Enhanced Binding and Reduced Immunogenicity of Glycoconjugates Prepared via Solid-State Photoactivation of Aliphatic Diazirine Carbohydrates

Article abstract of DOI:10.1021/acs.bioconjchem.0c00555

Biological conjugation is an important tool employed for many basic research and clinical applications. While useful, common methods of biological conjugation suffer from a variety of limitations, such as (a) requiring the presence of specific surface-exposed residues, such as lysines or cysteines, (b) reducing protein activity, and/or (c) reducing protein stability and solubility. Use of photoreactive moieties including diazirines, azides, and benzophenones provide an alternative, mild approach to conjugation. Upon irradiation with UV and visible light, these functionalities generate highly reactive carbenes, nitrenes, and radical intermediates. Many of these will couple to proteins in a non-amino-acid-specific manner. The main hurdle for photoactivated biological conjugation is very low yield. In this study, we developed a solid-state method to increase conjugation efficiency of diazirine-containing carbohydrates to proteins. Using this methodology, we produced multivalent carbohydrate-protein conjugates with unaltered protein charge and secondary structure. Compared to carbohydrate conjugates prepared with amide linkages to lysine residues using standard NHS conjugation, the photoreactive prepared conjugates displayed up to 100-fold improved binding to lectins and diminished immunogenicity in mice. These results indicate that photoreactive bioconjugation could be especially useful for in vivo applications, such as lectin targeting, where high binding affinity and low immunogenicity are desired.

Full text of DOI:10.1021/acs.bioconjchem.0c00555

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Article
Enhanced Binding and Reduced Immunogenicity of
Glycoconjugates Prepared via Solid-State Photoactivation of
Aliphatic Diazirine Carbohydrates
Molly D. Congdon and Jeffrey C. Gildersleeve*
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ABSTRACT: Biological conjugation is an important tool employed for many basic research and clinical applications. While useful,
common methods of biological conjugation suffer from a variety of limitations, such as (a) requiring the presence of specific surface-
exposed residues, such as lysines or cysteines, (b) reducing protein activity, and/or (c) reducing protein stability and solubility. Use
of photoreactive moieties including diazirines, azides, and benzophenones provide an alternative, mild approach to conjugation.
Upon irradiation with UV and visible light, these functionalities generate highly reactive carbenes, nitrenes, and radical intermediates.
Many of these will couple to proteins in a non-amino-acid-specific manner. The main hurdle for photoactivated biological
conjugation is very low yield. In this study, we developed a solid-state method to increase conjugation efficiency of diazirine-
containing carbohydrates to proteins. Using this methodology, we produced multivalent carbohydrate−protein conjugates with
unaltered protein charge and secondary structure. Compared to carbohydrate conjugates prepared with amide linkages to lysine
residues using standard NHS conjugation, the photoreactive prepared conjugates displayed up to 100-fold improved binding to
lectins and diminished immunogenicity in mice. These results indicate that photoreactive bioconjugation could be especially useful
for in vivo applications, such as lectin targeting, where high binding affinity and low immunogenicity are desired.
The preparation of biological conjugates is challenging for
several reasons. First, most biomolecules require aqueous
media, mild temperatures, and a narrow pH range, which
significantly limits the spectrum of reaction conditions that are
acceptable.1,14,15 Additionally, it is often difficult to obtain large
amounts of biomolecules, so reactions are typically conducted
at lower molar concentrations and/or on small scales.
Furthermore, they must be compatible with the many
functional groups found on proteins and other biomolecules
as well as a high molar excess of water.
INTRODUCTION
■
Biological conjugation, the process of covalently linking a
molecule of interest and a biomolecule, is a vital tool employed
in many basic and clinical applications.1−3 Bioconjugation
reactions are frequently used to attach a wide range of
molecules, such as affinity tags and fluorophores, to proteins,
DNA, and other biopolymers. In the field of glycoscience,
bioconjugations are used to produce multivalent glycoconju-
gates as probes/inhibitors of endogenous lectins, immunogens
for inducing antibody responses, and as reagents for various
assays.4−10 Bioconjugates are also valuable agents for a variety
of clinical applications.11,12 Some prime examples are anti-
body−drug conjugates such as Gemtuzumab ozogamicin
(acute myelogenous leukemia), Trastuzumab emtansine
(HER-2 positive metastatic breast cancer), and Sacituzumab
govitecan (triple-negative breast cancer). Additionally, carbo-
hydrate−protein conjugate vaccines such as Prevnar (Strepto-
coccus pneumoniae) as well as Menactra and Menveo (Neisseria
meningitides) have been administered to millions of people and
have significantly improved human health.13
Bioconjugation reactions are especially challenging when
producing conjugates with higher valency, i.e., the number of
units linked to the biomolecule. High valency can be critical for
Received: October 6, 2020
Revised: November 25, 2020
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bioactivity. For example, carbohydrate−protein conjugates
with multivalent displays are more immunogenic6,16−18 and
display higher avidity to lectins due to the ability to
simultaneously engage multiple binding sites on a protein.8,19
To achieve high valency with a residue specific conjugation
method, however, the target protein must have a sufficient
number of reacting residues on its surface. In addition, driving
a conjugation to favor a higher valency can result in protein
destabilization and denaturation and can increase the like-
lihood that one will modify residues in a protein active sites, or
generate conjugates that possess diminished activities.20,21
Additionally, increased valency can substantially alter the
protein’s isoelectric point (pI) and net charge. For example,
coupling molecules with carboxylic acids to the side chain
amines of lysine residues produces an amide bond and a charge
change at the conjugation site. As multiple sites are modified,
the net charge of a protein can be substantially perturbed
leading to destabilization, reduced activity, and increased
nonspecific adsorption/adherence.
Despite these challenges, many effective methods have been
developed for bioconjugations.1,10,15,22−24 Some commonly
employed reactions include reductive amination, amide
formation, Michael addition with maleimide-modified bio-
molecules, use of isothiocyanates, and azide−alkyne click
chemistry.1,14,25 While powerful, there are some important
limitations for these methods. The majority of the reactions
require surface-exposed lysine and/or cysteine residues as
nucleophilic sites.14,15,22,25 Some target proteins lack the
necessary residue(s), while others have a competing
nucleophile in a critical location, such as the active site of an
enzyme. Modification can be deleterious to biophysical
features of a protein/biomolecule too, such as solubility, net
charge, stability, and overall structure.
Photochemical methodologies offer a complementary
approach. They are used extensively for photoinduced cross-
linking and photoaffinity labeling, which have become valuable
tools to identify molecular binding sites, protein binding
partners, and identify protein−protein interactions within
heterogeneous systems.26−29 With this approach, light is used
to convert photoreactive functional moieties such as azides,
benzophenones, and diazirines, into highly reactive intermedi-
ates (e.g., carbenes, nitrenes, or radicals) that can covalently
link with the biological target through N−H, O−H, and C−H
insertions or cycloaddition mechanisms.26,30−35 Some photo-
phores have high residue specificity, such as benzophenone,
which displays a strong preference for methionine residues,36
while others are not amino-acid-specific. The incorporation of
a nonspecific photophore into a molecule of interest can
theoretically modify a protein at any surface-exposed area
without modifying the protein charge. As a result, this
approach is not dependent on any specific residue and can
theoretically modify a protein at any surface-exposed area
without modifying the protein charge. Additionally, photo-
reactions are typically compatible with common buffers near
physiological pH and are often completed within minutes to a
few hours.34,37,38 While offering several beneficial features, one
serious disadvantage of photoconjugation is extremely low
yield. This problem severely limits the use of photoactivation
for producing bioconjugates, especially in applications that
require high valency conjugates such as carbohydrate−protein
conjugates. We note that elegant approaches have been
developed for carbohydrate-based photoaffinity labeling33,38−42
and attachment of glycans to surfaces,33,43−46 but this approach
is not typically used to prepare glycoconjugates.
In this study, we developed a solid-state approach to
improve the efficiency of photoactivated conjugation of
aliphatic diazirine-linked carbohydrates to proteins and then
used it to prepare a set of glycoconjugates. This solid-state
methodology significantly improves efficiency compared to
standard solution-state protocols. The high-valency conjugates
prepared with the solid-state photoreactive conditions
displayed little or no alteration to protein structure, protein
charge, and pI. When the carbohydrate conjugates were
employed in biological assays, they exhibited up to 100-fold
increased binding by mammalian and plant lectins and
displayed reduced immunogenicity in mice.
RESULTS AND DISCUSSION
■
Our goal was to compare the biophysical properties and
biological activities of glycoconjugates prepared via photo-
conjugation to analogous glycoconjugates prepared by tradi-
tional amide bond formation. The first phase of the project
involved the design and synthesis of carbohydrate derivatives,
and optimization of the photoconjugation reaction, while the
second phase focused on the evaluation of the binding
properties and immunogenicity of the glycoconjugates.
Design and Synthesis of Conjugates. To test the effects
of photoreactive conjugation, we designed a matched pair of
haptens (compounds 8 and 9), each incorporating a single
GalNAc residue attached to a phenol. These structures mimic
a newly discovered form of glycosylation wherein GalNAc is
attached to the side chain oxygen of tyrosine residues of
proteins.47,48 GalNAc-tyrosine has been found on a variety of
proteins, is known to have altered expression in Alzheimer’s
patients, and has been reported to interact with lectins of the
immune system (i.e., the macrophage galactose type lectin;
MGL).49 While potentially very important, little is known
about its biosynthesis, expression, or biological effects.47,48,50
Access to defined glycopeptides and structurally related
haptens could be useful for studying this form of glycosylation.
For the reactive functional groups and coupling method-
ology, we compared the photoactivation of a diazirine to an
amide coupling with a carboxylic acid. Aliphatic diazirines have
been used extensively for photoaffinity labeling and have been
shown to interact with a wide range of amino acids including
Ala, Arg, Asp, Cys, His, Leu, Lys, Gln, Glu, Met, and Tyr
residues.35 This broad reactivity should assist in the generation
of multivalent conjugates. In addition, diazirines are small,
provide minimal perturbation of the glycan/hapten structure,
and are likely to have low immunogenicity, as opposed to
larger photoreactive groups like benzophenones. For photo-
reactive hapten 8, a secondary diazirine was used for synthetic
ease. The diazirine moiety was incorporated three carbons
away from the phenyl ring on a butyl chain to retain structural
similarity to the control hapten 9, such that the overall length
of the GalNAc haptens would be identical after conjugation.
For control hapten 9, we employed a 3-(4-hydroxyphenyl)-
propanoic acid linker that we have previously used to generate
a GalNAc-Tyr selective monoclonal antibody (manuscript in
preparation). Conjugates prepared with hapten 8 are
designated GalNAc-α/β-DZ-valency#-protein, while conju-
gates prepared with hapten 9 are designated GalNAc-α/β-
PA-valency#-protein. DZ indicates conjugation via photo-
reactive diazirine and PA indicates a phenyl amide linkage to
the protein.
B
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a
Scheme 1. Synthesis of Aliphatic Diazirine and GalNAc-Diazirine Compounds
a
(a) 7N NH3 in MeOH, 0 °C, 3 h; (b) Hydroxylamine-O-sulfonic acid, rt, overnight; (c) TEA, MeOH, 0 °C, 5 min, then I2 (55%); (d) Imidazole-
1-sulfonyl azide hydrochloride, K2CO3, CuSO4·5H2O, MeOH, rt, 2 h; (e) Ac2O, pyridine, rt, 16 h, 75%; (f) BiBr3, TMSiBr, DCM, rt, overnight
84%; (g) 2, DTBMP, AgOTf, 4 Å MS, DCM:Tol. (1.5, 1 v:v) −78 °C − rt, overnight, 86%, (1:1 α:β); (h) AcSH, pyridine, (1:1 v:v). 0 °C − rt,
overnight, 90% (1:1 α:β); (i) 0.5 MeOMe, MeOH, rt, 1 h, 97% (1:1 α:β).
a
Scheme 2. General Protein Conjugation Reactions of Haptens
a
(A) GalNAc-DZ-protein conjugation conditions and (B) GalNAc-PA-protein conjugation conditions.
For this study, both the α- and β-anomers of aliphatic
diazirine 8 were prepared as shown in Scheme 1. In short, the
phenol diazirine (2) was prepared using standard literature
conditions for installing the diazirine functionality.51 Azido-
bromide 5 was prepared from D-galactosamine hydrochloric
acid (3) using standard conditions.52 Glycosylation of 5 with 2
gave the desired protected sugar in good yield as a 1:1 mixture
of α:β-anomers.49 Conversion of the azide to the acetylated
amine was achieved with thioacetic acid and pyridine to
produce 7 in good yield.53,54 Finally, selective deacetylation
with 0.5 M sodium methoxide in methanol produced the
desired GalNAc derivative (8) in excellent yield.53 The α- and
β-anomers of 8 (8a and 8b, respectively) were separated and
purified by preparative HPLC.
Optimization of Photoreactive Conjugation Condi-
tions. One of the largest hurdles for photoactivated reactions
is efficiency. With the desired aliphatic diazirine containing
GalNAc haptens in hand, we attempted to prepare the
photoreactive protein conjugates (Scheme 2A). To determine
how much optimization of the reaction conditions were
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a
Table 1. Select Photoreactive Conjugation Optimization Conditions
entry
protein
buffer
pH
equiv 8
state
365 nm irradiation time (min.)
ave. number of 8 added
1
2
3
4
5
6
7
8
BSA
BSA
BSA
BSA
BSA
BSA
BSA
BSA
BSA
BSA
BSA
HSA
HSA
HSA
1× PBS
1× PBS
1× PBS
1× PBS
1× PBS
Borate
7.4
7.4
7.4
5.9
8.3
8.2
9.2
10.2
8.0
9.1
10.1
7.4
8
40
80
80
80
80
80
80
80
80
80
80
80
80
80
solution
solution
solid
solid
solid
solid
solid
solid
solid
solid
solid
solid
solid
10
40
40
40
40
40
40
40
40
40
40
40
40
40
1.0
1.3
7.9
4.1
5.4
9.7
8.9
8.7
9.6
8.5
9.5
9.0
3.5
11.3
Borate
Borate
9
Na2CO3
Na2CO3
Na2CO3
1× PBS
Borate
10
11
12
13
14
Na2CO3
9.1
solid
a
All reactions were protected from light exposure and incubated for 30 min at room temperature before irradiation or snap freezing and
lyophilization.
required, our initial conjugation attempts employed commonly
used diazirine photoaffinity labeling conditions with 40 equiv
of GalNAc−hapten per mole of protein.35,38,41,51 Upon
conjugation, the loading levels of the resulting glycoconjugates
were determined using MALDI-TOF analysis. Successful
conjugation of the aliphatic diazirine hapten was also verified
by ELISA with Helix pomatia Agglutinin (HPA) and Vicia
Villosa (VVL) lectins which recognize the GalNAc residue.
Unfortunately, the standard photoreaction conditions yielded
very low loading levels of only ∼1 unit per molecule of BSA
(Table 1, entry 1). Doubling the number of equivalents to 80
provided very little improvement (∼1.3 units per molecule of
BSA, entry 2). To increase the yield, numerous parameters
were varied, such as buffer, temperature, and irradiation time,
but no improvement was observed. Based on these results, we
concluded that it would be extremely difficult or impossible to
prepare a multivalent conjugate using this approach.
Carbenes and other highly reactive intermediates are rapidly
quenched by reaction with solvent, intramolecular rearrange-
ments, and intermolecular reactions. We hypothesized that one
could minimize these side reactions by removing the water and
conducting the reaction in the solid state. In principle, the
carbene would be generated in close proximity to the protein,
improving the probability that it would react with the protein
and decreasing the time that it has to undergo intramolecular
rearrangements. In addition, only minimal solvent would be
present, minimizing side reactions with water. To test this
hypothesis, the reactants were combined, snap frozen, and then
lyophilized. Irradiation of the resulting powder displayed a 6-
fold increase in loading efficiency (Table 1, entry 3). Thus, the
solid-state approach provided significant improvements over
the solution-phase reaction.
these buffers, we could obtain 8−10 units per molecule of BSA
using 80 equiv of diazirine-linked carbohydrate (Table 1,
entries 6−11). In addition to improved efficiency, sodium
carbonate also provided a consistent yield of ∼10% over a wide
range of equivalents (SI Table S2). At the highest ratio tested,
120 equiv, we obtained ∼14 units/BSA. Based on these results,
we concluded that the optimal conditions used sodium
carbonate between pH 8−10 for the initial reaction mixture
followed by solid-state photoconjugation at room temperature.
Having successfully identified conditions that could produce
a multivalent carbohydrate−protein conjugate with a valency
of at least 10, we next examined the generality of the method
by coupling to other proteins (Table 1, entries 12−14). Using
sodium carbonate buffer and the solid-state approach with 80
equiv of diazirine-linked GalNAc, we were able to successfully
couple to HSA (pH 9.1; 11.3 units/BSA) and CRM197 (pH
8.0; 17.5 units/CRM197, SI Table S3). These conditions were
then employed to prepare the various HSA and BSA
glycoconjugates used in the mice immunizations and biological
assays, including a GalNAc-α-DZ-10-HSA conjugate (average
of 10 units/HSA) for immunological studies and high and low
valency BSA conjugates for use in characterization studies and
biological assays.
The control GalNAc-phenyl amide linked glycoconjugates
were prepared via NHS−ester coupling (Scheme 2B) of
carboxylic acids 9a or 9b with BSA or HSA. For
immunological evaluation, a GalNAc-α-PA-10-HSA conjugate
was prepared. High and low valency BSA conjugates were
prepared for characterization studies and use in biological
assays.
Characterization of the Conjugates. To verify that the
photoconjugation approach was not disrupting the protein
structure or causing degradation, we evaluated the biophysical
properties of the conjugates using several methods. Both native
and SDS-PAGE gels revealed that there was no noticeable
degradation of BSA as a result of the solid-state conjugation
conditions (SI Figures S2−S3) or CRM197 (SI Figure S4).
The conjugates were also examined by circular dichroism
(CD) to evaluate potential effects on overall structure (SI
Figure S5). CD spectra of both the low and high loaded
diazirine and NHS-ester prepared conjugates retain the
indicative “W-shape” due to the hydrogen bonding α-helix
structure of BSA.
To further improve the yield, we evaluated a variety of
parameters using the solid-state approach. The effects of PBS
buffer concentration (0.1−10× PBS at pH 7.4), incubation
temperature, and incubation time were found to have minimal
effects on the efficiency of the reaction (SI Table S1).
Moreover, increasing or decreasing the pH in PBS led to lower
yields (Table 1, entries 4 and 5). Fortunately, improvements
could be obtained by altering the buffer. While ammonium
carbonate provided yields similar to PBS (SI Table S1), the use
of sodium borate or sodium carbonate buffers for the initial
reaction mixture both provided improved efficiency. With
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Figure 1. Lectin binding affinity to conjugates. (A) ASGPR, (B) HPA, (C) MGL, (D) VVL. Samples: Blue Triangles − GalNAc-α-DZ-7-BSA,
Green Diamonds − GalNAc-α-PA-7-BSA, Black Circles − BSA. Plots display the mean with SD of triplicate runs for each lectin.
Based on differences in the chemistry of the two conjugation
methods, we anticipated there would be differential effects on
overall charge and pI. The carbene generated from the
diazirine would likely insert into a wide variety of bonds on the
protein surface. As such, it is unlikely to significantly alter the
overall charge or pI of the carrier protein. In contrast, amide
formation converts a positively charged lysine side chain into a
neutral functional group; therefore, we expected substantial
changes to protein charge and pI. High levels of charge on a
protein can have significant effects on a variety of biophysical
properties, including nonspecific adsorption due to modified
electrostatic interactions.55 A change of as few as 3 charge
units can alter the physiochemical properties of the peptide
polymer chains and increase aggregation.56,57 Studies have
shown that more extensive modification of protein charge can
significantly alter other properties, such as resistance to
proteolysis and cell permeability.58
The effects of the solid-state and NHS-ester conjugation
methods on overall conjugate charge and size were examined
by native gel and isolelectric focusing (IEF; SI Figure S2A−C),
as well as size exclusion chromatography (SEC, SI Figure S5).
We observed a noticeable charge decrease with the GalNAc-β-
PA-11-BSA conjugate, verifying the neutralization caused by
formation of amide bonds. The size of the charge decrease was
correlated with valency and, for high loaded conjugates, was
easily distinguishable compared to the BSA standard. In
contrast, the diazirine GalNAc-β-DZ-12-BSA conjugate (SI
Figure S2A/B, lane 4) had a similar charge to BSA, and both
high and low valency conjugates had the same mobility on the
gel. These results were recapitulated in the IEF gel (SI Figure
S2C). The diazirine-prepared GalNAc-β-DZ-12-BSA conjugate
displays a pI of ∼4.7 (SI Figure S2C, lane 4) which is similar to
that of BSA (pI ∼4.7). As expected, the isoelectric points of
GalNAc-β-PA-11-BSA (pI ∼4.3, SI Figure S2C lane 2)
displayed a greater shift as a result of amide bond formation
and reduction of charged lysine residues. Despite the observed
shifts in pI as a result of the conjugation methods, GalNAc-β-
DZ-7-BSA and GalNAc-β-PA-11-BSA conjugates had similar
profiles via SEC (SI Figure S6), indicating no significant
differences in aggregation state.
Binding Affinity Evaluation of Conjugates. The most
important factor when considering bioconjugation methods is
bioactivity. To evaluate the effects of our solid-state
conjugation methodology on binding affinity, we evaluated
binding of a variety of terminal GalNAc-recognizing plant
(HPA and VVL) and mammalian [Asialoglycoprotein
Receptor (ASGPR) and MGL] lectins using ELISA. ASGPR
and MGL are C-type binding lectins found on liver
hepatocytes and dendritic cells, respectively. The ability to
selectively target these lectins could be beneficial avenues for
site-specific drug delivery and activating immune re-
sponse.59−61 To make comparisons between the diazirine
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and amide-linked conjugates, we measured binding at a series
of concentrations and then determined the apparent binding
constants (app KD).
In most cases, the photoconjugation method provided
significant benefits for binding over the amide-coupled
glycoconjugate (Figure 1A−D and Table 2). The biggest
negatively charged protein at physiological pH, with an
estimated net charge of −18 at pH 7.4. While the net charge
does not change for the diazirine conjugate, the estimated net
charge for the GalNAc-α-PA-7-BSA conjugate at pH 7.4
decreases to −25. This difference could manifest itself in
several ways. First, it could influence the total number of
glycoconjugate molecules that will stick to the surface of the
ELISA plate. The surface may be able to accommodate more
molecules of diazirine than amide conjugate due to less
charge−charge repulsion. Second, the difference in net charge
could influence overall binding via interactions between
protein surfaces. Three of the four lectins we studied are
also negatively charged at pH 7.4, with MGL at −10, VVL at
−10, and ASGPR at −13.6; HPA is close to neutral with a net
charge of +1.6. This effect could involve localized charge−
charge interactions near the binding pockets or secondary
interactions that influence the total number of lectins and
molecules that can be in close proximity to each other. In
addition to charge, the spacing and orientation of glycans on
the carrier protein could also be affected. Additional studies
will be needed to more fully understand these effects.
Immunological Evaluation of Conjugates. Immunoge-
nicity can be beneficial for the development of vaccines and
antibodies, but it can be detrimental in other cases such as
targeting endogenous lectins. To evaluate the effects of the
conjugation method on immunogenicity, HSA conjugates were
prepared with 8a and 9a (conjugates GalNAc-α-DZ-10-HSA
and GalNAc-α-PA-10-HSA, respectively). Two groups of 5
BALB/c mice were immunized with either GalNAc-α-DZ-10-
HSA or GalNAc-α-PA-10-HSA. Sera were collected from each
mouse pre-immunization and on days 24, 38, and 52 after
Table 2. Apparent KD Values of Select Lectins to Conjugates
lectin apparent KD
protein
HPA
N.D.
VVL
N.D.
MGL
N.D.
ASGPR
467 nM
BSA
GalNAc-α-phenylDZ-7
GalNAc-α-phenylamide-7 1.6 nM
0.93 nM 0.0067 nM 32.3 nM 585 nM
0.81 nM 74.1 nM 304 nM
effects were observed for VVL, which displayed 100-fold
stronger binding to the diazirine conjugate (apparent KD of
0.0067 nM for GalNAc-α-DZ-7-BSA and 0.81 nM for GalNAc-
α-PA-7-BSA). HPA and MGL also had improved binding to
the diazirine conjugate, although it was a smaller effect. In
addition to affinity, all 4 lectins displayed at least some increase
in total binding to the diazirine conjugate, with the largest
effects on the maximum signal observed for HPA and ASGPR.
Thus, in addition to affinity, the method of conjugation also
affects the maximum number of lectin molecules that will bind
to the glycoconjugate-modified surface. This improvement
could be beneficial for the development of diagnostics where
increased signal-to-noise is needed.
Although we do not yet know the mechanistic basis for the
improved binding to the diazirine conjugate, differences in
electrostatic interactions could be a key contributor. BSA is a
Figure 2. IgM and IgG titers for conjugates. (A) IgM titers, (B) IgG titers. Samples: green striped bars − GalNAc-α-PA-10-HSA final bleed sera
(day 52), dark blue bars − GalNAc-α-DZ-10-HSA final bleed sera (day 52). Boxes display the mean titer values (horizontal line) of each group of 5
mice
SD. The whiskers correspond to 2SD. IgM and IgG titers on day 52 were performed in triplicate for each mouse. P-values were
determined using the Mann−Whitney Test. *(p < 0.05) shows a statistically significant difference in IgG titers to the GalNAc-α-PA-7-BSA
conjugate between the serum antibodies on day 52 from the two groups of mice.
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Figure 3. IgG selectivity to GalNAc-α and GalNAc-β and GalNAc-a-S/T/Y peptides. Final bleed sera GalNAc-α-PA-10-HSA mice − dark green
striped bars; Final bleed sera GalNAc-α-DZ-10-HSA mice − dark blue bars. The final bleed (day 52) IgG selectivity for each mouse was evaluated
on the glycan microarray once at a 1:400 serum dilution. Plots display the mean with SD based on RFU of the 5 mice in each group. P-values
were determined using the Mann−Whitney Test. **(p < 0.01) and *(p < 0.05) show statistically significant differences in IgG response of day 52
serum antibodies between the two groups of mice to various array components..
immunization and subsequent boosters as shown in SI Figure
S7. Once the study was completed, serum antibody titers, as
well as serum antibody specificity, were evaluated.
cytokine response.62 Glycans linked to lysine residues may
provide a higher proportion of suitable glycopeptides relative
to a more random conjugation strategy.
Preimmunization, first draw (day 24), and post-immuniza-
tion (day 52) sera IgM and IgG titers from each mouse were
evaluated against the protein carrier (HSA), GalNAc-α-DZ-7-
BSA, GalNAc-α-PA-7-BSA, and BSA (Figure 2A,B, SI Figures
S8−S9). IgM responses were only evaluated in sera
preimmunization and on day 52, the final day of the study.
As expected, all mice possessed minimal IgM responses to the
various components before immunization. IgM responses
increased by the end of the immunization schedule but were
substantially lower than IgG responses in both groups of mice.
This response was anticipated due to general duration of IgM
immune response and class switching during the course of the
study. After 24 days, all mice developed IgG responses to both
GalNAc conjugates and proteins, and the titers increased
substantially after 52 days. Interestingly, the amide-linked
conjugates produced higher titers than the diazirine at both
time points (SI Figure S9). On day 24, the mice immunized
with GalNAc-α-PA-10-HSA possessed 2.4-fold and 2.8-fold
stronger responses for the GalNAc-α-DZ-7-BSA and GalNAc-
α-PA-7-BSAconjugates, respectively. On day 52, the IgG
responses from the mice immunized with GalNAc-α-PA-10-
HSA were 2.6-fold and 6.7-fold stronger for the GalNAc-α-DZ-
7-BSA and GalNAc-α-PA-7-BSA conjugates, respectively. IgG
titers for the carrier protein, HSA, were not significantly
different indicating that the effects discussed above were due to
the hapten/linker/conjugation method and not an unexpected
difference in immune capacity for the groups of mice. The
mechanistic basis for the difference in immunogenicity is not
yet clear. Prior studies have shown that certain glycopeptide
fragments can be presented on major histocompatibility class II
(MHCII), initiating a carbohydrate specific CD4+ T cell and
In addition to appraising the immunological strength of the
diazirene and NHS-ester conjugates, we evaluated the
selectivity and breadth of the IgM and IgG serum antibodies
pre-immunization and on day 52 using a 740-component
carbohydrate microarray (Supporting Information). Our
glycan microarray contains a diverse assortment of N-linked
and O-linked glycans, glycolipid glycans, glycopeptides, and
glycoproteins.7,63,64 In particular, it contained 154 components
with a terminal GalNAc residue.
Both haptens induced antibody responses with narrow
selectivity (Figure 3). As anticipated, both groups of mice did
not possess substantial IgM or IgG antibodies to GalNAc-α
and GalNAc-β-glycoconjugates or GalNAc-glycopeptides
before immunization with the glycoconjugates (SI Figures
S10−S11, SI Table S4). After 52 days, both groups displayed
robust IgG responses that were highly selective for GalNAc-α-
Tyr glycoconjugates. Modest IgM responses to GalNAc-α-
glycoconjugates and GalNAc-α-Ser/Thr/Tyr peptides were
also observed. Aside from the antibodies that are naturally
present in a mouse,18,65 little or no signal was observed for any
other glycans on the array, including GalNAc-α-Ser/Thr
glycopeptides, blood group A [GalNAcα1−3(Fucα1−2)Gal],
GalNAcα1−6Gal, and GalNAcα1−3Gal. Within the subset of
GalNAc-Tyr related glycoconjugates and glycopeptides,
induced antibodies displayed selectivity for α- over β-linked
GalNAc for both conjugates. Depending on the array
component, IgG antibodies were 3−15 times more selective
for GalNAc-α than the corresponding GalNAc-β construct.
The IgG response to the diazirine conjugated immunogen was
more selective for the hapten and GalNAc-Tyr peptides than
the response to the amide-linked immunogen or GalNAc
G
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Article
Author Contributions
residues linked through a non-phenyl-containing mercaptoe-
thylaminoglutarate linkage (GalNAc-α-22, GalNAc-β-21) to
the protein carrier.
All authors contributed to the writing of the manuscript and
have given approval of the final version. All experimental work
was carried out by MDC.
CONCLUSIONS
Funding
■
This work was supported by the intramural research program
of the National Institutes of Health.
Notes
Photoconjugation reactions offer numerous potential advan-
tages for the preparation of bioconjugates. The main
disadvantage has been extremely low yields, making it nearly
impossible to produce conjugates with high valency. By
devising a solid-state approach, we were able to reduce side
reactions and significantly improve the yield. While additional
improvements in efficiency would be beneficial, a 10−12%
yield is sufficient to prepare multivalent conjugates with a
valency of 10 units/protein while also using practical amounts
of carbohydrate hapten (80 equiv). The approach is
compatible with different proteins as well as multiple buffers
and pHs.
The conjugates produced via the photoreaction have several
beneficial features. First, they have minimal alteration to
protein charge, which may help reduce nonspecific adsorption
to surfaces, improve protein stability and solubility, and
minimize changes to other biophysical properties of the
protein. Second, the glycoconjugates exhibit up to 100-fold
enhanced binding to plant and mammalian lectins when
compared to a similar GalNAc-containing glycoconjugates
constructed with amide linkages to lysine residues. Third, the
photoreactive glycoconjugates were significantly less immuno-
genic in mice as compared to the corresponding amide-linked
glycoconjugate. The unique combination of high affinity and
low immunogenicity make glycoconjugates prepared via
photoconjugation especially useful for in vivo targeting of
endogenous lectins. They may also be useful as capture ligands
for various in vitro assays, such as glycan microarrays.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Consortium for Functional Glycomics
(GM62116; The Scripps Research Institute), X. Huang
(Michigan State University), T. Tolbert (University of
Kansas), Lai-Xi Wang (University of Maryland), J. Barchi
(National Cancer Institute), T. Lowary (University of
Alberta), Omicron Biochemicals Inc., GlycoHub, and Glycan
Therapeutics and for generously contributing glycans for the
array. This work was supported by the Intramural Research
Program of the National Cancer Institute, NIH.
ABBREVIATIONS
■
GalNAc, N-Acetylgalactosamine; ASGPR, Asialoglycoprotein
Receptor; HPA, Helix pomatia Agglutinin; MGL, Macrophage
Galactose Lectin; VVL, Vicia Villosa Lectin
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* Supporting Information
The Supporting Information is available free of charge at
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Corresponding Author
Jeffrey C. Gildersleeve − Chemical Biology Laboratory,
National Cancer Institute, National Institutes of Health,
Frederick, Maryland 21702, United States; orcid.org/
0000-0002-3744-6423; Phone: (301) 846-5699;
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Molly D. Congdon − Chemical Biology Laboratory, National
Cancer Institute, National Institutes of Health, Frederick,
Maryland 21702, United States; orcid.org/0000-0002-
9096-1344
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