A structured monodisperse PEG for the effective suppression of protein aggregation

Article abstract of DOI:10.1002/anie.201206563

Part of the solution: A PEG with a discrete triangular structure exhibits hydrophilicity/hydrophobicity switching upon increasing temperatures, and suppresses the thermal aggregation of lysozyme to retain nearly 80 % of the enzymatic activity. CD and NMR

Full text of DOI:10.1002/anie.201206563

.
Angewandte
Communications
DOI: 10.1002/anie.201206563
Protein Manipulation
A Structured Monodisperse PEG for the Effective Suppression of
Protein Aggregation**
Takahiro Muraoka,* Kota Adachi, Mihoko Ui, Shunichi Kawasaki, Nabanita Sadhukhan,
Haruki Obara, Hidehito Tochio, Masahiro Shirakawa, and Kazushi Kinbara*
Poly(ethylene glycols) (PEGs) are popular polyethers that
have been used in wide-ranging fields including chemical
biology, medical chemistry,[1] and materials science.[2] In
protein engineering, owing to their high water solubility,
low toxicity, and low antigenicity, PEGs have been used for
enhancing refolding, assisting crystallization, increasing water
solubility, and prolonging the blood circulation time of
proteins. In relation to these applications, the introduction
of PEGs by covalent-bond formation, that is, PEGylation,[1] is
an important method for improving the stability of proteins.
However, chemistry of pure PEG molecules remains unex-
plored, as most conventional PEGs are polydisperse.[3] For
example, it was not until very recently that the synthesis of
a discrete PEG 44-mer (M = 1956) was accomplished.[3b]
Moreover, PEGs are linear polymers and their structural
diversity is limited, although recent studies have indicated
that the properties of PEGs and their analogues change
drastically depending on the molecular weight and top-
ology.[1a,k]
sized trianglular PEG analogue 1, (M = 883) which consists of
three tetraethylene glycol (TEG) and three PE units
(Scheme 1a).[4] Interestingly,
1 undergoes dehydration,
which is associated with conformational switching between
Herein, we propose a practical approach to the formation
of PEGs with higher dimension structures having topological
diversity by using monodisperse oligoethylene glycol and
pentaerythritol (PE) as a junction unit. PE has four hydroxy
groups, and therefore allows multiple PEGs to be connected
with the introduction of minimal structural difference from an
ethylene glycol unit; the resulting structure has hydroxy
groups at the vertex, like the termini of linear PEGs. As the
first example of structured monodisperse PEGs, we synthe-
Scheme 1. a) Molecular structures of structured monodisperse PEGs
1 and 2. b) A schematic representation of the heat-responsive con-
formational change of 1. Left and right structures are molecular
models gauche-1 and anti-1, respectively, where all the ethylene units
adopt either gauche or anti conformation (C blue, O red, and H white).
The structures were optimized by a molecular mechanics calculation
with Merck molecular force field (MMFF) by using Spartan’08.
[*] Dr. T. Muraoka, K. Adachi, Dr. M. Ui, S. Kawasaki, Dr. N. Sadhukhan,
H. Obara, Prof. K. Kinbara
Institute of Multidisciplinary Research for Advanced Materials
Tohoku University, 2-1-1, Katahira, Aoba-ku, Sendai (Japan)
E-mail: kinbara@tagen.tohoku.ac.jp
Homepage: http://www.tagen.tohoku.ac.jp/labo/kinbara/
index.html
the gauche and anti forms (Scheme 1b), at lower temperature
than the corresponding linear version, 2 (Scheme 1a). This
unique feature could be applied for the manipulation of
proteins,[5] that is, 1 exhibits the ability to suppress protein
thermal aggregation, unlike 2 or PEG-1000, which is a con-
ventional linear PEG of a similar molecular weight (Mw =
993).
1 was synthesized from PE by the procedure shown in
Scheme 2. First, three hydroxy groups of PE were protected
selectively (3 and 4) and the residual free hydroxy groups
were coupled with tosylated TEGs to afford ditosylate 5 and
diol 6. Williamson ether synthesis between 5 and 6 and
subsequent stepwise cleavage of triisopropyl (TIPS) and
benzyl (Bn) groups successfully yielded compound 1.[6]
Similarly, 2 was also synthesized by Williamson ether syn-
Dr. H. Tochio, Prof. M. Shirakawa
Department of Molecular Engineering
Graduate School of Engineering, Kyoto University
Kyoto-Daigaku Katsura, Nishikyo-ku, Kyoto (Japan)
[**] We thank Prof. K. Tsumoto (University of Tokyo) for fruitful
discussions. This work was partially supported by the Ministry of
Education, Science, Sports and Culture (Japan), Grants-in-Aid for
Young Scientists S (21675003 for K.K.), Scientific Research on
Innovative Areas “Spying minority in biological phenomena (No.
3306)” (23115003 for K.K.), and Exploratory Research (24655112 for
T.M.) and the Management Expenses Grants for National Univer-
sities Corporations from the Ministry of Education, Culture, Sports,
Science and Technology of Japan (MEXT).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201206563.
2430
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2013, 52, 2430 –2434

Angewandte
Chemie
À
angles around the C O bonds between the PE and TEG units
(C2 and O3 atoms) change from an anti to a gauche relation-
ship upon increasing the temperature. These spectroscopic
studies indicate that dynamic changes in the conformation of
1 are triggered by heating.
As is the case with 1, 2 shows downfield shift of the
13C NMR signals upon an increase in temperature from 208C
to 808C, thus suggesting the gauche-to-anti conformational
change of the ethylene glycol units (see the Supporting
Information, Figure S5). As such gauche-to-anti conforma-
tional change is known to increase the hydrophobicity of
PEGs,[7a–e] we then investigated their hydrophobicity in terms
of dehydration. Hydrated PEGs are known to exhibit a linear
relationship between 1/h and T1/T, where h and T represent
the viscosity of water and temperature, respectively, since the
mobility of a PEG chain is governed mainly by the viscosity of
the solvent (Figure 1).[7a] Actually, 2 shows an almost linear
Scheme 2. Synthesis of 1. a) 1) CH3C(OEt)3, TsOH·H2O, toluene;
2) BnBr, KOH, DMSO; 3) HCl (aq.), MeOH, then Na2CO3; 4) TIPSCl,
imidazole, DMF, 41% over 4 steps. b) TrCl, Et3N, DMAP, CH2Cl2,
quant. yield. c) 1) NaH, TsO(CH2CH2O)4PMB, THF; 2) DDQ, CH2Cl2,
water; 3) TsCl, Et3N, CH2Cl2, 60% over 3 steps. d) 1) NaH, TsO-
(CH2CH2O)4Ts, THF; 2) ZnBr2, CH2Cl2, MeOH, 54% over 2 steps.
e) 1) NaH, THF; 2) TBAF, THF; 3) H2, Pd/C, EtOH, 32% over 3 steps.
See the Supporting Information for the details. Bn=benzyl, DMAP=4-
dimethylaminopyridine, DMF=N,N’-dimethylformamide, DMSO=di-
methyl sulfoxide, TBAF=tetra-n-butylammonium fluoride, TIPS=tri-
isopropylsilyl, Tr=trityl, Ts=p-toluenesulfonyl.
thesis between the PE and TEG units. 1 and 2 are colorless
oils, and readily dissolve in water at room temperature.
It is known that in water linear PEGs undergo a heat-
Figure 1. 1H NMR T1/T values of 1 (circles) and 2 (squares) in D2O
plotted as a function of the reciprocal of water viscosity (h) and
temperature (T). T1 values were measured for 1H NMR signals
corresponding to 1H at C5, C7, and C8 of 1 and C2, C4, C5, C7, C8,
C10, C18, C20, and C21 of 2.
À
responsive conformational change, whereby C C bonds in
the gauche form at room temperature change into the anti
form at high temperatures.[7] Infrared (IR) spectroscopic
analysis of a neat sample of 1 at 258C showed absorption
bands at 1458 and 1352 cmÀ1, which are characteristic to the
CH2 vibration of the ethylene glycol units in gauche con-
formation (see the Supporting Information, Figure S2, blue
line).[7f] Upon being heated to 1208C, 1 displayed an
intensified absorption band at 1328 cmÀ1, with a relatively
intensified band at 1473 cmÀ1 (see the Supporting Informa-
tion, Figure S2, red line), thus suggesting that the ethylene
glycol units change the conformation from gauche to anti
form.[7f] This conformational change is also indicated by
variable temperature 13C nuclear magnetic resonance (VT
13C NMR) spectroscopy, in which a downfield shift of all the
peaks in the spectrum of 1 in D2O (1.6 mgmLÀ1) occurs at
elevated temperatures (see the Supporting Information,
Figure S3).[7b]
correlation between T1/T and 1/h in the temperature range
between 20 and 808C, thus indicating that 2 is hydrated below
808C. In contrast, although T1/T values of 1 showed a linear
correlation with 1/h below 608C, this correlation did not occur
above 608C, thus strongly indicating that dehydration of
1 occurs around 608C. As well as 2, conventional linear PEGs
are known to dehydrate over 808C.[7a] Thus, the low dehy-
dration temperature of 1 is likely due to a distinct effect of
structuralization into the 2D trianglular geometry.
The unique thermo response of 1, whereby the hydro-
phobicity is increased at a rather lower temperature than for
linear PEGs inspired the following hypothesis. Namely, after
dehydration at high temperatures, 1 is able to interact with
thermally unfolded proteins, which have more exposed
hydrophobic residues on the surface than the folded ones,
and then is able to prevent their aggregation. This is
a practically important issue for the manipulation of ther-
apeutic proteins.
With respect to the conformation at the junction between
PE and TEG, variable temperature heteronuclear multiple-
bond correlation (VT HMBC) experiments also indicate that
À
rotation around C O bonds occurs. In the HMBC measure-
ment, 1H and 13C atoms in a gauche relationship display either
no cross peak or a weak cross peak because of the small 3JCH
1
value.[8] Indeed, at 258C, a cross peak between H at C4 and
13C at C2 was too weak to be observed, however a cross peak
appeared upon heating the sample up to 608C (see the
Supporting Information, Figure S4).[9] Hence, this marked
alteration of the cross peak intensity suggests that the torsion
When chicken-egg-white lysozyme was dissolved in phos-
phate-buffered saline (PBS, pH 7.4; 3.0 mgmLÀ1) in the
absence and presence of 1, 2, or PEG-1000 (30 mgmLÀ1), all
the resulting mixtures afforded clear solutions at 208C
Angew. Chem. Int. Ed. 2013, 52, 2430 –2434
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
2431

.
Angewandte
Communications
aggregation suppressor (Figure 3, black line), and its ester
derivative (Figure S7).[10]
The effect of 1 on the higher-order structures of lysozyme
were investigated by circular dichroism (CD) and VT
1H NMR spectroscopy. Upon being heated from 208C to
908C, for a PBS solution of lysozyme containing no additive
there was a significant decrease in the intensity of CD bands
around 210 nm and 225 nm, corresponding to the secondary
structures (b sheet and a helix, respectively); these signals did
not recover, but in fact decreased after cooling to 208C
(Figure 4a). In contrast, in the presence of 1, the intensity of
Figure 2. Photographs of lysozyme in PBS (3.0 mgmLÀ1) at 208C (top)
and 908C (bottom). a and b) no additive, c and d) with 2
(30 mgmLÀ1), and e and f) with 1 (30 mgmLÀ1).
(Figure 2). Upon heating the sample without any additives up
to 908C, white precipitates appeared owing to thermal
aggregation of denatured lysozyme (Figure 2b). The samples
containing either 2 or PEG-1000 also gave similar precipitates
shortly after heating (Figure 2d, and the Supporting Infor-
mation, Figure S6). In sharp contrast, the lysozyme solution
containing 1 remained colorless and transparent, even after
incubation at 908C for 30 min (Figure 2 f). These simple
experiments clearly demonstrate that only 1 effectively
suppresses thermal aggregation of lysozyme.
Figure 4. CD spectra of lysozyme (0.5 mgmLÀ1 =0.035 mm) in PBS
buffer with a) no additive and b) 1 (25 mgmLÀ1 =28 mm). Arrows
indicate the directions of spectral changes upon heating (left; 208C
(blue), 408C (pale blue), 608C (dark yellow), 808C (orange), to 908C
(red)) and cooling (right; 908C to 208C).
The enzymatic activity of lysozyme after heat treatment
were investigated to evaluate the ability of the additives to
suppress thermal aggregation (Figure 3). Lysozyme in PBS
the CD bands at 908C was higher than that in the absence of 1,
and the signal intensity was mostly recovered after cooling to
208C (Figure 4b). These CD spectral changes are consistent
with the changes in enzymatic activity of lysozyme after
heating (Figure 3). It should be mentioned that the CD signals
of 1 were recovered upon repeating the heat treatment cycles
(see the Supporting Information, Figure S8).
We investigated the conformational states of lysozyme
1
Figure 3. Enzymatic activity of lysozyme (3.0 mgmLÀ1 =0.21 mm) after
heating (988C, 30 min) with different concentrations of additives
(1 red; 2 orange; l-arginine hydrochloride black; PEG-1000 blue).
during heating in more detail by using VT H NMR spec-
troscopy. It is known that the protons of folded proteins
exhibit a wide range of chemical shifts owing to the
anisotropic magnetic fields of proximal aromatic or carbonyl
groups, whereas denatured proteins exhibit chemical shifts of
protons in a rather narrower range (d = 8.5 to 0.8 ppm), such
as short linear peptides adopting a random coil conforma-
(3.0 mgmLÀ1 = 0.21 mm) containing no additives completely
lost its activity after incubation at 988C for 30 min (Figure 3,
0 mm). In contrast, nearly 80% enzymatic activity was
retained even after such harsh thermal treatment when
1 (30 mm) was present (Figure 3, red line). Interestingly, the
presence of 2 barely helped lysozyme retain enzymatic
activity (Figure 3; orange line), thus clearly demonstrating
that the topology of PEGs drastically influences their proper-
ties. Also the presence of PEG-1000 barely improved the
outcome (Figure 3, blue line). Notably, the capability of 1 to
suppress thermal aggregation is significantly higher than that
of l-arginine, which is known to be a high-performance
tion.[11] In fact, a PBS solution of lysozyme (1.0 mgmLÀ1
=
0.070 mm) showed signals of protons from d = 11 to À2 ppm
at 408C (see the Supporting Information, Figure S9).[12] Upon
heating to 708C, the peak intensities decreased in the
magnetic field regions downfield of d = 8 ppm and upfield
of d = 0.6 ppm, and further heating up to 808C resulted in the
disappearance of peaks in those regions. At 808C, lysozyme
changes into a random coil state, in which the secondary
structures are no longer retained. At 908C, intensities of all
the signals significantly decreased as a result of the thermal
2432
www.angewandte.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2013, 52, 2430 –2434

Angewandte
Chemie
aggregation mentioned above, and the disappeared signals
did not recover even after cooling to 408C.[13] On the other
hand, a mixture of lysozyme (1.0 mgmLÀ1 = 0.070 mm) and
1 (33 mgmLÀ1 = 37 mm) also showed many signals in a wide
range of chemical shifts at 408C (Figure 5a), with a slightly
different spectral profile from intact lysozyme in the aromatic
region. Thus, 1 is likely to interact with lysozyme at this
temperature while preserving lysozyme’s ternary structure. It
is of great importance that several signals in the upfield and
downfield regions persisted at 808C and even at 908C with
significant intensities (Figure 5a, filled circles), thus strongly
indicating that lysozyme remained dissolved in the buffer at
such high temperatures and retained partial higher-order
structures. Moreover, the original signals mostly recovered
after cooling to 408C (Figure 5a, filled squares). The five
signals observed in the high magnetic field region can be
assigned to the alkyl protons of Leu8, Leu17, Thr51, Leu56,
Ile98, Met105, and Lys116 according to a literature,[12] where
the original secondary and ternary structures around those
residues are deemed to be recovered after the heating
process. As shown in Figure 5b, these residues are located
in the secondary structures such as a-helix (Leu8, Ile98 and
Met105), b-sheet (Thr51), and loop (Leu56) or close to a-
helix (Leu17 and Lys116), and are distributed in the whole
molecule. Of importance, Thr51, Leu56, Ile98, and Met105
are located beside a large cleft, that is, the active site.[14] Thus,
1 stabilizes lysozyme to preserve the partial higher-order
structures as well as the solubility in the buffer at elevated
temperatures, which in turn results in the suppression of
thermal aggregation and the recovery of the native confor-
mation after cooling.
Here, it is of importance to investigate the relevancy of
the thermal gauche/anti conformational change of 1 and 2 to
their capability to suppress aggregation. Fluorescence aniso-
tropy (FA) measurement of lysozyme displayed aggregation
features of lysozyme upon an increase in temperature
(Figure 6). A slowly tumbling molecule, namely a large
molecule or an aggregate, exhibits a large FA value compared
with small molecules. Actually, lysozyme, in the absence of
additives, showed a monotonic increase of FA values upon
elevation of temperatures above 308C (Figure 6, blue curve
and squares),[15] thus indicating that heating encourages
aggregation of lysozyme, and the size of the aggregates
increases with increasing temperature. The mixture of lyso-
zyme and 2 displayed a similar tendency (Figure 6, orange
curve and diamonds), that is, as expected, 2 hardly influences
the aggregation of lysozyme at temperatures above 308C. In
clear contrast, the mixture of lysozyme and 1 exhibited
a gradual decrease of FA values from 208C to 508C, followed
by a steep increment at 608C and a subsequent decrement
(Figure 6, red circles and curve). The decrease of FA values
from 208C to 508C is deemed to result from the increased
tumbling rate of lysozyme, without aggregation. The incre-
ment of the FA value around 608C is likely due to complex-
ation of lysozyme with 1, and not to protein aggregation as is
suggested by the subsequent FA values above 708C. As
mentioned above, dehydration of 1 accompanied by a gauche-
to-anti conformational change occurs around 608C, which
coincides with the temperature at which complexation occurs.
Figure 5. a) 1H NMR spectra of a mixture of lysozyme
(1.0 mgmLÀ1 =0.070 mm) and 1 (33 mgmLÀ1 =37 mm) in PBS at
408C, 708C, 808C, and 908C, followed by cooling to 408C. The high-
magnetic-field region (d<0.6 ppm) of the spectra are magnified three
times compared to those in the low-magnetic-field region
(d>6.6 ppm). Filled circles represent the detectable proton signals of
lysozyme at 808C and 908C, and suggest that partial higher-order
structures of lysozyme were retained. Proton signals recovered after
cooling to 408C are marked with filled squares. b) Side and top views
of the crystal structure of lysozyme (PDB ID: 2HUB). The residues for
which the corresponding 1H NMR signals were recovered after cooling
to 408C are labeled on the space-filling representation. The red parts
represent the binding site residues (Glu35, Asp52, Asn59, Trp62,
Trp63, Asp101).
Therefore, it is likely that the dehydration of 1 allows for
interaction with lysozyme at high temperature to suppress
Angew. Chem. Int. Ed. 2013, 52, 2430 –2434
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
2433

.
Angewandte
Communications
h) G. Kochendoerfer, Expert Opin. Biol. Ther. 2003, 3, 1253 –
1261; i) Y.-S. Wang, S. Youngster, M. Grace, J. Bausch, R.
Bordens, D. F. Wyss, Adv. Drug Delivery Rev. 2002, 54, 547 – 570;
j) P. Bailon, A. Palleroni, C. A. Schaffer, C. L. Spence, W.-J.
Fung, J. E. Porter, G. K. Ehrlich, W. Pan, Z.-X. Xu, M. W. Modi,
A. Farid, W. Berthold, Bioconjugate Chem. 2001, 12, 195 – 202;
k) F. M. Veronese, P. Caliceti, O. Schiavon, J. Bioact. Compat.
Polym. 1997, 12, 196 – 207.
[2] a) B. Scrosati, J. Garche, J. Power Sources 2010, 195, 2419 – 2430;
b) P. Arora, Z. Zhang, Chem. Rev. 2004, 104, 4419 – 4462; c) J. Y.
Song, Y. Y. Wang, C. C. Wan, J. Power Sources 1999, 77, 183 –
197.
[3] a) A. C. French, A. L. Thompson, B. G. Davis, Angew. Chem.
2009, 121, 1274 – 1278; Angew. Chem. Int. Ed. 2009, 48, 1248 –
1252; b) S. A. Ahmed, M. Tanaka, J. Org. Chem. 2006, 71, 9884 –
9886.
[4] The molecular design of 1 based on molecular modeling is
explained in the Supporting Information, Figure S1.
[5] For examples of amphiphilic polymer assemblies for the
manipulation of proteins, see: Y. Sasaki, K. Akiyoshi, Chem.
Rec. 2010, 10, 366 – 376.
[6] Interestingly, without any template, the reaction between 5 and 6
preferentially gave cyclic products 13 and 14 (40% yield), and
the polymerized products were obtained only in < 3% yield, see
the Supporting Information.
[7] a) M. J. Hey, S. M. Ilett, G. Davidson, J. Chem. Soc. Faraday
Trans. 1995, 91, 3897 – 3900; b) M. Bjçrling, G. Karlstrçm, P.
Linse, J. Phys. Chem. 1991, 95, 6706 – 6709; c) H. Matsuura, K.
Fukuhara, J. Mol. Struct. 1985, 126, 251 – 260; d) G. Karlstrçm, J.
Phys. Chem. 1985, 89, 4962 – 4964; e) S. Saeki, N. Kuwahara, M.
Nakata, M. Kaneko, Polymer 1976, 17, 685 – 689; f) H. Matsuura,
T. Miyazawa, J. Polym. Sci. Part A 1969, 7, 1735 – 1744.
[8] N. E. Jacobsen in NMR Spectroscopy Explained: Simplified
Theory, Applications and Examples for Organic Chemistry and
Structural Biology, Wiley-InterScience, Hoboken, 2007, pp. 489 –
550.
Figure 6. Fluorescence anisotropy changes of lysozyme (0.70 mm) in
the absence (blue) and presence of 1 (red, 0.20 mm) or 2 (orange,
0.20 mm) in PBS buffer (pH 7.4) upon an increase in temperature
from 208C to 988C. Excitation and emission wavelengths are 295 and
335 nm, respectively.
aggregation. It should be noted here that because 2 stays
hydrated below 808C, denatured lysozyme is likely to
aggregate before being bound by 2.
In summary, we have demonstrated that the structured
monodisperse PEG analogue 1, having a triangular structure,
has a low dehydration temperature compared with the
corresponding linear PEGs, and suppresses protein thermal
aggregation with high efficiency. Thus, the structuring of PEG
from a linear geometry into higher-dimensional ones affects
the physicochemical nature of PEGs to bring about the
distinctive biochemical effect of 1. We believe that detailed
studies using monodisperse PEG analogues with diverse
structures offer a better and reliable understanding of the
characteristics of PEGs and these characteristics can be
exploited to explore new applications of PEG-related com-
pounds. Further studies on the capability of 1 to suppress the
aggregation of other proteins and on other structured PEGs
are now in progress.
[9] As 13C NMR signals corresponding to C2 and C4 of 1 overlap at
808C, the VT HMBC experiment was performed at 608C.
[10] a) C. Lange, R. Rudolph, Curr. Pharm. Biotechnol. 2009, 10,
408 – 414; b) T. Arakawa, D. Ejima, K. Tsumoto, N. Obeyama, Y.
Tanaka, Y. Kita, S. N. Timasheff, Biophys. Chem. 2007, 127, 1 – 8;
c) K. Shiraki, M. Kudou, S. Nishikori, H. Kitagawa, T. Imanaka,
M. Takagi, Eur. J. Biochem. 2004, 271, 3242 – 3247; d) T.
Arakawa, K. Tsumoto, Biochem. Biophys. Res. Commun. 2003,
304, 148 – 152.
[11] J. Cavanagh, W. J. Fairbrother, A. G. Palmer III, M. Rance, N. J.
Skelton, Protein NMR Spectroscopy: Principles and Practice,
2nd ed., Academic Press, San Diego, 2006.
[12] Y. Wang, T. C. Bjorndahl, D. S. Wishart, J. Biomol. NMR 2000,
17, 83 – 84.
Received: August 14, 2012
Published online: January 30, 2013
Keywords: enzymes · macrocycles · polyethylene glycol ·
.
protein manipulation · supramolecular chemistry
[1] a) K. Knop, R. Hoogenboom, D. Fischer, U. S. Schubert, Angew.
Chem. 2010, 122, 6430 – 6452; Angew. Chem. Int. Ed. 2010, 49,
[13] White precipitates were observed in the NMR tube.
[14] a) J. C. Cheetham, P. J. Artymiuk, D. C. Phillips, J. Mol. Biol.
1992, 224, 613 – 628; b) N. C. J. Strynadka, M. N. G. James, J.
Mol. Biol. 1991, 220, 401 – 424.
[15] An FA value greater than 0.4 is likely due to the presence of
scattered light by the precipitates of lysozyme. J. R. Lakowicz, in
Principles of Fluorescence Spectroscopy, 3rd ed., Springer, New
York, 2006.
ˇ
6288 – 6308; b) S. Jevsevar, M. Kunstelj, V. G. Porekar, Biotech-
nol. J. 2010, 5, 113 – 128; c) M. J. Joralemon, S. McRae, T.
Emrick, Chem. Commun. 2010, 46, 1377 – 1393; d) D. da Silva
Freitas, J. Abrah¼o-Neto, Int. J. Pharm. 2010, 392, 111 – 117; e) J.-
B. Park, Y. M. Kwon, T.-Y. Lee, R. Brim, M.-C. Ko, R. K.
Sunahara, J. H. Woods, V. C. Yang, J. Controlled Release 2010,
142, 174 – 179; f) S. M. Ryan, G. Mantovani, X. Wang, D. M.
Haddleton, D. J. Brayden, Expert Opin. Drug Delivery 2008, 5,
371 – 383; g) C. S. Fishburn, J. Pharm. Sci. 2008, 97, 4167 – 4183;
2434
www.angewandte.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2013, 52, 2430 –2434