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nprot.2017.109

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protocol
Preparation of glycoconjugates from unprotected
carbohydrates for protein-binding studies
Christian T Hjuler1,2, Nicolai N Maolanon1,2, Jørgen Sauer1,2, Jens Stougaard3,4 , Mikkel B Thygesen1,2 &
Knud J Jensen1,2 1Centre for Carbohydrate Recognition and Signaling, Copenhagen University, Frederiksberg, Denmark. 2Department of
Chemistry, University of Copenhagen,
Frederiksberg, Denmark. 3Centre for Carbohydrate Recognition and Signaling, Aarhus University, Aarhus, Denmark. 4Department of Molecular Biology and Genetics,
Aarhus University, Aarhus, Denmark. Correspondence should be addressed to M.B.T. (mbth@chem.ku.dk) or K.J.J. (kjj@chem.ku.dk).
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Published online 26 October 2017; doi:10.1038/nprot.2017.109
Glycobiology, in particular the study of carbohydrate–protein interactions and the events that follow, has become an important
research focus in recent decades. To study these interactions, many assays require homogeneous glycoconjugates in suitable
amounts. Their synthesis is one of the methodological challenges of glycobiology. Here, we describe a versatile, three-stage protocol
for the formation of glycoconjugates from unprotected carbohydrates, including those purified from natural sources, as exemplified
here by rhizobial Nod factors and exopolysaccharide fragments. The first stage is to add an oligo(ethylene glycol) linker (OEGlinker) that has a terminal triphenylmethanethiol group to the reducing end of the oligosaccharide by oxime formation catalyzed by
aniline. The triphenylmethyl (trityl) tag is then removed from the linker to expose a thiol (stage 2) to allow a conjugation reaction
at the thiol group (stage 3). There are many possible conjugation reactions, depending on the desired application. Examples shown
in this protocol are as follows: (i) coupling of the oligosaccharide to a support for surface plasmon resonance (SPR) studies, (ii)
fluorescence labeling for microscale thermophoresis (MST) or bioimaging, and (iii) biotinylation for biolayer interferometry (BLI)
studies. This protocol starts from unprotected carbohydrates and provides glycoconjugates in milligram amounts in just 2 d.
INTRODUCTION
The study of carbohydrate–protein interactions has become
an important research focus in recent decades1. Proteins that
bind carbohydrates are ubiquitous and include lectins, glycosidases, toxins, cancer-related antibodies, carbohydrate receptors
in plants, viral proteins that bind to glycans, and many more.
Glycoconjugates, which in addition to the carbohydrate (glycan)
contain a functional moiety, are very important tools for studying
interactions with proteins. Glycoconjugates can contain a fluorophore for bioimaging and MST, or a selectively reactive group
that allows anchoring to surfaces, which enables studies of carbohydrate–protein interactions by SPR or BLI. Surface-anchored
glycoconjugates can be used to obtain binding affinities or to
elucidate the effects of multivalency, whereas fluorescently tagged
glycoconjugates can be used to localize carbohydrate receptors.
It is essential that the carbohydrates be displayed in a functional
manner, for example, the reducing-end monosaccharide unit
must remain in a cyclic form. Furthermore, it is very attractive to
directly convert unmodified, native carbohydrates into glycoconjugates without the requirement for excessive protecting-group
chemistry. This protocol describes the easy functionalization of
carbohydrates for the efficient preparation of homogeneous glycoconjugates in milligram amounts for studying carbohydrate–
protein interactions.
Our research groups are interested in studying legume–rhizobium
symbiosis, in particular to identify carbohydrate receptors that regulate this process. In legume–rhizobium symbiosis, various rhizobial carbohydrates have pivotal roles as signal molecules2. These
include lipochitin oligosaccharides, such as nodulation (Nod) factors3, and other complex carbohydrates, such as rhizobial exopolysaccharide4 fragments (EPSs), which we have reported on in recent
years by exploiting chemoselective reagents5,6.
In our recent publication on the regulatory role of EPSs7, several
lines of evidence suggest a signaling role for EPSs. Through generation of transgenic rhizobia and mutant-variant plant lines, it
was shown that the transmembrane receptor kinase EPR3 binds to
EPSs directly, and disruption of this perception causes a suppression of infection-thread development, which in turn inhibits the
formation of infected root nodules. Direct carbohydrate–protein
interaction between EPSs and EPR3, as well as kinetics measurements, was ultimately determined using BLI8.
We have also previously reported on the chemical modification of Nod factors3,9,10, which are signal molecules responsible
for initiating the development of symbiotic nodules on the roots
of many legume plants. These were applied for SPR studies and
fluorescence labeling for use in MST3. We showed that Nod factors
from Mesorhizobium loti bound Lotus japonicus receptors NFR1
and NFR5 with high affinity and specificity3.
The chemical modifications and labeling of carbohydrate
ligands are described in greater detail for more general applicability. The protocol presented includes two common steps to
provide a glycoconjugate with a free thiol (−SH) group (Fig. 1).
There are many possible reactions that could be performed at the
free −SH group; we show three options in Figure 1, and detailed
steps for these are described in the PROCEDURE (Step 17).
Using a combination of vendor-purchased chemicals and a synthesized OEG-linker (Box 1), this protocol enables the modification of a wide array of different carbohydrates. The use of highly
chemoselective reactions makes it possible to use small quantities of starting carbohydrate and obtain high conversion rates.
Furthermore, the chemical steps are conveniently performed
in aqueous solutions. Conjugation at the reducing end of the
carbohydrate means the conjugates will retain the ring-closed
form in equilibrium with the open-chain configurations. This
protocol relies on the aniline-catalyzed 6 reaction between the
reducing-end aldehyde of an unprotected carbohydrate with a
reagent that carries an amino-oxy moiety in one end. The resulting glycoconjugate carries an oxime linkage. This reaction and
the reagents developed for this purpose have proven very useful
nature protocols | VOL.12 NO.11 | 2017 | 2411
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protocol
O
O
O
OH
Carbohydrate of interest
O
O
O
NH
O
Linker
S-Trt
O
O
O
NH
O
Linker
NH
O
Linker
HN
O
NH
O
Linker
S-Trt
O
O
SO3
O
NH
O
Cl
Linker
SO3
NH
COOH
Cl
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Free thiol for
surface plasmon resonance
SH
OH
N
Biotin label for
biolayer interferometry
or surface plasmon resonance
NH
S
Cl
Fluorophore label for
bioimaging
or microscale thermophoresis
Figure 1 | Overview of the general procedure. A carbohydrate of interest is reacted with the oligo(ethylene glycol) linker (OEG-linker) to form an oxime linkage
at the reducing end. The resulting conjugate exists in equilibrium between the open- and closed-ring forms (lower left). In the second stage, a free thiol
(yellow circle, right side) is liberated and this product enables the covalent attachment of a fluorophore label (lower right) or a biotin moiety (upper right).
Key examples that show the application of the method are listed next to each compound. However, these compounds could potentially also find many other
applications. Trt, triphenylmethyl.
in our laboratory for the robust assembly of complex glycoconjugates under mild conditions.
For an overview of alternative reactions that can be used to
transform unprotected carbohydrates into glycoconjugates, the
reader is directed to our very recent comprehensive review 11.
These alternative reactions differ widely with respect to reaction
conditions, generality, and the accessible conjugates. Table 1 lists
the advantages and limitations of some key methods. Notably,
Shoda and coworkers have developed electrophilic reagents to
react with the 1-OH group of unprotected carbohydrates12.
Derivatives of Shoda’s reagent have been used to introduce an
azide group at the reducing end, which can then be used for couplings in subsequent steps13.
Protocol overview
This protocol describes the two-step chemoselective modification of unprotected carbohydrates with the bifunctional OEGlinker (Box 1; the analytical data can be found in Supplementary
Data 1–3).
The OEG-linker has a triphenylmethanethiol group on one end,
and each linker will react with only one oligosaccharide. This
group was chosen, in part, because the triphenylmethyl (trityl,
Trt) protecting group can be easily removed to expose a thiol
group. The trityl group has additional beneficial properties; its
hydrophobicity helps retain the glycoconjugate on reversed-phase
HPLC columns for analysis and purification, and its UV activity makes it detectable using a conventional HPLC instrument.
After the trityl group has been removed, the compound can be
conjugated in a variety of ways. In the PROCEDURE, we describe
how the unprotected thiol of the carbohydrate-linker conjugate
can be conjugated to SPR chip surfaces (Step 15, option A) using
a thiol-binding kit, modified with Alexa Fluor 546 C5 maleimide
(Step 15, option B) for MST and bioimaging studies, or modified
with a biotin-iodoacetamide reagent (Step 15, option C) for BLI
studies. The PROCEDURE shows only the chemical modification
steps. Brief descriptions of the biochemical assays are given as
Supplementary Methods.
Within our group, we have used this methodology to
modify many different carbohydrate ligands, such as lipochitin oligosaccharides3,6,10, chito-oligosaccharides3,10, glycans5,7,
and glycosidase inhibitors14. By far, most carbohydrates carry
an aldehyde functionality at the reducing end and exist in
an equilibrium between open- and closed forms, and the
first reaction (oxime formation) with the bifunctional OEGlinker uses the aldehyde moiety of the open-chain form.
After formation of the oxime, the carbohydrate is still in an
equilibrium between the open-chain and closed-ring forms5.
Here, the different reactions are demonstrated through the
chemical modification of two different carbohydrates: Nod factors (Step 15, options A and B) and exopolysaccharide fragments
(Step 15, option C).
This protocol does not cover how to obtain the purified carbohydrates or the proteins used in these studies. For this, we refer
to the experimental section and supplementary information of
our previous work3,7.
Experimental design
The protocol can be used for carbohydrates with one reducing
end. This protocol offers examples for two very different carbohydrates. The hydrophilic EPS is very soluble in water, but it
is insoluble in some organic solvents. By contrast, the Nod factors are amphiphilic molecules that require the presence of both
water and an organic solvent. In our experience, a mixture of
acetonitrile and water (1:1) is a suitable solvent for a wide range
of reactions. However, should solubility problems arise, the ratio
of these two solvents can be changed to help solubilize a difficult
molecule, and the reaction will still occur. If the ratio between
the solvents is changed markedly, longer reaction times may be
needed to compensate for this.
The reactions described in this protocol are performed on a
milligram scale, starting at ~1 mg. The reaction can be scaled up
to a 20-mg scale without further modification of the protocol,
and it can also be scaled down to ~0.3 mg. It must be noted, however, that the efficiency of the HPLC purification processes will
2412 | VOL.12 NO.11 | 2017 | nature protocols
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protocol
Box 1 | Synthesis of bifunctional OEG-linker ● TIMING 3 d
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
This box describes the synthesis of the OEG-linker.
Additional Materials
Acetonitrile (Sigma-Aldrich, cat. no. 34967)
Argon (acquired from Air Liquid) atmosphere is applied above the reaction mixtures by fitting an argon-filled balloon, by a syringe and
a needle, to a rubber septum on the reaction flask.
Celite 545 (Sigma-Aldrich, CAS. no. 68855-54-9)
Dichloromethane (Sigma-Aldrich, cat. no. 439223)
Diethyl ether (Sigma-Aldrich, cat. no. 309966)
Diisopropylazodicarboxylate (Sigma-Aldrich, cat. no. 225541)
Ethyl acetate (Sigma-Aldrich, cat. no. 34972)
Heptane (Sigma-Aldrich, cat. no. 34873)
Hydrazine monohydrate (Sigma-Aldrich, cat. no. 207942). ! CAUTION Hydrazine hydrate is toxic and carcinogenic.
N-Hydroxyphthalimide (Sigma-Aldrich, cat. no H53704)
Silica gel, 60 0.035–0.070 mm (Sigma-Aldrich, cat. no. 60738)
Silica gel, 60 0.015–0.040 mm (Merck-Millipore, cat. no. 115111)
Tetra(ethylene glycol) (Sigma-Aldrich, cat. no. 110175)
Tetrahydrofuran (Sigma-Aldrich, cat. no. 109-99-9), dried over 3-Å molecular sieves (Sigma-Aldrich, cat. no. 69839)
Synthesis of N-(2-{2-[2-(2-Hydroxyethoxy)ethoxy]ethoxy}ethoxy)-phthalimide (1)
1. Dissolve tetra(ethylene glycol) (0.97 g, 5.0 mmol) in dry tetrahydrofuran (80 ml) in a dry 250- ml round-bottom flask. Equip the
flask with a rubber septum. Evacuate the flask using a pump and apply argon atmosphere using a balloon.
2. Add solid-polymer-supported triphenylphosphine (4.69 g, 7.5 mmol and 1.6 mmol/g) followed by N-hydroxyphthalimide (0.82 g, 5.0 mmol).
3. Under magnetic stirring, add a solution of diisopropylazodicarboxylate (1.52 g, 7.5 mmol) in dry tetrahydrofuran (20 ml) over
30 min using a syringe dropwise. Stir the mixture for 4 h, and then leave the reaction mixture unstirred for 16 h at room temperature.
4. Filter the reaction mixture through a pad of celite (H×D=2 × 5 cm) to remove the polymer-supported reagent, wash the reagent with
additional tetrahydrofuran, and concentrate the filtrate to dryness using a rotary evaporator (30 °C bath temperature, ~50-mbar pressure).
5. Purify the intermediate on silica gel by dissolving the residue in ethyl acetate (10 ml) and filtering it through silica gel 60, particle
size 0.035–0.070 mm (H×D=8 × 4 cm). Wash the column extensively with ethyl acetate (~1.5–2 liter in 100-ml fractions; the desired
intermediate elutes after ~500 ml). Make sure that the silica does not dry out during the filtration. Combine the fractions containing
the product (check by TLC using ethyl acetate as the eluent, Rf=0.15), and evaporate the solvent to dryness on a rotary evaporator
(30 °C bath temperature, ~50-mbar pressure).
 PAUSE POINT Intermediate 1 can be stored at −18 °C for at least 6 months.
Synthesis of N-(2-{2-[2-(2-Tritylsulfanylethoxy)ethoxy]ethoxy}ethoxy)phthalimide (2)
1. Suspend the polymer-supported triphenylphosphine (1.88 g, 3.0 mmol, and 1.6 mmol/g) in dry tetrahydrofuran (25 ml) at 0 °C in a
dry 100-ml round-bottom flask. Equip the flask with a rubber septum. Evacuate the flask using a pump and apply an argon atmosphere
using a balloon.
2. Add diisopropylazodicarboxylate (0.607 g, 3.0 mmol) to the mixture using a syringe, and stir the mixture using a magnetic stirrer for
30 min at 0 °C. Add more tetrahydrofuran if the suspension solidifies.
3. Under magnetic stirring, add a solution of intermediate 1 (0.509 g, 1.5 mmol) and triphenylmethanethiol (0.829 g, 3.0 mmol) in dry
tetrahydrofuran (20 ml) at 0 °C dropwise using a syringe.
4. Allow the suspension to reach room temperature, and stir the suspension for an additional 4 h at room temperature.
5. Filter the reaction mixture through a pad of celite (H×D=2 × 5 cm) to remove the polymer-supported reagent, wash the column
with additional tetrahydrofuran, and concentrate the filtrate to dryness using a rotary evaporator (30 °C bath temperature,
~50-mbar pressure).
6. Purify the residue by vacuum liquid chromatography (VLC on silica gel 60, particle size 0.015–0.030 mm, H×D=7 × 3 cm) using
diethyl ether–heptane 50:50 as the starting eluent, taking 15-ml fractions. Increase the diethyl ether content stepwise by 5% for
each fraction until its content reaches 100% (10 steps). Combine the fractions containing the product (check by TLC using ethyl
acetate–heptane 1:1 as the eluent, Rf=0.50) and evaporate the solvent to dryness on a rotary evaporator (30 °C bath temperature,
~50-mbar pressure).
 PAUSE POINT Intermediate 2 can be stored at −18 °C for at least 6 months.
Synthesis of O-(2-{2-[2-(2-Tritylsulfanylethoxy)ethoxy]ethoxy{ethyl)hydroxylamine (3)
1. Dissolve intermediate 2 (0.598 g, 1.0 mmol) in acetonitrile (20 ml) in a 50-ml round-bottom flask.
2. Add hydrazine hydrate (230 µl, 5.0 mmol) using a syringe, and stir the mixture vigorously for 2 h at room temperature using a
magnetic stirrer. ! CAUTION Hydrazine hydrate is toxic and carcinogenic and should be handled in a fume hood.
nature protocols | VOL.12 NO.11 | 2017 | 2413
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protocol
3. Concentrate the resulting white suspension to dryness by rotary evaporation (30 °C bath temperature, ~50-mbar pressure).
! CAUTION Hydrazine hydrate is toxic and carcinogenic and should be handled in a fume hood.
4. Suspend the residue in dichloromethane (20 ml), and filter the suspension through a pad of celite (H×D=2 × 5 cm). Wash the column
with additional dichloromethane.
5. Evaporate the filtrate to dryness using a rotary evaporator (30 °C bath temperature, ~50-mbar pressure). Dry the product using an
oil pump for 24–48 h.
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
 PAUSE POINT The product can be stored at −18 °C for at least 6 months. The oil solidifies upon storage.
Anticipated Results:
A total of 0.454 g (97% yield) of the product, bifunctional OEG-linker, can be isolated as a clear oil. The identity and purity can be
confirmed by NMR and HPLC-MS, respectively (Fig. 5).
Analytical data for compound 3 are given as Supplementary Data 3.
A total of 0.622 g (37% yield) of the desired monosubstituted intermediate 1 can be isolated as a clear oil. The identity and purity can
be confirmed by NMR and HPLC-MS, respectively (Fig. 5).
Analytical data for compound 1 are given as Supplementary Data 1.
A total of 0.699 g (78% yield) of intermediate 2 can be isolated as a clear oil. The identity and purity can be confirmed by NMR and
HPLC-MS, respectively (Fig. 5).
Analytical data for compound 2 are given as Supplementary Data 2.
typically decrease at a lower scale. Table 2 describes the expected
conversion yields for the chemical reactions before HPLC purification. In our experience, the overall yields are expected to be
~25%, including final purification.
The second step, in which the trityl moiety is removed using
trifluoroacetic acid (TFA) under anhydrous conditions, does not
give rise to acidic hydrolysis of other functional groups. A range
of acid-labile functional groups have been evaluated using this
protocol, including O-acetates, O-carboxamides and O-sulfates,
as well as highly acid-labile glycosidic linkages, including glucuronides and riburonides. In no case did we observe any hydrolysis
or degradation using this protocol, as determined by HPLC-MS.
Furthermore, potential thiol oxidation to the disulfide or other
byproducts has generally not occurred with this protocol. This is
probably due to the acidic conditions for unmasking of the thiol.
Handling of conjugates containing free thiol groups under neutral
or basic conditions before the reaction with electrophiles should
be minimized to avoid thiol oxidation. Free-thiol concentrations
can be determined using the Ellman reagent. Peracetylation of
the carbohydrates or glycoconjugates is not necessary with this
protocol. The purified glycoconjugates show excellent stability in
aqueous solution at pH >5.
Table 1 | Advantages and limitations of various methods for glycoconjugate formation.
Method
Reaction steps
Advantages
Limitations
O-Glycosylation
(i) Peracetylation, (ii) O-glycosylation, Native O-glycoside is formed
and (iii) deacetylation
Anhydrous and harsh
conditions. Low conversion yields for oligosaccharides
—
Reductive amination
(i) Reductive amination and (ii) acylation
Simple, many amines available
Permanent open-chain
structure formed at the
reducing end. Side reactions possible
19
C-Glycosylation
(i) Knoevenagel condensation and (ii)
hydrazone formation
Stable anomeric linkage
Harsh conditions, not
compatible with some
carbohydrates and substitutions. Hydrazone is
prone to hydrolysis
20
Azide–alkyne
cycloaddition
(i) 1-Azidation and (ii) azide–alkyne
cycloaddition
Often highly efficient
Requires suitably functionalized alkyne and
bulky triazole substituent
at the reducing end
This protocol
(i) Oxime formation, (ii) trityl deprotection, and (iii) thiol reaction
Simple, versatile, and highly
efficient
Prone to hydrolysis at
low pH
2414 | VOL.12 NO.11 | 2017 | nature protocols
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Reference
12,13
3,5–7,14
protocol
Table 2 | Expected conversion yields for the different transformations, before HPLC purification after the final step.
Reaction with OEG-linker
Trityl-group removal
Reaction with free thiol
Nod factor
70–80%
95–100%
90–100%
EPS
40–60%
90–100%
80–100%
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
MATERIALS
REAGENTS
! CAUTION Most reagents used in this protocol are toxic and/or harmful
upon inhalation, ingestion, or skin contact. Laboratory coat, gloves, and
safety goggles should be worn, and all synthetic operations should be performed in a chemical fume hood.
• Acetic acid (Sigma-Aldrich, cat. no. 695092)
• Acetonitrile (Sigma-Aldrich, cat. no. 34967)
• Aniline (Sigma-Aldrich, cat. no. 242284)
• Biotin polyethylene oxide iodoacetamide (Sigma-Aldrich, cat. no. B2059)
• Borax (Sigma-Aldrich, cat. no. B3545)
• Diethyl ether (Sigma-Aldrich, cat. no. 309966)
• Ellman’s reagent (optional; Thermo Fisher, cat. no. 22582)
• Formic acid (Sigma-Aldrich, cat. no. 94318)
• Heptane (Sigma-Aldrich, cat. no. 34873)
• Hydrochloric acid (Sigma-Aldrich, cat. no. 320331)
• Ultrapure H2O (from Evoqua Ultra Clear UV system; see Equipment)
• NaOAc (Sigma-Aldrich, cat. no. S2889)
• NaOH (Sigma-Aldrich, cat. no. S8045)
• Nitrogen (Alphagaz 1; Air Liquide)
• Pierce Biotin Quantitation Kit (Thermo Fisher, cat. no. 28005)
• Polymer-supported triphenylphosphine (Sigma-Aldrich, cat. no. 93094)
• Purified exopolysaccharide (EPS) octasaccharide
• Sodium phosphate dibasic heptahydrate (Sigma-Aldrich, cat. no. S9390)
• Sodium phosphate monobasic monohydrate (Sigma-Aldrich, cat. no. S9638)
• Thiol-coupling kit (GE Healthcare, cat. no. BR100557)
• Triethylsilane (Sigma-Aldrich, cat. no. T1375)
• Trifluoroacetic acid (TFA; Sigma-Aldrich, cat. no. 302031) ! CAUTION TFA
is highly corrosive and causes burns.
EQUIPMENT
• Analytical HPLC column (Proto 300 Å, C4, 5 µm, 150 × 4.6 mm; Biotage)
• Analytical HPLC-MS (optional; Dionex, model no. UltiMate
3000, including Thermo Fisher Scientific MSQ Plus Mass Spectrometer)
• Biacore T100 system (GE Healthcare) or similar
• CM5 sensor chips (GE Healthcare, cat. no. BR100012)
• Dip and Read Streptavidin Biosensors (Pall, cat. no.18-502)
• Evoqua Ultra Clear UV water purification system or similar
• Freeze drier (Telstar LyoQuest)
• HPLC vials (Mikrolab Aarhus, cat nos. ML33134 and ML33117L)
• Heat shaker (IKA, model no. KS-125 or similar)
• Vortex shaker (IKA, model no. Vortex Genius 3 or similar)
• Biolayer interferometer (Pall FortéBio Octet Red96)
• pH meter (Knick Portamess)
• Preparative HPLC column (Jupiter 300 Å, C4, 5 µm, 250 × 21.2 mm;
Phenomenex)
• Preparative HPLC system (Dionex, model no. UltiMate 3000, including
variable-wavelength detector)
• Rotary evaporator (Büchi)
• Semipreparative HPLC column (Jupiter 300 Å, C4, 5 µm, 250 × 10 mm,
Phenomenex)
• Spectrophotometer (Jasco, model no. V-630)
REAGENT SETUP
100 mM Acetic acid buffer with 100 mM aniline (pH 4.5) Combine 0.21 g of
NaOAc (2.5 mmol), 0.15 ml of acetic acid (5 mmol), and 40 ml of ultrapure
water. Add 0.46 ml of aniline (5 mmol) to the solution, and adjust the pH to
4.5 with acetic acid. Adjust the volume of the solution to 50 ml with ultrapure
water and re-adjust the pH to 4.5, if required. Store the buffer at 5 °C and use
it within 2 weeks.
50 mM Borax buffer in H2O (pH 8.5) Dissolve 0.95 g of borax
(Na2B4O7·10H2O, 2.5 mmol) in 40 ml of H2O. Adjust the pH to 8.5 with a
100 mM solution of hydrochloric acid (HCl), adjust the volume to 50 ml
with ultrapure water, and re-adjust the pH to 8.5 if required. Store the solution at 5 °C and use it within 2 weeks.
Sodium hydroxide (100 mM) Dissolve 200 mg of NaOH pellets in 50 ml of
ultrapure water. ! CAUTION NaOH is corrosive. Wear a lab coat, nitrile gloves,
and safety goggles and stir the solution with a magnetic Teflon bar. Store the
NaOH solution at room temperature (~20 °C). The solution is stable for at
least 6 months.
Hydrochloric acid (100 mM) Add 0.41 ml of concentrated hydrochloric
acid (37%, 12 M) to 49.59 ml of ultrapure water. ! CAUTION HCl is corrosive.
Add acid to water, but do not add water to the acid. Wear a lab coat, nitrile
gloves, and safety goggles and stir the solution with a magnetic Teflon bar.
Store the HCl solution at room temperature. The solution is stable for at least
6 months.
Phosphate buffer (100 mM, pH 7.0) Dissolve 0.34 g of sodium
phosphate monobasic monohydrate (2.5 mmol) and 0.67 g of
sodium phosphate dibasic heptahydrate (2.5 mmol) in 45 ml of H2O.
Adjust the pH to 7.0 with a 1.0 M solution of hydrochloric acid (HCl)
or 1.0 M sodium hydroxide as needed, adjust the volume to 50 ml,
and re-adjust the pH to 7.0 if required. Store the phosphate buffer at
5 °C and use it within 2 weeks.
Saccharide starting materials In general, we buy monosaccharides and
short oligosaccharides from Sigma-Aldrich, Carbosynth, or Isosep. More
complex oligosaccharides such as lipochitin oligosaccharides and EPS fragments are more typically isolated in-house after bacterial expression. For the
preparation of EPSs and Nod factors, refer to the experimental section and
supplementary information of our previous work3,7.
OEG-linker Prepare the OEG-linker as described in Box 1.
Protein receptors for biochemical assays Purified protein receptors used in
these studies were isolated from optimized bacterial expression systems3,7.
EQUIPMENT SETUP
Nitrogen flow Clean, dry N2 is used from a tank, which should be fitted with
tubing that allows it to reach inside a fume hood. The tip of the tubing is fitted
with a syringe, which in turn is fitted with a needle. The valve of the N2 tank is
slowly opened, until a soft flow of gas is released from the needle. The needle is
then suspended above the sample to be dried. Be careful that the sample is not
spilled—the flow should perturb the surface of the liquid in the sample, but it
should not spray it over the sides of the container.
Analytical HPLC Use a flow rate of 1 ml/min (do not exceed instrument
pressure limit) for analytical HPLC. Use degassed water (solvent A) and
acetonitrile (solvent B), both containing 0.1% (vol/vol) formic acid. Run the
program gradient as follows:
Time (min)
% Acetonitrile
0–1
5
1–10
5–100
10–12
100
 CRITICAL Do not run pure water on the column, as this can result in the
collapse of the separation material.
nature protocols | VOL.12 NO.11 | 2017 | 2415
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protocol
Semipreparative HPLC Use a flow rate of 4 ml/min (do not exceed
instrument pressure limit) for semipreparative HPLC. Use acid-free,
degassed water (solvent A) and acetonitrile (solvent B). Run the program
gradient as follows:
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Time (min)
pressure limit) for preparative HPLC. Use acid-free, degassed water (solvent
A) and acetonitrile (solvent B). Run the program gradient as follows:
Time (min)
% Acetonitrile
0–5
5
5–29
5–100
29–35
100
Collect the fractions throughout the entire run, as the retention time may
vary according to the physicochemical properties of the carbohydrates. If additional runs are needed to purify the whole amount of the compound, then
fraction collection can be adjusted in these subsequent runs to collect only
the desired product. The identity of the product can be confirmed by analytical HPLC-MS.  CRITICAL Do not run pure water on the column, as this can
result in the collapse of the separation material.
Preparative HPLC Use a flow rate of 10 ml/min (do not exceed instrument
% Acetonitrile
0–4
5
4–29
5–60
29–30
60–100
30–35
100
Collect the fractions throughout the entire run, as the retention time may
vary according to the chemical properties of the carbohydrates. If additional
runs are needed to purify the whole amount of the compound, then fraction
collection can be adjusted in these following runs to collect only the desired
product. The identity of the product can be confirmed by analytical HPLCMS.  CRITICAL Do not run pure water on the column, as this can result in
the collapse of the separation material.
PROCEDURE
Modification of hydrophilic carbohydrates with OEG-linker ● TIMING 17 h
1| Weigh out the carbohydrate, e.g., either 1.44 mg of EPS octasaccharide or 1.50 mg of M. loti R7A Nod factor (1.0 µmol,
1 equiv.), and OEG-linker (0.94 mg, 2.0 µmol, 2 equiv.) in two separate 1.5-ml glass vials. Use of excess OEG-linker is unproblematic, as it can be removed by washing with diethyl ether before performing the deprotection reaction.
2| Dissolve the carbohydrate in 100 µl of aniline buffer and the OEG-linker in 100 µl of acetonitrile. Mix the two solutions
by adding the OEG-linker solution to the aqueous carbohydrate solution, and shake the solution vigorously using an IKA Vortex Genius 3. The final concentration of the sample should be 5 mM carbohydrate, 10 mM OEG-linker, 50 mM NaOAc, 50 mM
aniline, pH 4.5 and 1:1 water–acetonitrile.
? TROUBLESHOOTING
a
b
O
HO
O
HO O
O HO
O
HO
O
HO
RO OR
OH
O
O
HO
HO
RO
Linker
O
H2N
HO
O
O
O
OH
HO
HO
O
O
OR RO
OH
O
SH
Thiol-functionalized carbohydrate
N
O
HO
O
OH
O
O
NH HO
O
OH
O
OH
O
O
OR HO
H2N O
HO
RO
OR
O
O
OH
O
OH
O
OH
HO
O
O
O
NH HO
O
O
HO
O
HO
RO OR
RO OR
O
O
O
OH
NH
Linker
SH
Thiol-functionalized carbohydrate
OH
H2N O
O
O
S
O
50 mM aniline, 50 mM acetate buffer, pH 4.5, 40 °C, 16 h
S
O
OH
O
RO
OR
50 mM aniline, 50 mM acetate buffer, pH 4.5, 40 °C, 16 h
O
HO
HO
RO
O
H2N
O
OH
HO
HO
O
O
OR RO
OH
O
O
OR HO
OH
O
OH
O
OH
OH
OH
OH
O
HO
O
O
N
O
HO
O
OH
O
O
NH HO
O
N O
HO
O
O
O
S
O
OH
O
OH
O
O
O
O
NH HO
OH
O
OH
O
NH HO
NH
N O
O
O
O
S
O
5% triethylsilane in trifluoroacetic acid, 15 min
5% triethylsilane in trifluoroacetic acid, 15 min
HO
O
O
O
HO O
O HO
O
HO O
O HO
O
OH
O
O
NH HO
O
OH
O
OH
O
O
O
HO
O
HO
OH
O
HO
HO
RO
RO
OR
O
O
OH
HO
HO
H2N
O
O
O
OR RO
OH
O
O
OR HO
OH
O
OH
HO
O
O
OH
O
OH
OH
HO
OH
O
N
O
O
HO
OH
O
O
NH HO
O
N O
O
O
O
SH
O
OH
O
OH
O
O
O
O
NH HO
O
NH HO
OH
O
OH
NH
N O
O
O
O
SH
O
Figure 2 | Overview of the two-step carbohydrate-linker conjugation. (a,b) The process leads to the formation of a free thiol from EPS octasaccharide (a) or
M. loti R7A Nod factor (b). Conditions: (Steps 1–8) OEG-linker conjugation in 50 mM aniline, 50 mM acetate buffer, at pH 4.5, 40 °C, 16 h; (Steps 9–16). Thiol
deprotection using 5% triethylsilane in trifluoroacetic acid, 15 min. R-groups indicate partially acetylated positions (R = H or Ac). A mixture of acetylated
species is present in the native carbohydrate and glycoconjugates.
2416 | VOL.12 NO.11 | 2017 | nature protocols
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
protocol
3| Place the sample in an IKA KS-125 shaker at 40 °C for 16 h. The reaction occurs as shown in Figure 2.
4| Place the sample under a flow of N2 and let it dry completely (see Equipment Setup).
5| To remove excess linker, perform Steps 5–7. Add 3 ml of diethyl ether to the sample. Shake the sample vigorously using
the IKA Vortex Genius 3.
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
6| Using a glass Pasteur pipette, carefully remove the supernatant to a separate flask.
7| Repeat Steps 5 and 6 three times.
 CRITICAL STEP Make sure not to scrape the sides of the sample tube. The excess OEG-linker is in solution, but the carbohydrate product (and any unreacted starting material) is precipitated on the walls of the sample tube.
 CRITICAL STEP Do not discard the diethyl ether, but collect it the first time you perform this reaction. Place the total volume of diethyl ether from Steps 5 to 7 under a N2 flow until it is completely dry. Check whether the concentrated supernatant
contains any of your desired product or unreacted starting carbohydrate by re-dissolving a fraction of the dry compound and
running it on an analytical HPLC system. If it does contain the desired glycoconjugate product then it is necessary to replace
Steps 5–7 with preparative HPLC purification.
 PAUSE POINT The dry, precipitated sample can be stored at −18 °C for at least 6 months. Any unreacted carbohydrate will
not interfere with the subsequent steps and can be carried over to the next stage of the protocol without removal.
8| (Optional) Analyze the OEG-linker glycoconjugate by HPLC-MS using the method described in the Equipment Setup.
Deprotection of the linker to liberate the thiol group ● TIMING 2 h
9| To remove the trityl group and thus to deprotect the thiol, prepare a deprotection solution: with a 5-ml syringe, add
4.75 ml of trifluoroacetic acid to a 15-ml flask. Using a 1-ml syringe, add 250 µl of triethylsilane (95% trifluoroacetic acid
and 5% triethylsilane).
! CAUTION TFA is highly corrosive and causes burns. All work with TFA should be performed in a fume hood. Wear eye
protection, gloves, and a lab coat at all times, and be careful not to spill.
10| Add the freshly prepared deprotection solution to the dried sample and let it react for 15 min.
 CRITICAL STEP It is very important that the sample does not contain water at this stage, as this can lead to degradation
of the sample.
11| Place the sample under a N2 flow to evaporate the trifluoroacetic acid for 10 min. Repeat this step if necessary until the
sample is completely dried.
12| To remove the detached trityl protecting group, perform Steps 12–14: add 3 ml of heptane to the sample. Shake the
sample vigorously using the IKA Vortex Genius 3.
13| Using a glass Pasteur pipette, carefully remove the supernatant to a separate flask.
14| Repeat Steps 12 and 13 three times.
 CRITICAL STEP Make sure not to scrape the sides of the sample tube. The trityl tag is in the heptane solution, but the
carbohydrate is precipitated on the walls of the sample tube.
15| Dry the sample under a N2 flow.
 CRITICAL STEP Do not add water to the sample at this stage before storage, as this can destroy the conjugate.
 PAUSE POINT The dry, deprotected conjugate can be stored at 2–8 °C (but preferably at −18 °C) for at least a month.
16| (Optional) Analyze the OEG-linker glycoconjugate containing a free thiol group by HPLC-MS using the method described
in the Equipment Setup.
Example reactions ● TIMING 2–24 h
17| The resulting glycoconjugate containing a free thiol can be used in a variety of conjugation reactions. We have included
three examples based on publications involving EPSs and Nod factor. For the reaction with SPR surfaces to enable biochemical
measurements using a Biacore instrument, follow the steps in option A. Here, the example shown is for M. loti R7A Nod
nature protocols | VOL.12 NO.11 | 2017 | 2417
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protocol
RO
OR
H2N
OH
O
HO
O
OH
O
O
HO
N
O
NH HO
O
O
O
NH HO
O
OH
O
OH
O
OH
O
O
NH HO
O
NH
O
OH
NH
O
N O
O
O
O
SH
O
–
O
SO3
+
Linker
S
–
NH
COOH
Cl
Fluorescently labeled carbohydrate
–
–
SO3
H
N
SO3
O
O
Cl
H
N
O
SO3
Cl
Cl
O
N
O
H+
N
COOH
S
Cl
O
Cl
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
50 mM phosphate buffer, DMSO, pH 7, RT, 16 h
–
H2N
OH
O
HO
O
OH
O
O
HO
N
O
O
OH
O
O
NH HO
O
NH HO
O
OH
O
OH
O
O
NH HO
O
O
OH
NH
O
O
O
S
O
Cl
H
N
N
O
O
SO3
O
O
N O
–
SO3
H
N
RO
OR
H+
N
COOH
S
O
Cl
Cl
Figure 3 | Fluorescence labeling of Nod factor by reaction between free thiol and maleimide-functionalized Alexa 546. R-groups indicate partially acetylated
positions (R = H or Ac). A mixture of acetylated species is present in the native carbohydrate and glycoconjugates. RT, room temperature.
factor–OEG-thiol conjugate. For the reaction of an OEG-thiol-functionalized Nod factor with a maleimide fluorophore, follow
the steps in option B (Fig. 3). This derivative could be used for MST or bioimaging. For the reaction of an OEG-thiol-functionalized EPS octasaccharide with biotin for BLI kinetics studies using a FortéBio instrument, follow the steps in option C (Fig. 4).
(A) Application of the free thiol for surface immobilization for SPR ● TIMING 2 h
(i) Dissolve the dry M. loti R7A Nod factor–OEG-thiol conjugate in a small volume of 100 mM phosphate buffer.
Check the pH to make sure that the resulting solution is at pH 7.0. The free-thiol compound can be quantified
O
HO
HO O
O HO
O
RO OR
O
O
OH
O
HO
O
HO
O
O
HN
Linker
O
HO
HO
RO
O
NH
S
Biotinylated carbohydrate
O
OH
HO
HO
O
O
OR RO
OH
O
OH
O
O
OR HO
OH
OH
OH
O
OH
N O
O
O
HO
SH
O
O
HN
O
HO
HO O
O HO
O
RO OR
I
O
O
HO
O
HO
OH
O
HO
HO
RO
O
N
H
O
NH
H
H
H
N
S
O
50 mM borate buffer, pH 8.5, 15 min
O
O
OH
HO
HO
O
O
O
O
OR RO
OH
O
O
OR HO
OH
O
OH
OH
O
OH
OH
HO
HN
O
N O
O
O
O
S
N
H
O
O
H
H
N
S
O
Figure 4 | Biotinylation of EPS octasaccharide by a reaction between a free thiol and iodoacetamide-functionalized biotin. R-groups indicate partially
acetylated positions (R = H or Ac). A mixture of acetylated species is present in the native carbohydrate and glycoconjugates.
2418 | VOL.12 NO.11 | 2017 | nature protocols
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
NH
H
protocol
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
using Ellman’s reagent. A protocol for the Ellman’s test is supplied by Thermo Fisher with the reagent. Be careful not
to use too much of the conjugate.
(ii) In a Biacore instrument, immobilize the thiol-functionalized glycoconjugate by using a thiol-coupling kit (GE Healthcare) on CM5 sensor chips (GE Healthcare), following the protocol supplied by the manufacturer15.
(iii) Apply a solution of 1 mM thiol conjugate dissolved in 100 mM phosphate buffer at pH 7 at a flow rate of 10 µl/min
for 420 s.
Determine the degree of immobilization using the Biacore instrument. The immobilization level of Nod factor–OEG-thiol conjugate should be ~1,000 response units (RU). The Biacore chips should be used for binding assays immediately
after preparation (Supplementary Methods).
(B) Fluorescence labeling of carbohydrate ligand for MST or bioimaging ● TIMING 24 h
(i) Dissolve the dry M. loti R7A Nod factor–OEG-thiol conjugate in a small volume of 100 mM phosphate buffer. Check the
pH to make sure that the resulting solution is at pH 7.0. The free-thiol compound can be quantified using Ellman’s
reagent. A protocol for the Ellman’s test is supplied by Thermo Fisher with the reagent. Be careful not to use too much
of the conjugate.
(ii) Dilute the Nod factor–linker conjugate with 100 mM phosphate buffer to a concentration of 4 mM (concentration based
on the Ellman’s test described above). Add an equal volume of a 2 mM solution of Alexa 546 C5 maleimide in DMSO. The
final concentration is thus 2 mM Nod factor–thiol conjugate, 1 mM Alexa maleimide and 50 mM phosphate at pH 7.
(iii) Vortex the solution thoroughly; in our laboratory, we use an IKA Vortex Genius 3 and leave the solution to
react for 16 h at room temperature on an IKA KS-125 shaker.Formation of fluorescence-labeled conjugates will occur
according to the scheme in Figure 3, and the target
compound can be checked on an HPLC-MS using the
a
Nod factor conjugation
method described in the Equipment Setup.
Nod
Linker
(iv) Purify the reaction mixture using a semipreparative
factor
HPLC system (see Equipment Setup).
(v) Freeze-dry the HPLC fractions containing the
Trityl
pure glycoconjugate. The product is a bright,
Nod
Linker
SH
factor
pink solid, which is well-separated from the
starting materials and intermediates by HPLC (Fig. 5a).
 PAUSE POINT The fluorophore conjugates should
Nod
Linker
factor
be stored at −20 °C; they are typically stable for
at least 6 months.
(vi) Quantify the Alexa 546 conjugates using a
spectrophotometer according to the protocol
0
1.3
2.5
3.8
5.0
6.3
7.5
8.8
10.0
11.2
(min)
provided by Invitrogen16 (λabs,max = 555.5 nm,
EPS conjugation
b
λem,max = 568 nm).
(vii) (optional) Use the fluorescent glycoconjugates
for binding assays using MST. Refer to the
EPS
Linker
Excess linker *
Supplementary Methods for a procedure to do this.
(C) Biotinylation of carbohydrate ligand for BLI studies
● TIMING 17 h
(i) Dissolve the dry EPS octasaccharide–OEG-thiol
conjugate in a small volume of 50 mM borax buffer.
Check the pH to make sure that the resulting solution
is at pH 8.5. The free-thiol compound can be
quantified using Ellman’s reagent. A protocol for the
Ellman’s test is supplied by Thermo Fisher with the
reagent. Be careful not to use too much of the conjugate.
(ii) Dilute the EPS octasaccharide–OEG thiol with 50 mM
borax buffer to a concentration of 5 mM. Add an equal
volume of a 10 mM solution of biotin polyethylene
oxide iodoacetamide in borax buffer. Check the pH—it
should not drop below 8.3. The final concentration is
thus 2.5 mM EPS octaose thiol, 5 mM biotin
O
HN
EPS
2.5
3.0
4.0
Linker
5.0
(min)
NH
S
6.0
7.0
7.5
Figure 5 | Chromatographic analysis of glycoconjugates (absorbance at
215 nm). (a) The Nod factor–linker conjugate (top) elutes at ~7.7 min,
the deprotected linker (middle) elutes at 6.5 min, and the detached tritylprotecting group (¤) elutes at 8.3 min. The fluorescently labeled Nod factor
(bottom) elutes at ~9.5 min. (b) The EPS-linker conjugate (top) elutes at
~6.8 min as a double peak due to varying acetylations, whereas the excess
unreacted OEG-linker (*) elutes at 4.5 min. The final product, biotinylated
EPS (bottom), elutes at 4.0 min.
nature protocols | VOL.12 NO.11 | 2017 | 2419
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© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
protocol
polyethylene oxide iodoacetamide and 50 mM borax at pH 8.5. Let the mixture react for 15 min at room temperature
on an IKA KS-125 shaker.
(iii) (Optional) Use analytical HPLC-MS to confirm the formation of the product that occurs according to the scheme in
Figure 4. The product elutes at ~4.0 min (Fig. 5b).
? TROUBLESHOOTING
(iv) Purify the product by preparative HPLC (Equipment Setup). The desired products elute in the range of 25–40%
(vol/vol) (percentage of acetonitrile). Collect the desired fractions in a round-bottom flask.
? TROUBLESHOOTING
(v) Place the flask under an N2 flow for 1 h to remove acetonitrile.
(vi) Lyophilize the sample by cooling it to −86 °C and placing the flask on a lyophilizer.
 PAUSE POINT The lyophilized solid can be stored for at least 6 months at −18 °C.
(vii) Resuspend the sample in 5 ml of H2O.
 PAUSE POINT This solution can be stored at −18 °C for 6 months.
(viii) Quantify the sample using a Pierce Biotin Quantitation Kit17 with a spectrophotometer.
(ix) Perform BLI studies8 as described in the instructions supplied with the Pall FortéBio18. When a ligand is used in a
200 nM concentration, immobilization levels (quantified using the BLI instrument) of ~2 nm (optical thickness) are
achieved by using 5 min of immobilization time. The BLI sensor tips should be used for binding assays immediately
after preparation (Supplementary Methods).
? TROUBLESHOOTING
Troubleshooting advice can be found in Table 3.
Table 3 | Troubleshooting table.
Step
Problem
Possible cause
Solution
2
The compounds are not fully
dissolved
Different carbohydrates may have varying
hydrophilic/hydrophobic properties
Use a higher or lower ratio of MeCN/water,
if needed Alternatively, replace MeCN with
MeOH
17C(iii)
No observation of thioether
conjugate
Reaction rate is too low for the given carbohydrate because of particular ring-opening
properties of the reducing sugar unit
Leave the reaction overnight to increase
the time for the reaction to occur
The compound does not elute
at the specified retention
time
Different carbohydrates may have varying
hydrophilic/hydrophobic properties and therefore elute differently
Make sure to collect fractions from the
entire run until the desired product has
been identified by MS and you know its
elution profile
No peaks are observed in the
25- to 40% MeCN range
The compound is washing through the column
Try to inject a smaller volume of analyte
17C(iv)
● TIMING
Steps 1–8, modification of carbohydrate ligands with OEG-linker: ~17 h
Steps 9–16, deprotection of the linker to liberate the thiol group: ~2 h
Table 4 | Mass spectrometry data for the three end products3,7
Compound
Nod factor–thiol
Nod factor–fluorophore
EPS–biotin, 3Ac
Ion
Mol. formula
Calculated
Observed
[M+H]+
C74H130N7O35S
1,708.83
1,708.5
[M+2H]2+
C117H176Cl3N11O47S4
1,360.49
1,360.4
[M+H]+
C79H130N5O53S2
2,060.7067
2,060.7066
2420 | VOL.12 NO.11 | 2017 | nature protocols
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
protocol
a
SPR
WGA–(GlcNAc)5
NFR1–NF
NFR5–NF
WGA–NF
100
RU (%)
Figure 6 | Measurements of Nod factor binding to legume-plant receptors
NFR1 and NFR5. (a) Binding data from Nod factor–thiol immobilized on CM5
sensor chip gold surface in a Biacore system. ‘x’ and ‘+’ indicate Nod factor–
receptor-binding data points, whereas ‘’ and ‘’ mark the data points for
the WGA lectin positive-control binding to chitopentaose (GlcNAc)5 and Nod
factor, respectively. (b) MST-binding data from the fluorophore-labeled Nod
factor and the receptors NFR1 and NFR5 (‘x’ and ‘+’ data points, respectively).
‘’ and ‘’ indicate data points for the negative controls, linker and
chitopentaose, respectively. Data shown for protein concentration range from
10–10 to 10–4 M in panel a, and from 10–12 to 10–7 M in panel b.
Measurements were repeated with at least three independent protein
preparations. Error bars indicate the 95% confidence interval. Image
reproduced with permission from ref. 3, National Academy of Sciences.
50
0
–10
b
–8
–6
–4
–10
–8
NF
–4
x
Protein 10 (M)
Linker
NF
Protein 10 (M)
MST
–6
x
(GlcNAc)5
∆Fnorm (%)
Step 17A, application of the free thiol for surface
immobilization for SPR: ~2 h
Step 17B, fluorescence labeling of carbohydrate ligand
for MST or bioimaging: ~24 h
Step 17C, biotinylation of carbohydrate ligand for
BLI studies: ~17 h
Box 1, synthesis of bifunctional OEG-linker: 3 d
0.5
0.0
–12
–11
–10
–9
–8
–7
–12
–11
NFR1 10x (M)
–10
–9
–8
–7
NFR5 10x (M)
ANTICIPATED RESULTS
The carbohydrate conjugates and their intermediate products can be analyzed by HPLC (Fig. 5) and by mass spectrometry
(Table 4; the spectra are shown in Supplementary Figures 1–3).
The Nod factor–thiol can be applied in SPR-binding studies (Fig. 6a), whereas the fluorophore-labeled Nod factor may be
used with an MST system (Fig. 6b). Binding constants for both techniques gave values in the nM range, showing a slightly
higher affinity for NFR1 as compared with NFR5 (see Kd values in Table 5), thus confirming that this protocol constitutes a
powerful tool for analyzing complex carbohydrate–protein interactions.
The biotinylated bacterial EPS octasaccharide (EPS–biotin) can be applied in a FortéBio system for BLI studies (Fig. 7), in
which biotin enables the immobilization of the EPS to a streptavidin-coated surface. By this approach, the Kd value can be
determined for the interaction between the EPS and its legume-plant receptor, EPR3 (Table 5).
b
2
40 µM
1
20 µM
10 µM
5 µM
2.5 µM
100 300 500 700
Time (s)
Optical thickness (nm)
a
Optical thickness (nm)
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
1.0
Table 5 | Summary of binding experiments3,7
2
Carbohydrate ligand
Receptor
Method
Kd value
Nod factor–thiol
NFR1
SPR,
Biacore
4.9 ± 1.3 nM
Nod factor–thiol
NFR5
SPR,
Biacore
10.1 ± 2.5 nM
Nod factor–fluorophore
NFR1
MST
0.61 ± 0.25 nM
Nod factor–fluorophore
NFR5
MST
4.0 ± 1.5 nM
EPS–biotin
EPR3
BLI,
FortéBio
2.7 ± 0.2 µM
1
100 300
500 700
Time (s)
Figure 7 | BLI experiments showing the interaction between the EPS
fragment and the EPR3 receptor. (a) Sensorgrams resulting from EPS binding
to EPR3 for varying concentrations of the EPR3 receptor. (b) Negativecontrol experiment with immobilized maltohexaose and injection of the same
concentration series of EPR3. Values shown (optical thickness in nanometers)
are from triplicate measurements. Error bars indicate S.E.M. Image adapted
from ref. 7, Nature Publishing Group.
Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.
Acknowledgments Generous support by the Danish National Research
Foundation (grant no. DNRF79) and by the Villum Foundation (grant no.
VKR022710) is gratefully acknowledged. The authors thank C.W. Ronson and
J.T. Sullivan for bacterial production of the EPS octa-saccharide and
M. loti R7A Nod factors.
AUTHOR CONTRIBUTIONS M.B.T., J. Sauer and K.J.J. designed the study. C.T.H.,
N.N.M., J. Sauer and M.B.T. performed the experiments. C.T.H., N.N.M.,
J. Stougaard, M.B.T. and K.J.J. wrote the manuscript.
COMPETING FINANCIAL INTERESTS The authors declare no competing financial
interests.
Reprints and permissions information is available online at http://www.nature.
com/reprints/index.html. Publisher’s note: Springer Nature remains neutral with
regard to jurisdictional claims in published maps and institutional affiliations.
1. Larsen, K., Thygesen, M.B., Guillaumie, F., Willats, W.G.T. & Jensen, K.J.
Solid-phase chemical tools for glycobiology. Carbohydr. Res. 341,
1209–1234 (2006).
2. Oldroyd, G.E.D. Speak, friend, and enter: signalling systems that promote
beneficial symbiotic associations in plants. Nat. Rev. Microbiol. 11,
252–263 (2013).
nature protocols | VOL.12 NO.11 | 2017 | 2421
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
protocol
3. Broghammer, A. et al. Legume receptors perceive the rhizobial lipochitin
oligosaccharide signal molecules by direct binding. Proc. Natl. Acad. Sci.
USA 109, 13859–13864 (2012).
4. Kelly, S.J. et al. Conditional requirement for exopolysaccharide in the
mesorhizobium–lotussymbiosis. Mol. Plant-Microbe Interact. 26, 319–329
(2013).
5. Thygesen, M.B., Sauer, J. & Jensen, K.J. Chemoselective capture of
glycans for analysis on gold nanoparticles: carbohydrate oxime tautomers
provide functional recognition by proteins. Chem. Eur. J. 15, 1649–1660
(2009).
6. Thygesen, M.B. et al. Nucleophilic catalysis of carbohydrate oxime
formation by anilines. J. Org. Chem. 75, 1752–1755 (2010).
7. Kawaharada, Y. et al. Receptor-mediated exopolysaccharide perception
controls bacterial infection. Nature 523, 308–312 (2015).
8. Abdiche, Y., Malashock, D., Pinkerton, A. & Pons, J. Determining kinetics
and affinities of protein interactions using a parallel real-time label-free
biosensor, the Octet. Anal. Biochem. 377, 209–217 (2008).
9. Bek, A. et al. Improved characterization of nod factors and genetically
based variation in lysM receptor domains identify amino acids expendable
for Nod factor recognition in Lotus spp. Mol. Plant-Microbe Interact. 23,
58–66 (2010).
10. Maolanon, N.N. et al. Lipochitin oligosaccharides immobilized through
oximes in glycan microarrays bind LysM proteins. ChemBioChem 15,
425–434 (2014).
11. Villadsen, K., Martos-Maldonado, M.C., Jensen, K.J. & Thygesen, M.B.
Chemoselective reactions for the synthesis of glycoconjugates from
unprotected carbohydrates. ChemBioChem 18, 574–612 (2017).
12. Tanaka, T., Nagai, H., Noguchi, M., Kobayashi, A. & Shoda, S. One-step
conversion of unprotected sugars to beta-glycosyl azides using
2-chloroimidazolinium salt in aqueous solution. Chem. Commun. 45,
3378–3379 (2009).
13. Lim, D., Brimble, M.A., Kowalczyk, R., Watson, A.J. & Fairbanks, A.J.
Protecting-group-free one-pot synthesis of glycoconjugates directly from
reducing sugars. Angew. Chem. Int. Ed. Engl. 53, 11907–11911 (2014).
14. Sauer, J., Hachem, M.A., Svensson, B., Jensen, K.J. & Thygesen, M.B.
Kinetic analysis of inhibition of glucoamylase and active site mutants via
chemoselective oxime immobilization of acarbose on SPR chip surfaces.
Carbohydr. Res. 375, 21–28 (2013).
15. GE Healthcare. Thiol Coupling Kit. Instruction 22-0618-10 AB, 1–9
(2003) https://www.gelifesciences.com/gehcls_images/GELS/
Related%20Content/Files/1384878377977/litdoc22061810_
20161015124116.pdf.
16. Molecular Probes Inc. Thiol-Reactive Probes. 1–4 (2006) https://tools.
thermofisher.com/content/sfs/manuals/mp00003.pdf.
17. Thermo Scientific. Pierce Biotin Quantitation Kit. 1–5 (2014) https://
tools.thermofisher.com/content/sfs/manuals/MAN0011484_Pierce_Biotin_
Quantitation_UG.pdf.
18. Pall FortéBio. Octet® RED96 System http://www.fortebio.com/documents/
Octet_RED96_datasheet_1216.pdf.
19. Xia, B.Y. et al.Versatile fluorescent derivatization of glycans for glycomic
analysis Nat. Methods 2, 845–850 (2005).
20. Price, N.P.J. et al. Functionalized C-glycoside ketohydrazones: carbohydrate
derivatives that retain the ring integrity of the terminal reducing sugar.
Anal. Chem. 82, 2893–2899 (2010).
2422 | VOL.12 NO.11 | 2017 | nature protocols
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