solution containing 2 was added to the
reaction mixture. Acetone was added dropwise to the reaction mixture to
dissolve the undissolved reactants, and the reaction mixture was stirred
at room temperature for 2 h. Excess acetone was added to the reaction
mixture to precipitate out the crude product. The precipitated solid was
washed thoroughly with acetone and dried under high vacuum to yield
the pure compound 6 (7.60 mg, 64%) as a pale yellow solid. 1H NMR
(500 MHz, CDCl3): δ 7.19À7.15 (t, 1H, JHÀH = 10.0 Hz), 6.79À6.64
(m, 2H), 5.13À5.01 (m 7H), 4.04À3.91 (m, 24H), 3.87À3.68 (m, 18H).
Analyzed by MALDI and matched [M + Na] + at 1364.64. Expected mass
of sodium adduct: 1364.38.
’ MATERIALS AND METHODS
Chemicals. Chicken egg white lysozyme (catalog number L6876)
and RNase A (catalog number R6513) were obtained from Sigma
Chemical Company (St. Louis, MO) and were used without further
purification. Sinapinic acid was obtained from ProteoChem (Denver,
CO). Phosphate buffered saline, 1Â PBS (2.7 mM potassium chloride,
137 mM sodium chloride, 8 mM sodium phosphate dibasic, 1.48 mM
potassium phosphate monobasic), was used for all protein reactions
unless mentioned (pH of the prepared buffers was adjusted using 1 N
HCl and 1 N NaOH). Cameo 3A syringe filters, acetate 0.22 micron,
3 mm, were obtained from GE Water & Process Technologies (Trevose,
PA). All other chemicals were obtained from Aldrich Chemicals
(Milwaukee, WI). Deionized water used for preparing all buffers and
solutions had a resistivity of 18 MΩ cm (Millipore, Billerica, MA).
General Information for Synthesis of βCD Amino Squa-
rate Reagents. Reagents and solvents were reagent grade and used
as supplied. EMD Silica Gel 60 F254 precoated plates (0.25 mm
thickness) were used for TLC. Flash column chromatography was
Modification of Lysozyme with βCD. Compound 6 (4.68 mg,
50 equiv) was added to a solution of lysozyme (1 mg/mL) prepared in
1Â PBS pH 8.2 (reaction buffer). The reaction mixture was stirred in a
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Scheme 2
28 °C incubator shaker for 3 days. The reaction mixture was dialyzed in
250 mL 1Â PBS, pH 7.4. MALDI-TOF was used to characterize the
number of modifications on the protein. The modified protein sample
was freeze-dried to obtain a light yellow solid.
comparing the SPR intensity before and after the gold spots were
exposed to the protein solution.
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophor-
esis (SDS-PAGE). SDS-PAGE was conducted using BIO-RAD 7.5%
Mini-PROTEAN TGX Precast Gel (BIO-RAD, Hercules, CA). A total
of 15 μL of protein sample (0.5 mg/mL or 0.05 mg/mL) in 1Â PBS
buffer was mixed with 5 μL of deionized water and 5 μL of 5 Â SDS-
loading dye (Tris-Cl 0.25 M, pH 6.8, 10% SDS, 50% glycerol, and 0.25%
bromophenol blue). The mixture was heated to 90 °C for 5 min, and
20 μL of each sample was loaded on to the gel. A protein ladder,
Precision Plus Dual Color from BIO-RAD (BIO-RAD, Hercules, CA),
was added in one of the wells as a molecular weight reference. Tris-
glycine (1Â) was used as the running buffer for gel electrophoresis. The
gel was run at 100 V for 80 min, and was washed with deionized water
three times and stained with GelCode blue stain reagent (Thermo
Scientific, Rockford, IL) for 1 h and then destained in deionized water
overnight. The gel image was taken on the Alpha DigiDoc system (Alpha
Innotech, San Leandro, CA).
Dialysis. The protein samples were dialyzed in 1Â PBS to remove
unreacted small-molecule reagents or in deionized water (18 M Ω cm)
to promote aggregation of unmodified proteins using Slide-A-Lyzer
dialysis cassettes 7K MWCO purchased from Thermo Scientific
(Rockford, IL). Before use, the Slide-A-Lyzer dialysis cassette was
hydrated in the dialysis media for 5 min. Sample was injected in the
cassette which was placed in 250 mL of 1Â PBS. The 1Â PBS was
changed twice every 2 h and then left overnight. The solution was further
dialyzed in a new dialysis bag in 250 mL of deionized water. The
deionized water was changed twice every 2 h and then left overnight. The
solution was then passed through a syringe filter (0.22 μm) to remove
aggregated protein.
Aging of Unmodified Lysozyme or RNase A. A solution of
lysozyme or RNase A (1 mg/mL) in 1Â PBS buffer pH 8.2 was placed
in a 28 °C incubator shaker for 3 days. The sample was then dialyzed in
water overnight and then freeze-dried to obtain a white solid.
Matrix Assisted Laser Desorption Ionization Mass Spec-
trometry (MALDI-MS). A saturated solution of sinapinic acid
(matrix) was prepared in a 1:2 mixture of acetonitrile and aqueous
0.1% trifluoroacetic acid (TFA). The protein solution was mixed with
aqueous 0.1% TFA solution and sinapinic acid solution in a 1:1:1 ratio
and spotted on a MTP 384 target ground steel plate (Bruker Daltonics,
Billerica, MA). Mass spectra were obtained on a Bruker AutoFlex III
instrument (Bruker Daltonics, Billerica, MA). Protein mass was mea-
sured using a positive ion linear mode.
Dynamic Light Scattering (DLS). DLS experiments were per-
formed on a MALVERN zetasizer nanoseries (MALVERN Instruments
Ltd. Worcestershire, U.K.) instrument at a scattering angle of 173°.
Solutions (1 mg/mL) of modified and unmodified proteins in 1Â PBS
buffer (pH 7.4) at controlled temperature were used for all DLS
experiments.
Surface Plasmon Resonance (SPR). All SPR experiments
were performed on a SPRimagerII Array system (GWC Technologies,
Madison, WI). Protein solution (0.5 mg/mL) of lysozyme-(βCD)n or
RNase A-(βCD)n and lysozyme or RNase A in 1Â PBS buffer, pH 7.4 at
25 °C was used for all SPR experiments. SpotReady 16 chip (GWC
Technologies, Madison, WI) were mounted in a dual channel flow cell,
and the experiment was performed by first flowing 1Â PBS, pH 7.4
through the cell followed by solutions of modified and unmodified
protiens in the same buffer (in separate channels) and PBS. The amount
of protein which adsorbs irreversibly on the gold was estimated by
’ RESULTS AND DISCUSSION
Water-Driven Reaction for Protein Modification. Amino
squarates are stable in water against hydrolysis, but they have a
unique reactivity for substitution that is driven by the solvent
water; both the yield and the rate decrease significantly in organic
solvents.31 This unique stability and reactivity of amino squarate
reagents in water provides a great opportunity to modify proteins
with cyclodextrins with well-defined bond identity. To enable the
efficient conjugation of proteins with βCD, we synthesized βCD
amino squarates 6, 7, and 8 (Scheme 1) to conjugate the lysine
residues in proteins with βCD. This reaction resulted in a stable
and neutral conjugation between proteins and βCD (Scheme 2).
We introduced fluorine substitutions on the phenyl moiety of
the squarate-βCD reagent to study if electron withdrawing
groups can promote the modification of the lysine residues.
We used MALDI-TOF mass spectrometry to characterize the
degree of modification of the lysine residues of lysozyme with
reagents 6, 7, and 8 at 5, 10, 24, 48, and 72 h. Mass spectra
showed that all three squarate reagents modified the same
amount of lysine residues on the proteins, but the intensity of
peaks in the MALDI spectra corresponding to each degree of
modifications was different. After 5 h, two lysine residues of
lysozyme were modified with βCD using 6, 7, and 8, but the inten-
sity of peaks corresponding to different degrees of modification
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Figure 1. MALDI-TOF spectra for lysozyme-(βCD)n before (A) and after (B) dialysis in deionized water.
using reagent 6 was higher than that by using reagents 7 or 8. After
10 h, the number of lysine residues modified with 6, 7, and 8
increased to three, and the intensity of the product peaks corre-
sponding to different degrees of modification was again highest for
6, followed by 7 and 8. Prolonged reaction time (72 h) increased
the degree of modification of lysine residues to four, but the
intensity of peaks corresponding to different degrees of modifica-
tion became similar for all three compounds 6, 7, and 8. To further
characterize the products of lysozyme modification with βCD,
SDS-PAGE was conducted on lysozyme modified with 6, 7, and 8.
We observed separate bands corresponding to proteins with one,
two, and three lysine residues modified with βCD on the SDS-gel
(see the Supporting Information). This observation was consistent
Figure 2. Number distribution of DLS showing two replicates each for
1 mg/mL lysozyme-(βCD)n (A) and lysozyme (B) in PBS buffer pH 7.4
at 25 °C.
with the results from MALDI-TOF analysis, which showed multi-
ple modifications of proteins with βCD.
Prevention of Protein Aggregation. Different conditions
cause protein to aggregate. Aggregation of lysozyme and RNase
A is reported to be mainly influenced by pH and temperature.34À37
Earlier reports using equilibrium sedimentation have shown that
1 mM hen egg white lysozyme (HEWL) forms dimers in the pH
range 5À9 and higher oligomers at pH 10À11.36 Ribonuclease A
(RNase A) has also been shown to form aggregates when stored in
the freeze-dried form at elevated temperatures.35
After the reaction of lysozyme with βCD-squarate reagent 6,
the unreacted reagents and other small molecules were removed
by dialysis in phosphate buffered saline (PBS), pH 7.4. Interest-
ingly, we found that further dialyzing the samples in deionized
water instead of in a buffer resulted in significant reduction in
the mass peak of unmodified proteins (Figure 1). The effect of
ionic strength on protein stability depends on the identity of the
protein.34,38 Our results here suggest that unmodified lysozyme
and RNase A are induced to form aggregates by the low ionic
strength of deionized water. These aggregates formed during
dialysis in water were removed by filtration, whereas the βCD
modification stabilized the folding and prevented the modified
proteins from aggregating. A similar degree of modification was
obtained with RNase A. We note that MALDI showed only small
amount of unmodified RNase A relative to the βCD modified
RNase A after the modification reaction, and that dialysis in
deionized water only reduced a small amount of the unmodified
RNase A (see the Supporting Information).
βCD-modified proteins remained monomeric under the same
conditions (Figure 2). After dialysis in pure water, lysozyme-
(βCD)n showed only monomeric proteins with a hydrodynamic
diameter of about 3.8 nm in number distribution (Figure 2A) and
volume distribution (see the Supporting Information), which is
consistent with the reported hydrodynamic diameter for unmo-
dified lysozyme.39 Under the same conditions of dialysis in pure
water, the entire sample of unmodified lysozyme formed aggre-
gates, with a hydrodynamic diameter about 698 nm (Figure 2B).
We estimate that this hydrodynamic diameter corresponds to
more than 6 Â 106 unmodified protein molecules in the
aggregate. We obtained similar results for RNase A. While RNase
A-(βCD)n showed no aggregates in number and volume dis-
tribution, unmodified RNase A showed only aggregates (see the
Supporting Information).
Increase in Thermal Stability. Past studies have shown that
trypsin modified with α, β, and γCD are stable when heated
up to 70 °C, whereas native trypsin was totally inactivated at
60 °C.40 To study the thermoinduced aggregation of unmodified
lysozyme and lysozyme-(βCD)n using DLS, we observed that
heating unmodified lysozyme (0.5 mg/mL in 1Â PBS buffer at
pH 7.4) at 67 °C for 25 min resulted in complete aggregation,
whereas treating lysozyme-(βCD)n under the same conditions
gave only monomeric proteins. After heating lysozyme-(βCD)n
at 77 °C for 25 min, aggregates started to emerge, while the
sample contained mostly monomers. Complete aggregation of
lysozyme-(βCD)n was observed only when the same sample was
heated at 87 °C for 25 min (see the Supporting Information).
These results suggest an increase in the thermal stability of lyso-
zyme by nearly 20 °C.
To measure the aggregation behavior of βCD-modified pro-
teins, we examined the dynamic light scattering (DLS) of both
modified and unmodified proteins under the same conditions,
using different stress factors including aging, freeze-drying, and
dialysis. We discovered that dialysis in pure water was the major
cause for complete aggregation of unmodified lysozyme, but
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Figure 3. Four replicates of SPR for the adsorption of (A) lysozyme and lysozyme-(βCD)n and (B) RNase A and RNase A-(βCD)n (0.5 mg/mL of
protein in 1Â PBS, pH 7.4). The SPR intensity (pixels) is plotted against time (s) as protein solutions are flowed over a SPR microarray.
Reduction of Surface Adsorption of Proteins. We believe
that aggregation and surface adsorption of proteins share some
common mechanism. Thus, modification that results in prevent-
ing aggregation should also reduce or prevent surface adsorption.
To measure the effect of βCD modification on surface adsorp-
tion of proteins, we used SPR to directly monitor the adsorption
of modified and unmodified proteins on gold surfaces of 1 mm
diameter spots (on a 16-spot microarray). Buffer (PBS, pH 7.4)
first introduced to the gold spots, followed by solutions of
proteins (0.5 mg/mL) in the buffer. The protein solution was
then replaced by pure PBS, to remove proteins in the carrier
buffer and possibly any reversibly attached proteins on the
surface (Figure 3). The amount of irreversibly adsorbed proteins
was calculated from the difference in the SPR intensity in pixel
intensity units (ΔPIU), before and after the gold surface was
exposed to the protein solutions. The amount of adsorbed
proteins on the surface was calculated as % monolayer (%ML),
where %ML = [ΔPIU(modified lysozyme)/ΔPIU(unmodified lysozyme)] Â
100.41 Relative to unmodified lysozyme, %ML for adsorbed
lysozyme-(βCD)n on the gold surface was found to be 6.75 (
2.44% over eight replicates, suggesting that adsorption of lysozyme
on gold surfaces was reduced by ∼93% due to covalent grafting of
βCD groups (Figure 3A). For RNase A, %ML for adsorbed RNase
A-(βCD)n on gold surface was found to be 21.26 ( 2.62% over
eight replicates, corresponding to a reduction of adsorption by 80%
(Figure 3B). We note that whereas the adsorption of lysozyme-
(βCD)n appeared to have reached equilibrium, adsorption of
RNase A did not. This result suggests that, at least for RNase A,
modification of proteins with βCD can reduce only the rate of
protein adsorption.
Unlike PEG, cyclodextrins have well-defined rigid conforma-
tions and molecular weights. Cyclodextrins comprise both
hydrogen bond acceptors and donors, whereas PEG comprises
only acceptors. Thus, the molecular solvation of βCD and PEG
in water will be different. When grafted on proteins, βCD has a
fixed conformation with only orientational variations, whereas
PEG comprises a large ensemble of conformations and orienta-
tional variations. These differences suggest that both the en-
thalpy and entropy for aggregation and surface adsorption will be
different between CD-grafted and PEG-grafted proteins, and so
the mechanisms will be different for these two types of protein
modifications at reducing protein aggregation and surface ad-
sorption. Considering the effect of additives on protein folding,
recent studies suggest that while kosmotropic anions stabilize
protein structures by direct interactions,15,16,42,43 the effect of
nonionic organic osmolytes on proteins is likely indirect.15
Particularly for sugar molecules that stabilize protein
structures,11 the hydrogen bonding for the molecular solvation
is likely multivalent in nature. For instance, sucrose gradients
commonly used for purification consist of visible layers of
different density that can be easily prepared and do not readily
become a homogeneous solution.44 Thus, we believe that the
relatively rigid structure of cyclodextrin may present a solvation
with multiple water molecules that contribute to two plausible
mechanisms for preventing aggregation and adsorption. First, the
folding stability is increased by grafting βCD on protein surfaces
and thus reduces the denaturation-initiated aggregation and
surface adsorption. Second, βCD creates less dynamic hydration
shells on proteins that prevent proteins from getting in close
contact with each other. There is a possibility that βCD on the
proteins forms intramolecular inclusion complexes with nearby
hydrophobic residues on the same protein, and thus reduces
aggregation and surface adsorption. Both mechanisms are
possible and may be effective under different conditions. This
intramolecular inclusion mechanism would be best explored by
specific protein or peptide engineering, in which βCD is de-
signed to be at specific distance from hydrophobic groups.
’ CONCLUSIONS
The water-driven ligation provides an effective and efficient
method to modify proteins with cyclodextrins by a neutral
covalent bond, which has been challenging for other existing
methods. Cyclodextrin-modified proteins exhibit significant re-
duction in aggregation and surface adsorption as compared to
that by unmodified proteins. Because both lysozyme and RNase
A contain different amounts of α-helices and β-sheets (lysozyme,
41% α-helix, 10% β-sheet; RNase A, 20% α-helix, 35% β-sheet),45
this work likely presents a general approach for preventing
proteins from forming aggregates and from adsorbing onto sur-
faces. This chemistry opens a new chemical window to under-
stand and control protein stability, aggregation, and surface
adsorption. Because of the gross structural and functional
differences, the mechanisms for influencing protein properties
are likely different for βCD modification and pegylation. Further-
more, cyclodextrin can form noncovalent hostÀguest inclusion
complexes with hydrophobic groups in water. Tethering the
hydrophobic groups with small molecules or drugs enables com-
bined drug therapeutics as well as potential targeted drug
delivery. This noncovalent derivatization of proteins and
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mechanistic studies of aggregation and surface adsorption of
modified proteins are the subjects of ongoing research in
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’ ASSOCIATED CONTENT
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S
Supporting Information. Details for the synthesis and
b
characterization of compounds 7 and 8 and other experimental
details. This material is available free of charge via the Internet at
http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: yluk@syr.edu.
’ ACKNOWLEDGMENT
We thank NSF-CAREER (#0845686), and the Syracuse CoE
for the CARTI award supported by the U.S. EPA (#X-83232501-0)
for financial support. We also thank Prof. P. N. Borer, M. M.
Maye, and Dabrowiak for instrumentation and Prof. Sponsler for
a critical read of the manuscript.
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