Site-selective glycosylation of hemoglobin with variable molecular weight oligosaccharides: Potential alternative to PEGylation

Article abstract of DOI:10.1021/ja300893t

Poly(ethylene glycol) (PEG) conjugation (i.e., PEGylation) is a commonly used strategy to increase the circulatory half-life of therapeutic proteins and colloids; however, few viable alternatives exist to replicate its functions. Herein, we report a method for the rapid site-selective glycosylation of proteins with variously sized carbohydrates, up to a molecular weight (MW) of 10 000, thus serving as a potential alternative for PEGylation. More importantly, the method developed has two unique features. First, traditional protecting group strategies that typically accompany the modification of the carbohydrate fragments are circumvented, allowing for the facile site-selective glycosylation of a desired protein with variously sized glycans. Second, the methodology employed is not limited by oligosaccharide size; consequently, glycans of MW similar to that of PEG, used in the PEGylation of therapeutic proteins, can be employed. To demonstrate the usefulness of this technology, hemoglobin (Hb) was site-selectively glycosylated with a series of carbohydrates of increasing MW (from 504 to ～10 000). Hb was selected on the basis of the vast wealth of biochemical and biophysical knowledge present in the literature and because of its use as a precursor in the synthesis/formulation of artificial red blood cell substitutes. Following the successful site-selective glycosylation of Hb, the impact of increasing the glycan MW on Hb's biophysical properties was investigated in vitro.

Full text of DOI:10.1021/ja300893t

Article
pubs.acs.org/JACS
Site-Selective Glycosylation of Hemoglobin with Variable Molecular
Weight Oligosaccharides: Potential Alternative to PEGylation
Thomas J. Styslinger,† Ning Zhang,‡,∥ Veer S. Bhatt,†,§ Nicholas Pettit,†,‡ Andre F. Palmer,*,∥,‡
and Peng G. Wang*,†,‡,§
‡
§
∥
†Department of Chemistry, Biochemistry Program, Biophysics Program, and William G. Lowrie Department of Chemical and
Biomolecular Engineering, The Ohio State University, Columbus, Ohio 43210, United States
S
* Supporting Information
ABSTRACT: Poly(ethylene glycol) (PEG) conjugation (i.e.,
PEGylation) is a commonly used strategy to increase the
circulatory half-life of therapeutic proteins and colloids;
however, few viable alternatives exist to replicate its functions.
Herein, we report a method for the rapid site-selective
glycosylation of proteins with variously sized carbohydrates,
up to a molecular weight (MW) of 10 000, thus serving as a
potential alternative for PEGylation. More importantly, the
method developed has two unique features. First, traditional
protecting group strategies that typically accompany the modification of the carbohydrate fragments are circumvented, allowing
for the facile site-selective glycosylation of a desired protein with variously sized glycans. Second, the methodology employed is
not limited by oligosaccharide size; consequently, glycans of MW similar to that of PEG, used in the PEGylation of therapeutic
proteins, can be employed. To demonstrate the usefulness of this technology, hemoglobin (Hb) was site-selectively glycosylated
with a series of carbohydrates of increasing MW (from 504 to ∼10 000). Hb was selected on the basis of the vast wealth of
biochemical and biophysical knowledge present in the literature and because of its use as a precursor in the synthesis/formulation
of artificial red blood cell substitutes. Following the successful site-selective glycosylation of Hb, the impact of increasing the
glycan MW on Hb’s biophysical properties was investigated in vitro.
Furthermore, glycosylation has been shown to increase thermal
stability while decreasing precipitation and aggregation.11,12
Consequently, due to the importance of glycoproteins in
nature, the glycosylation of proteins in the laboratory
(glycoprotein synthesis) has long been an area of interest
among researchers, and many elegant methods exist in the
literature.13,14
Unfortunately, two limiting factors are frequently associated
with the ability to chemically glycosylate a protein in the
laboratory. The first is the rate at which the carbohydrate
derivatives, or carbohydrate linkers, used in protein glyco-
sylation can be synthesizeda bottleneck mainly due the
tedious protecting group strategies that accommodate carbohy-
drate modification, as shown in Figure 1A.13,14 The second
limiting factor is that current methods for protein glycosylation
are conceivably confined to small oligosaccharides due, in part,
to the low solubility of large oligosaccharides and poly-
saccharides in many organic solvents. This low solubility, or
lack thereof, significantly reduces the routes available for glycan
modification necessary to employ them in glycoprotein
synthesis. Consequently, on the basis of these limitations, we
sought to develop a method that would circumvent
carbohydrate protecting group chemistry altogether and be
INTRODUCTION
■
The covalent coupling of poly(ethylene glycol) (PEG) to
therapeutic proteins and colloids, PEGylation, has long
provided an effective means to increase the pharmacological
effectiveness of such biopharmaceuticals.1−4 PEGylation serves
to increase the hydrodynamic radius of the protein/colloid,
effectively shielding its surface and preventing recognition by
the reticuloendothelial system. Benefits of PEGylation include
increased circulatory half-life, decreased degradation by
metabolic enzymes, and reduced protein immunogenicity.
However, despite the numerous benefits of PEGylation,
questions regarding its universal application have arisen in the
literature.5,6 In certain cases, PEGylation has been observed to
promote decreased biological activity and can accumulate over
long periods of treatment, giving rise to macromolecular
syndrome.3
Currently, various groups are exploring potential alternatives
to PEGylation for improving the pharmaceutical effectiveness
of therapeutic proteins.7 In certain cases, inspiration has been
taken from nature, in which the glycosylation of proteins, with
even small oligosaccharides, functions in much the same way as
PEGylation.8,9 Similar to PEGylation, glycosylation provides a
large hydration shell capable of protecting proteins from
proteolysis.10 Interestingly though, glycosylation has the added
benefit of being able to stabilize the protein by forming
hydrogen bonds with a protein’s amino acid residues.
Received: January 31, 2012
Published: April 10, 2012
© 2012 American Chemical Society
7507
dx.doi.org/10.1021/ja300893t | J. Am. Chem. Soc. 2012, 134, 7507−7515

Journal of the American Chemical Society
Article
Figure 1. (A) Traditional route toward glycoprotein synthesis involving protecting group manipulation. (B) New oxime-ligation approach avoiding
the need for protecting group manipulation.
aqueous-friendly, thus allowing a desired protein to be
glycosylated with a wide variety of carbohydrates, regardless
of size.
PEGylation of similar therapeutics. To the best of our
knowledge, this is the first reported method for the site-
selective chemical glycosylation of proteins with oligosacchar-
ides of such high MWs.
Herein, we report a versatile method for the site-selective
glycosylation of proteins that requires no protecting group
chemistry on the carbohydrate fragment and, importantly,
allows for the use of large molecular weight (MW)
oligosaccharides. By taking advantage of the aldehyde available
in the open-chain conformation on the reducing end
carbohydrate, we were able to employ oxime-ligation chemistry
for the installation of a linker fragment (Figure 1B). It is
important to note that this method enabled us to employ
glycans of MW comparable to that of PEG, commonly used in
the PEGylation of therapeutic proteins, to be conjugated site-
selectively to proteins. Thus, this method of site-selectively
glycosylating proteins with high MW oligosaccharides may
provide a new viable alternative to site-selective PEGylation, as
carbohydrates are biodegradable, whereas PEG is not, and carry
with them further advantageous properties. In addition, the
ease by which this method can be employed may allow for the
facile fine-tuning of the pharmacokinetic and pharmacodynamic
properties of therapeutic proteins, as is done with the
To demonstrate the usefulness of this methodology,
hemoglobin (Hb) was site-selectively glycosylated with three
different maltose-type sugars (MWs ranging from 504 to 1153)
and a dextran sugar (MW ∼10 000). After determination that
the glycosylation reactions were successful and indeed site-
selective, the biophysical properties of the oligosaccharide-
conjugated Hb's were subsequently studied in vitro. Hb was
chosen as the test protein for the conjugation chemistry on the
basis of several factors, including (1) the significant amount of
information present in the literature regarding Hb’s bio-
chemistry, (2) the feature of bovine Hb (bHb) containing one
cysteine residue on the β chain at position 93 and none on the
α chain, making site-selective modification possible, and (3) the
potential of Hb to serve as an oxygen therapeutic for use in
transfusion medicine to treat moderate to severe blood
loss.15−17 Thus, using thiol-targeting chemistry, we were able
to successfully conjugate various MW glycans to the two
cysteine residues on the Hb tetramer (α2β2) and, subsequently,
7508
dx.doi.org/10.1021/ja300893t | J. Am. Chem. Soc. 2012, 134, 7507−7515

Journal of the American Chemical Society
Article
Figure 2. (A) Synthesis of the aminooxy-bearing linker and subsequent synthesis of maltotriose, maltopentaose, maltoheptaose, and dextran linkers.
(B) Ring−chain tautonomers of the oximine-ligation products, including (E)-oxime, (Z)-oxime, β-pyranose, and α-pyranose.24,36 (C) H NMR of
1
dextran linker 6 showing the presence of the (E)-oxime (0.6H) and (Z)-oxime (0.19H) H-1, with the integration of the C1−H of the repeating
glucose residues being (61.18H).37
investigate the biophysical effects of glycosylation. This
methodology not only allowed us to explore the potential of
glycosylated Hb to serve as an oxygen therapeutic, but also
holds the promising ability to be applied to a wide variety of
other therapeutic proteins bearing naturally occurring and
accessible cysteine residues or to therapeutic proteins that have
undergone site-directed mutagenesis to incorporate cysteine
residues.
various glycans and subsequent investigation of the biophysical
effects of glycosylation.
Thus, in an attempt to abstain from the use of this lengthy
process, we searched for a method that would circumvent the
requirement of protecting the desired glycan and be aqueous-
friendly, allowing for the use of high MW oligosaccharides not
soluble in most organic solvents. As a result, we chose to
employ an oxime ligation for the installation of a linker and
“click” chemistry for its subsequent modification to allow for
site-selective glycosylation (Figure 1B). Employing an oxime
ligation allowed us to take advantage of the ability of an
aminooxy group to react with a ketone or aldehydein our
case, the aldehyde present in the open-chain conformation on
the reducing end carbohydrate of the oligosaccharideforming
an oxime bond. Notably, oxime-ligation conjugates have been
shown to be stable under physiological conditions and a wide
pH range.19−21 This type of oxime-ligation chemistry has
previously been applied to the development of carbohydrate
microarrays,22,23 glyconanoparticles,24,25 and solution-phase
tagging of glycans.26−28 Furthermore, at the time of conception,
it was our belief that the addition of aniline to the oxime
ligation would catalyze the reaction, functioning as a
nucleophilic catalyst under slightly acidic conditions, as noted
by Dawson and co-workers, but which had yet to be applied to
carbohydrate chemistry.29 Confirming our suspicions, while we
RESULTS AND DISCUSSION
■
Design of the Carbohydrate Linkers. Typically, site-
selective protein glycosylation occurs in a fashion similar to that
depicted in Figure 1A in which the chosen glycan is chemically
modified and then allowed to react with the desired
protein.13,14,18 Chemical modification of the glycan typically
involves initial protection of the hydroxyl groups followed by
activation at the anomeric position, thus allowing for the
subsequent installation of a substituent (referred to as the linker
herein) responsible for linking the glycan moiety to the desired
protein. Lastly, the carbohydrate must be deprotected and the
linker modified to accommodate a functional group capable of
reacting site-selectively with the protein. Consequently, when
new glycans are chosen for protein glycosylation, an
exponentially greater number of synthetic steps are required,
slowing the rate at which a protein can be glycosylated with
7509
dx.doi.org/10.1021/ja300893t | J. Am. Chem. Soc. 2012, 134, 7507−7515

Journal of the American Chemical Society
Article
Figure 3. Synthesis of maltotriose−bHb, maltopentaose−bHb, maltoheptaose−bHb, and dextran−bHb.
were carrying out the synthetic portion of this project, Jensen
and co-workers published a paper diligently detailing the effects
of nucleophilic catalysis on carbohydrate oxime formation by
aniline.30 Accordingly, the linker was designed to incorporate
an aminooxy functional group on one end and an azido group
on the opposite end, with the two being separated by a short
oligoethylene glycol spacer. The aminooxy group was selected
to partake in the aforementioned oxime ligation, while the
azido group was selected to remain inert during the oxime
ligation and to subsequently allow for the installation of a
maleimide groupcapable of site-selectively reacting with the
cysteine residues of a proteinvia a CuAAC (copper(I)-
catalyzed azide−alkyne cycloaddition) reaction. It is important
to note that the overall design of the synthesis was constructed
to allow for easy scale-up with a minimal number of synthetic
steps.
Four differently sized carbohydrates, spanning a MW range
of 504−10 000, were chosen for the glycosylation of bHb. Of
the four chosen carbohydrates, three were maltose sugars,
maltotriose, maltopentaose, and maltoheptaose, with the other
being a dextran sugar with an average MW of 10 000. Both the
maltose and dextran sugars are oligomers of glucose with
differing linkages, the maltoses being composed of α(1→4)
linkages and the dextran being composed of α(1→6) linkages.
Although the maltose carbohydrates have a lower MW range
(504−1153), conjugation of smaller oligosaccharides could still
be advantageous on the basis of reports that PEGylation with
low MW PEG (300−2500) effectively increases the pharma-
cokinetics and immunogenicity profiles of therapeutic pro-
teins.31 The dextran glycan, in addition to being of a much
higher MWcomparable to that of PEG commonly used in
therapeutic protein conjugationbelongs to a family of
carbohydrates (dextrans) that are utilized as plasma expanders,
coatings for certain biomedical devices, and components of
biomedical hydrogels.32−35
Following the removal of the phthalimide protecting group
using hydrazine monohydrate, oxime ligations between linker 2
and the desired glycans were carried out under slightly acidic
conditions (pH 4.7) in the presence of 100 mM aniline, giving
the carbohydrate−azide linkers 3−6. All reactions proceeded
with relatively high yields and were purified by size exclusion
chromatography and subsequently characterized by LC−MS,
1H NMR, and 13C NMR. The oxime-ligation products 3−6 can
exist as several ring−chain tautonomeric forms (Figure 2B), of
which the (E)-oxime and (Z)-oxime predominate over the α-
1
and β-pyranoses, with each giving rise to distinctive H NMR
and 13C NMR peaks for H-1 and C-1 of the conjugated
glycan.24,30 Importantly, the H NMR of the dextran linker 6
1
(Figure 2C) confirmed that the aminooxy linker 2, once
deprotected, was capable of reacting with the high MW dextran
oligosaccharide as shown by the presence of the (E)-oxime and
1
(Z)-oxime H-1 H NMR peaks. With the assembly of the four
carbohydrate−azide linkers complete, a maleimide functional
group was installed via a CuAAC reaction between linkers 3−6
and compound 7, which itself was synthesized by a short one-
step procedure (see the Supporting Information), giving the
desired carbohydrate maleimide linkers 8−11.
Preparation and Glycosylation of bHb. bHb is a readily
available type of Hb capable of being purified on a
multikilogram scale, making it an attractive precursor for the
synthesis/formulation of oxygen therapeutics.38,39 Like other
forms of Hb, bHb is a tetramer consisting of two identical α
chains and two identical β chains. More importantly for our
purposes, the two β chains both display one cysteine residue
(Cys-93), which has been previously shown to be capable of
modification with maleimide-modified compounds.18,40
Using a minimal excess of the synthesized glycan linkers 8−
11, bHb was successfully site-selectively glycosylated via a
maleimide conjugation reactionintroducing a novel chiral
center on the resultant succinimide thioethergiving the
desired maltotriose−bHb, maltopentaose−bHb, maltohep-
taose−bHb, and dextran−bHb glycoconjugates (Figure 3).
After purification, the glycosylated bHb samples were
submitted for analysis by MALDI, LC−MS, and tryptic digest
followed by LC−MS/MS. Initial analysis by MALDI for the
linker-treated bHb showed successful glycosylation of the β
subunit (Figure 4A). Glycosylation of the β subunit was evident
Synthesis of the Carbohydrate Linkers. Synthesis of the
carbohydrate linkers is depicted in Figure 2A and began with 2-
(2-[2-chloroethoxy]ethoxy)ethanol to which a protected
aminooxy functional group was installed via a Mitsunobu
reaction with N-hydroxyphthalimide, giving substrate 1.
Subsequent azide displacement of the chloro group gave linker
2, which was capable of being synthesized on a multigram scale.
7510
dx.doi.org/10.1021/ja300893t | J. Am. Chem. Soc. 2012, 134, 7507−7515

Journal of the American Chemical Society
Article
correlating to native bHb (Figure S1, Supporting Information),
maltotriose−bHb (Figure S2, Supporting Information), malto-
pentaose−bHb (Figure S3, Supporting Information), and
maltoheptaose−bHb (Figure S4, Supporting Information),
respectively. Additionally, trypsin digestion, followed by LC−
MS/MS analysis, was used to confirm that Cys-93 of the β
subunit was indeed the site of glycosylation (Tables S1−S3,
Supporting Information), with the results indicating that the
maleimide moiety was present in the open maleimic acid form
due to the partial hydrolysis of the succinimidyl thioether that
occurred during the procedurea side reaction that has been
reported in the literature.44
Evaluation of the Biophysical Properties of bHb. After
confirming that bHb was indeed glycosylated at the Cys-β93
position, we proceeded to study the effect of glycosylation on
the biophysical properties of bHb. In particular, we were
interested in examining the effect of glycosylation on the
equilibria between oxygen and bHb, the secondary structure
and heme environment of bHb, the kinetics of haptoglobin
(Hp) binding to bHb, and the 3-dimensional structure of bHb.
Oxygen−bHb/Glycosylated bHb Equilibria. Hb is primarily
responsible for gaseous ligand storage and transport in the
circulatory system and thus has received significant attention as
a precursor for the synthesis/formulation of oxygen therapeu-
tics, i.e., Hb-based oxygen carriers (HBOCs).15−17 Conse-
quently, in this study, the equilibria between oxygen and bHb/
glycosylated bHb was measured to investigate the effects of
glycosylation on the oxygen affinity (P50) and cooperativity
coefficients (n) of bHb. P50 is an indicator of oxygen affinity
Figure 4. (A) MALDI-ESI of bHb, maltotriose−bHb, maltopentaose−
bHb, maltoheptaose−bHb, and dextran−bHb. (B) SDS−PAGE of
bHb and dextran−bHb. (C) Western blot of bHb, dextran−bHb,
dextran, and dextran−bHb.
through the complete disappearance of the MW peak
corresponding to the native β subunit (15 955) and the
emergence of a new peak corresponding to that of the
carbohydrate-modified β subunit. For instance, when bHb was
treated with the maltotriose linker (MW 811), the detected
mass shifted from the original mass of 15 955 Da to 16 766 Da
(β subunit + linker). For bHb treated with the dextran linker,
the disappearance of the native β subunit MW was observed;
however, the presence of the dextran-modified β subunit could
not be detected by MALDI due to the interference of ionization
imposed by the large oligosaccharide dextran, a trait commonly
seen with proteins bearing large oligosaccharides.41 Thus, to
further analyze the dextran-modified Hb and confirm
glycosylation, SDS−PAGE and Western blot analysis, with an
antidextran antibody, were performed (parts B and C,
respectively, of Figure 4).42,43 Both the SDS−PAGE and
Western blot analysis showed a large shift in the MW of the β
subunit, with the Western blot staining positive for dextran in
the region corresponding to the modified β subunit. Further
analysis of the glycosylated bHb by LC−MS demonstrated that
the glycosylation reaction went to completion, leaving no
unmodified β-subunit, and showed an increase in MW
corresponding to the linker that the bHb was treated with, as
was observed by MALDI. Furthermore, completion of the
glycosylation reaction was evident in the RP-HPLC chromato-
grams of the LC−MS analysis, in which only two peaks were
present, corresponding to the α and modified β subunits.
Deconvolution of the ESI mass spectra for the peaks
corresponding to the modified β subunits yielded molecular
ions with masses of 15 955, 16 767, 17 091, and 17 415 Da,
and refers to the partial pressure of oxygen (pO ) at which half
2
the HBOC is saturated with oxygen. The cooperativity
coefficient (n) of Hb is associated with the allosteric transition
from the tense (T) to the relaxed (R) quaternary state upon
oxygenation.45 For native bHb, P50 was determined to be 27.61
mmHg, while the glycosylated Hb's had values ranging from 12
to 16 mmHg (Table 1). It is apparent from the oxygen
equilibrium curves (Figure 5A), decreased cooperativity
coefficients, and decreased P50 values (Table 1) that the
glycosylated Hb's possess a higher affinity for oxygen compared
to native Hb. The observed reduction in cooperativity upon
glycosylation of Cys-β93 indicates a reduction in the available
motion of the α1β2 interface in which the Cys-β93 is located.
Similar modifications of Cys-β93 by other chemical agents have
been shown to have an analogous effect by influencing the
heme pocket of the β chain.46−48
Interestingly, the low P50 of glycosylated Hb's may be
beneficial in any future development of glycosylated Hb's as
potential HBOCs. This lies in the fact that one of the few
HBOCs to reach phase III clinical trials, Hemospan (PEGylated
human Hb), has a low P50 value of 5−6 mmHg, whereas other
HBOCs that have failed in phase II clinical trials typically lie in
the range of 26−38 mmHg.49,50 It has been postulated that
lower P50 values may be beneficial by limiting arteriole oxygen
transport, thus targeting oxygen transport to tissues and organs
with low partial pressure and avoiding induced high blood
Table 1. Oxygen Affinity (P50) and Cooperativity Coeffient (n) of Native bHb and Glycosylated bHb's
bHb
maltotriose−bHb
maltopentaose−bHb
maltohaptaose−bHb
dextran−bHb
P50 (mmHg)
27.61 0.02
2.862 0.010
15.75 0.01
2.106 0.004
12.57 0.01
1.835 0.004
14.09 0.01
2.002 0.004
14.91 0.01
2.065 0.004
n
7511
dx.doi.org/10.1021/ja300893t | J. Am. Chem. Soc. 2012, 134, 7507−7515

Journal of the American Chemical Society
Article
Figure 5. (A) Oxygen equilibrium curves of bHb, maltotriose−bHb, maltopentaose−bHb, maltoheptaose−bHb, and dextran−bHb. CD spectra of
native and glycosylated Hb's in the (B) far-ultraviolet region and (C) near-ultraviolet region. (D) SPR analysis of native and glycosylated bHb's.
Table 2. Association/Dissociation Rate Constants and Equilibrium Dissociation Constant of Native and Glycosylated bHb's
bHb
maltotriose−bHb
maltopentaose−bHb
maltohaptaose−bHb
dextran−bHb
ka (M−1·s−1
kd (s−1
KD (M)
)
(1.30 0.01) × 105
(5.23 0.04) × 10−5
(4.02 0.03) × 10−10
(1.83 0.13) × 105
(10.00 0.05) × 10−5
(5.46 0.10) × 10−10
(1.49 0.07) × 105
(8.85 0.06) × 10−5
(5.94 0.07) × 10−10
(1.12 0.05) × 105
(6.45 0.04) × 10−5
(5.76 0.05) × 10−10
(1.00 0.04) × 105
(8.23 0.07) × 10−5
(8.23 0.06) × 10−10
)
pressure caused by an autoregulatory response to high arteriole
O2 levels, resulting in vasoconstriction.51
Overall, glycosylation does not appear to alter the secondary
structure and heme environment of bHb, as indicated by
examination of the CD spectra.
Circular Dichroism (CD) Analysis. To investigate the effects
of glycosylation on the structural characteristics of Hb and its
glycosylated variants, the CD spectra were obtained, in the far-
ultraviolet (200−250 nm) region (Figure 5B) and near-
ultraviolet (250−500 nm) region (Figure 5C). An initial
concern in performing CD spectroscopy was the potential of
the conjugated carbohydrates themselves to alter the obtained
spectra of the modified Hb; however, it is known that the
absorbance of unsubstituted polysaccharides does not lie above
wavelengths greater than 200 nm, and thus, the spectra
obtained were expected to yield accurate information about the
secondary structure and heme environment of glycosylated
Hb's.52 Comparison of the glycosylated and native Hb spectra
in the far-ultraviolet region reveals that glycosylation does not
adversely affect the structure of bHb as indicated by the
analogous spectra. In the near-ultraviolet spectra, the L band
(in the vicinity of 260 nm) provides information indicative of
the heme ligand, and examination of this region shows no
significant change in intensity upon glycosylation. Likewise,
investigation of the Soret region (around 420 nm), which
provides information regarding the interactions of the heme
prosthetic group with surrounding aromatic residues, shows
little deviation for the glycosylated Hb compared to native Hb.
Surface Plasmon Resonance (SPR) Analysis. Haptoglobin−
hemoglobin (Hp−Hb) complexation is an important biological
process in which Hp binds to free Hb in the blood to effectively
remove it from systemic circulation.53 Thus, SPR analysis was
used to investigate the effect of glycosylation at Cys-β93 on the
formation of Hp−Hb complexes. The binding−dissociation
curves (Figure 5D) indicate that glycosylation at Cys-β93 has
only a slight influence on the interaction between glycosylated
bHb and Hp. Compared to bHb with an association rate
constant (ka) of 1.30 × 105 M−1·s−1, maltotriose and
maltopentaose modification resulted in a slightly higher
association rate constant (1.83 × 105 and 1.49 × 105
M−1·s−1, respectively), while maltoheptaose and dextran
modification resulted in a lower association rate constant,
1.12 × 105 and 1.30 × 105 M−1·s−1, respectively (Table 2).
Furthermore, glycosylation increased the dissociation rate
constant (kd) from 5.23 × 10−5 s−1 for native bHb to a value
greater than 6.45 × 10−5 s−1 for all four forms of glycosylated
bHb (Table 2). Collectively, glycosylation increased the
equilibrium dissociation constant (KD) from 4.02 × 10−10
M
to a value above 5.46 × 10−10 M for the lower MW glycosylated
forms of bHb, with the dextran−bHb reaching as high as 8.23 ×
7512
dx.doi.org/10.1021/ja300893t | J. Am. Chem. Soc. 2012, 134, 7507−7515

Journal of the American Chemical Society
Article
Figure 6. (A) Superposition of bHb (gray), maltotriose−bHb (yellow), maltopentaose−bHb (green), and maltoheptaose−bHb (purple). The
overall tertiary and quaternary structures of all three glycosylated bHb's are quite similar to those of native bHb. Gray rectangles enclose two regions
exhibiting conformational change: the CE elbow on the α subunit (top left rectangle) and the EF elbow on the β subunit (bottom right rectangle).
(B) Variable deviation from native bHb in the α and β subunits of maltotriose−bHb (yellow) and maltoheptaose−bHb (purple). Five residues at the
N- and C-termini of each of the four subunits were deleted, and the Cα atoms of all of the remaining residues were used to generate an optimal
superposition. The peaks represent the separation of a Cα atom of each of the glycosylated Hb's from the corresponding Cα atom of native bHb. (C)
Overall preservation of the side chain conformations in the “EF” region of the β1 subunit. The side chains of bHb (gray), maltotriose−bHb (yellow),
maltopentaose−bHb (green), and maltoheptaose−bHb (purple) are depicted as thin wires. (D) Axial displacement of E and F helices of the β1
subunit upon glycosylation. Axes were computed through the geometric centroid for Cα atoms of three residues on the E helix and Cα atoms of
residues selected within the EF elbow, middle of the F helix, and the carboxy end of the F helix. The axes passing through the maltotriose−bHb
(yellow) and maltoheptaose−bHb (purple) are in a very similar position, especially on the F helix. This position is distinct from the axes passing
through the corresponding position of native bHb (gray).
10−10 M (Table 2). Consequently, it can be inferred that
glycosylation appears to slightly interfere with the formation of
Hp−Hb complexes, which may be expected considering the
glycans rest on the surface of Hb and likely interfere with Hp’s
recognition of Hb.
structures of maltotriose−bHb, maltopentose−bHb, and
maltoheptose−bHb were obtained and reveal that the
tetrameric state was preserved upon glycosylation. More
importantly, a superposition of only the α1β1 subunits of
tetrameric bHb and glycosylated bHb's shows that the
quaternary state of the glycosylated bHb's corresponds exactly
to the quaternary state of native bHb (data not shown). A
complete structural alignment of the 572 Cα atoms of the α2β2
subunits of bHb and the glycosylated bHb's shows that their
tertiary and quaternary features correspond well (Figure 6A).
The average root-mean-square deviations from bHb are 0.337
Å for maltotriose−bHb and 0.371 Å for maltoheptose−bHb.
These analyses strongly suggest that the overall tertiary and
quaternary features of bHb are preserved upon glycosylation.
The comparison between the structures of glycosylated
bHb's and the structure of native bHb reveals that the
deviations from the structure of native bHb are not uniformly
distributed throughout the bHb molecule, but instead localized
(Figure 6B). These deviations were found to be present mostly
in the β1 and β2 subunits. However, this was expected to a
X-ray Crystallography. Hb is a α2β2 tetramer having one
true 2-fold rotational symmetry axis and two 2-fold
pseudosymmetry axes. Functional properties of Hb, such as
its equilibrium oxygen binding characteristics, are regulated at
the structural level by the relative orientation and interaction of
its constituent subunits.54,55 For example, interactions at the
α1β1 (and the corresponding α2β2) intersubunit interface as
well as the interactions at the α2β1 (and the corresponding
α1β2) interface launch Hb into quaternary conformations that
have distinct oxygen affinities.56−59 Furthermore, in the absence
of certain salt bridges Hb’s “central cavity” assumes a more
relaxed, high oxygen affinity state referred to as the R state.55,60
Consequently, the quaternary structure of Hb directly reflects
its functional behavior, making it paramount to investigate any
significant changes upon glycosylation. Accordingly, the crystal
7513
dx.doi.org/10.1021/ja300893t | J. Am. Chem. Soc. 2012, 134, 7507−7515

Journal of the American Chemical Society
Article
Table 3. Dimensions of the Heme Pocket for Selected Hb's
bHb
maltotriose−bHb
maltoheptaose−bHb
α1
α2
β1
β2
α1
α2
β1
β2
α1
α2
β1
β2
heme pocket surface area (Å2)
heme pocket volume (Å3)
800
960
680
950
600
760
600
740
730
920
640
900
580
790
530
740
730
880
820
530
700
520
710
1010
certain degree, since it is the β Cys-93 residue located near the
carboxy edge of the F helix which is glycosylated. Although the
electron density for the glycan linker is not defined, the
position of structural deviations suggests that the glycan linker
interacts closely with the β subunit. The α2β1 interface is
defined by a close interaction between the C helix and the CE
elbow on the α1 subunit and the carboxy edge of the F helix
and the FG elbow on the β2 subunit (Figure 6A). Given the
minor structural deviations observed in C-helices on α subunits,
it is possible that the interaction influences the conformation of
the α2β1 interface. Inspection of the β subunit region of
glycosylated bHb's for structural deviation from the corre-
sponding region in native bHb reveals the region is comprised
mainly of the E helix, the EF elbow, the F helix, and the FG
elbow. Interestingly, the side chain conformations in this region
closely resemble each other (Figure 6C). This raises the
intriguing possibility of a global movement of the helices in the
region that accommodates glycosylation, which likely serves as
a buffer to restrict transmission of these changes to other
regions of the quaternary structure of the glycosylated bHb's.
The Cα atoms of three residues contained in the E helix of
the β1 subunit, namely, Pro-57, Lys-66, and Lys-76, were
selected for calculating an axis passing through the geometric
centroid of the atoms. This procedure was repeated for bHb,
maltotriose−bHb, and maltoheptaose−bHb. In a similar
fashion, axes were computed through the geometric centroid
of Cα atoms of three residues on the β1 subunit, namely, His-77
that is located at the EF elbow, Ala-87 that is located in the
middle of the F helix, and Asp-94 that is located at the carboxy
end of the F helix. These two sets of axes revealed some
interesting features (Figure 6D) that may have profound
implications. First, the axes drawn through the β1 subunits of
maltotriose−bHb and maltoheptaose−bHb are in almost
identical positions. Second, the axes computed for the β1
subunit of native bHb are in a position that is close to, yet
distinctly apart from, the corresponding axes in maltotriose−
bHb and maltoheptaose−bHb. Third, these analyses reveal the
structure contained within the EF helices undergoes a “global”
movement farther away from the α2β1 interface. Finally, the β1
superposition in the region of the FG elbow and amino edge of
the G helix as well as the β1 superposition in the region of the
D helix and the DE elbow indicates that these conformational
changes are not transmitted further. Conformational changes in
the F helix of β subunits, especially near its carboxy edge, are
deemed important in determining the oxygen affinity of Hb.61
Thus, upon oxygen binding, this region is displaced farther
from the α2β1 interface, which in turn “relaxes” Hb and
increases its oxygen affinity. The fact that glycosylation at Cys-
β93 also results in a small displacement farther from the α2β1
interface rationalizes the observation that glycosylated Hb's
have a higher oxygen affinity than native Hb. A measurement of
the surface area and volume of the heme pocket of bHb,
maltotriose−bHb, and maltopentose−bHb further reveals these
values are quite similar across all three structures (Table 3),
suggesting the conformational changes upon glycosylation are
accommodated and compensated for in such a manner that
allows the quaternary structure to be preserved.
CONCLUSIONS
■
In this work, we have presented a novel method allowing for
the facile site-selective glycosylation of proteins with carbohy-
drates of variable MWs. To the best of our knowledge, this is
the first reported methodology for the site-selective glyco-
sylation of proteins with oligosaccharides of such a high MW.
To demonstrate the feasibility of the reported methodology,
Hb was site-selectively glycosylated with four glycans of
increasing MW, up to 10 000. Although the present study
was limited to oligosaccharides with a maximum MW of 10 000,
it is envisioneddue to the aqueous nature of the reactions
employed in both the synthesis of the carbohydrate linkers and
the bioconjugation reactionthat larger saccharides could be
utilized. Furthermore, upon examination of the biophysical
properties of the glycosylated Hb, it was discovered that
glycosylation of Hb lowered P50 to levels similar to that of the
commercially developed oxygen therapeutic Hemospan, now in
phase III clinical trials. It has been postulated that a low P50
value may be critical to the development of HBOCs because it
facilitates oxygen transport to tissues with low levels of oxygen
and decreases arteriole oxygen transport, potentially eliminating
undesired cardiovascular effects such as induced high blood
pressure.51 Consequently, the presented methodology may be
of significant importance for the future development of
glycosylated Hb's as potential HBOCs. Specifically, the
dextran-conjugated bHb has a significant potential to serve as
a “red blood cell substitute”, for through site-specific
glycosylation it combines Hb, an oxygen carrier, and dextran,
a plasma expander.
Moreover, the method developed has the ability to be
applied toward the site-specific glycosylation of other
therapeutic proteins with high MW oligosaccharides/poly-
saccharides, thus serving as a viable alternative to site-specific
PEGylation. Glycans carry with them a vast array of unique
biological properties spread out over a wide range of MWs;
consequently, the method reported herein, which easily allows
for high MW glycan conjugation to a desired therapeutic
protein, could be of significant usefulness. Furthermore, glycans
are quite biodegradable in comparison to PEG, making their
use advantageous when administration must occur over long
periods of time or in large doses, thus preventing long-term
accumulation. Future potential applications range from the
conjugation of high MW polysacchardies to carrier proteins
used in vaccine conjugationa task that has been daunting in
the pastto the conjugation of large oligosaccharides/
polysaccharides to therapeutic proteins as a means to extend
the circulation half-life and thus their pharmacological
effectiveness.
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures, compound characterization, biophys-
ical characterization methods, and data and crystallographic
■
S
7514
dx.doi.org/10.1021/ja300893t | J. Am. Chem. Soc. 2012, 134, 7507−7515

Journal of the American Chemical Society
Article
(29) Dirksen, A.; Hackeng, T. M.; Dawson, P. E. Angew. Chem., Int.
Ed. 2006, 45, 7581.
information files. This material is available free of charge via the
Internet at http://pubs.acs.org
́
(30) Thygesen, M. B.; Munch, H.; Sauer, J. r.; Clo, E.; Jørgensen, M.
R.; Hindsgaul, O.; Jensen, K. J. J. Org. Chem. 2010, 75, 1752.
(31) Novikov, B. N.; Grimsley, J. K.; Kern, R. J.; Wild, J. R.; Wales,
M. E. J. Controlled Release 2010, 146, 318.
AUTHOR INFORMATION
■
Corresponding Author
palmer.351@osu.edu; pwang11@gsu.edu
(32) Clay, J. G.; Zierold, D.; Grayson, K.; Battistella, F. D. J. Surg. Res.
2009, 155, 89.
(33) Moriyama, K.; Yui, N. J. Controlled Release 1996, 42, 237.
(34) Ferreira, L.; Rafael, A.; Lamghari, M.; Barbosa, M. A.; Gil, M. H.;
Cabrita, A. M. S.; Dordick, J. S. J. Biomed. Mater. Res., Part A 2004,
68A, 584.
(35) Sun, G.; Shen, Y.-I.; Kusuma, S.; Fox-Talbot, K.; Steenbergen,
C. J.; Gerecht, S. Biomaterials 2010, 32, 95.
(36) Finch, P.; Merchant, Z. J. Chem. Soc., Perkins Trans. 1 1975,
1682.
(37) Integrations for the (E)-oxime and (Z)-oxime were based on
previous reports which showed a ratio of approximately 0.6:0.20
following 2D NMR experiments. See refs 21 and 33.
(38) Palmer, A. F.; Sun, G.; Harris, D. R. Biotechnol. Prog. 2009, 25,
189.
(39) Sun, G.; Palmer, A. F. J. Chromatogr., B: Anal. Technol. Biomed.
Life Sci 2008, 867, 1.
(40) Manjula, B. N.; Tsai, A.; Upadhya, R.; Perumalsamy, K.; Smith,
P. K.; Malavalli, A.; Vandegriff, K.; Winslow, R. M.; Intaglietta, M.;
Prabhakaran, M.; Friedman, J. M.; Acharya, A. S. Bioconjugate Chem.
2003, 14, 464.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by National Institutes of Health
Grants R01HL078840 and R01DK070862 to A.F.P. This work
was also supported by the CounterACT program, NIH Office
of the Director, and NINDS with grant U54 NS058183 to
P.G.W.
REFERENCES
■
(1) Fishburn, C. S. J. Pharm. Sci. 2008, 97, 4167.
(2) Harris, J. M.; Chess, R. B. Nat. Rev. Drug Discovery 2003, 2, 214.
(3) Veronese, F. M.; Pasut, G. Drug Discovery Today 2005, 10, 1451.
(4) Otsuka, H.; Nagasaki, Y.; Kataoka, K. Adv. Drug Delivery Rev.
2003, 55, 403.
(5) Gaberc-Porekar, V.; Zore, I.; Podobnik, B.; Menart, V. Curr. Opin.
Drug Discovery Dev. 2008, 11, 242.
(6) Veronese, F. M. Biomaterials 2001, 22, 405.
(7) Pisal, D. S.; Kosloski, M. P.; Balu-Iyer, S. V. J. Pharm. Sci. 2010,
99, 2557.
(41) Harvey, D. J. J. Chromatogr., A 1996, 720, 429.
(42) The broad nature of the band in Figure 4c arose primarily due
to a combination of (1) the “smearing” effect seen when
polysaccharides are analyzed via SDS−polyacrylamide gel electro-
phoresis and (2) the heterogeneity of the dextran used. See ref 43 for a
description of the smearing effect seen when utilizing SDS−
polyacrylamide gel electrophoresis for the analysis of protein
glycosylation.
(8) Russell, D.; Oldham, N. J.; Davis, B. G. Carbohydr. Res. 2009,
344, 1508.
(9) Sato, M.; Sadamoto, R.; Niikura, K.; Monde, K.; Kondo, H.;
Nishimura, S.-I. Angew. Chem., Int. Ed. 2004, 43, 1516.
(10) Dwek, R. A. Chem. Rev. (Washington, DC, U.S.) 1996, 96, 683.
(11) Sola, R. J.; Griebenow, K. BioDrugs 2010, 24, 9.
(12) Sola, R. J.; Rodriguez-Martinez, J. A.; Griebenow, K. Cell. Mol.
Life Sci. 2007, 64, 2133.
(13) Davis, B. G. Chem. Rev. (Washington, DC, U.S.) 2002, 102, 579.
(14) Gamblin, D. P.; Scanlan, E. M.; Davis, B. G. Chem. Rev.
(Washington, DC, U. S.) 2009, 109, 131.
(15) Riess, J. G. Chem. Rev. (Washington, DC, U.S.) 2001, 101, 2797.
(16) Buehler, P. W.; D’Agnillo, F.; Schaer, D. J. Trends Mol. Med.
2010, 16, 447.
(17) Awasthi, V. Curr. Drug Delivery 2005, 2, 133.
(18) Zhang, Y.; Bhatt, V. S.; Sun, G.; Wang, P. G.; Palmer, A. F.
Bioconjugate Chem. 2008, 19, 2221.
(43) Jacobs, P. P.; Geysens, S.; Vervecken, W.; Contreras, R.;
Callewaert, N. Nat. Protoc. 2009, 4, 58.
(44) Kalia, J.; Raines, R. T. Bioorg. Med. Chem. Lett. 2007, 17, 6286.
(45) Vasquez, G. B.; Karavitis, M.; Ji, X.; Pechik, I.; Brinigar, W. S.;
Gilliland, G. L.; Fronticelli, C. Biophys. J. 1999, 76, 88.
(46) Manjula, B. N.; Tsai, A. G.; Intaglietta, M.; Tsai, C. H.; Ho, C.;
Smith, P. K.; Perumalsamy, K.; Kanika, N. D.; Friedman, J. M.;
Acharya, S. A. Protein J. 2005, 24, 133.
(47) Khan, I.; Dantsker, D.; Samuni, U.; Friedman, A. J.;
Bonaventura, C.; Manjula, B.; Acharya, S. A.; Friedman, J. M.
Biochemistry 2001, 40, 7581.
(48) Juszczak, L. J.; Manjula, B.; Bonaventura, C.; Acharya, S. A.;
Friedman, J. M. Biochemistry 2002, 41, 376.
(49) Vandegriff, K. D.; Malavalli, A.; Wooldridge, J.; Lohman, J.;
Winslow, R. M. Transfusion 2003, 43, 509.
(50) Natanson, C.; Kern, S. J.; Lurie, P.; Banks, S. M.; Wolfe, S. M.
JAMA, J. Am. Med. Assoc. 2008, 299, 2304.
(51) Rohlfs, R. J.; Bruner, E.; Chiu, A.; Gonzales, A.; Gonzales, M. L.;
Magde, D.; Magde, M. D., Jr.; Vandegriff, K. D.; Winslow, R. M. J. Biol.
Chem. 1998, 273, 12128.
(52) Parra, A.; Stevens, E. S. Carbohydr. Polym. 2000, 41, 111.
(53) Wintrobe, M. M.; Lee, G. R. Wintrobe’s Clinical Hematology,
10th ed.; Williams & Wilkins: Baltimore, MD, 1999.
(54) Perutz, M. F. Annu. Rev. Biochem. 1979, 48, 327.
(55) Perutz, M. F.; Fermi, G.; Poyart, C.; Pagnier, J.; Kister, J. J. Mol.
Biol. 1993, 233, 536.
(56) Tsai, C. H.; Ho, C. Biophys. Chem. 2002, 98, 15.
(57) Baldwin, J.; Chothia, C. J. Mol. Biol. 1979, 129, 175.
(58) Zhang, J.; Hua, Z. Q.; Tame, J. R. H.; Lu, G. Y.; Zhang, R. J.;
Gu, X. C. J. Mol. Biol. 1996, 255, 484.
(59) Mueser, T. C.; Rogers, P. H.; Arnone, A. Biochemistry 2000, 39,
15353.
(19) Liu, Y.; Feizi, T.; Carnpanero-Rhodes, M. A.; Childs, R. A.;
Zhang, Y. N.; Muiioy, B.; Evans, P. G.; Osborn, H. M. I.; Otto, D.;
Crocker, P. R.; Chai, W. C. Chem. Biol. 2007, 14, 847.
(20) Forget, D.; Renaudet, O.; Boturyn, D.; Defrancq, E.; Dumy, P.
Tetrahedron Lett. 2001, 42, 9171.
(21) Forget, D.; Boturyn, D.; Defrancq, E.; Lhomme, J.; Dumy, P.
Chem.Eur. J. 2001, 7, 3976.
(22) Liu, Y.; Feizi, T.; Campanero-Rhodes, M. A.; Childs, R. A.;
Zhang, Y.; Mulloy, B.; Evans, P. G.; Osborn, H. M.; Otto, D.; Crocker,
P. R.; Chai, W. Chem. Biol. 2007, 14, 847.
(23) Lee, M. R.; Shin, I. Org. Lett. 2005, 7, 4269.
(24) Thygesen, M. B.; Sauer, J.; Jensen, K. J. Chem.Eur. J. 2009, 15,
1649.
(25) Thygesen, M. B.; Sorensen, K. K.; Clo, E.; Jensen, K. J. Chem.
Commun. (Cambridge, U.K.) 2009, 6367.
(26) Pauly, M.; York, W. S.; Guillen, R.; Albersheim, P.; Darvill, A. G.
Carbohydr. Res. 1996, 282, 1.
(27) Ramsay, S. L.; Freeman, C.; Grace, P. B.; Redmond, J. W.;
MacLeod, J. K. Carbohydr. Res. 2001, 333, 59.
(28) Namavari, M.; Cheng, Z.; Zhang, R.; De, A.; Levi, J.; Hoerner, J.
K.; Yaghoubi, S. S.; Syud, F. A.; Gambhir, S. S. Bioconjugage Chem.
2009, 20, 432.
(60) Shaanan, B. J. Mol. Biol. 1983, 171, 31.
(61) Baldwin, J.; Chothia, C. J. Mol. Biol. 1979, 129, 175.
7515
dx.doi.org/10.1021/ja300893t | J. Am. Chem. Soc. 2012, 134, 7507−7515