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Microbe-focused glycan array screening platform

Andreas Geissner, Anika Reinhardt, Christoph Rademacher, Timo Johannssen,

João Monteiro, Bernd Lepenies, Michel Thépaut, Franck Fieschi, Jana Mrázková,

Michaela Wimmerova, Frank Schuhmacher, Sebastian Götze, Dan Grünstein,

Xiaoqiang Guo, Heung Sik Hahm, Jeyakumar Kandasamy, Daniele Leonori,

Christopher E. Martin, Sharavathi G. Parameswarappa, Sandip Pasari, Mark K. Schlegel,

Hidenori Tanaka, Guozhi Xiao, You Yang, Claney L. Pereira, Chakkumkal Anish,

and Peter H. Seeberger

SI Appendix

www.pnas.org/cgi/doi/10.1073/pnas.1800853116

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1. Experimental Procedures

Microarray printing

Glycans were dissolved at a final concentration of 100 µM in sterile printing buffer (50 mM

sodium phosphate, pH 8.5). For some rare exceptions of glycans containing base labile groups

(indicated in SI Appendix Table S2), printing solutions were prepared in phosphate buffered

saline (PBS, pH 7.4). Approximately 400 pL of the substances per spot were printed in 16 field

format (overall 284 spots per field) with two replicate spots per field on NHS activated hydrogel

glass slides (CodeLink slides, Surmodics) using a contact-free piezoelectric microarray spotter

(SciFLEXARRAYER S3, Scienion, Berlin, Germany) equipped with one type 4 coated Piezo

Dispense Capillary (PDC80). Relative humidity was kept constant at 40% during the entire

print run. After printing, slides were incubated for 16 to 24 h in a humidity saturated chamber.

Remaining reactive NHS esters were subsequently quenched by incubation in 100 mM

ethanolamine, 50 mM sodium phosphate pH 9 for 1 h at room temperature (RT). Slides were

washed twice with water and dried by centrifugation at 300 x g using the CombiSlide system

(Eppendorf). Slides were stored dry at 4 °C until blocking.

Plant lectins

Slides were blocked for 30 min at RT with plant lectin binding buffer (1% (w/v) bovine serum

albumin (BSA, VWR Product ID 422361V) in 20 mM Hepes, 2 mM calcium chloride, 2 mM

magnesium chloride, 0.1 mM manganese chloride pH 7.0), rinsed with water and dried by

centrifugation. A 16 well incubation grid (FlexWell Incubation chamber, Grace BioLabs, Bend,

OR, USA) was attached to the surface. Fluorescein isothiocyanate (FITC) labeled plant lectins

(see SI Appendix Table S1; obtained from Vector Laboratories, Burlingame, CA, USA) were

diluted in plant lectin binding buffer. The diluted solutions (100 µL per well) were applied to the

slides and incubated overnight at 4 °C. Wells were washed for 3 min with plant lectin binding

buffer, the multiwell grid was removed, and the whole slide washed for 3 min with 10 mM

Hepes, 1 mM calcium chloride pH 7.0. After rinsing with water and drying by centrifugation,

slides were scanned using a GenePix 4300A microarray scanner (Molecular Devices,

Sunnyvale, CA, USA) at a detector voltage that prohibits detector signal saturation and at

10 µm resolution that is adequate for the size of the spots. Binding analysis: binding was

considered positive when a signal-to-noise ratio (SNR) of 7 was reached for at least three of

four spots from two independent experiments when the circle diameter was manually adjusted

to the spot size. Intensity evaluation: intensities were evaluated using GenePix Pro 7.2

(Molecular Devices) with a spot diameter of 70 μm at the position of greatest intensity, even if

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spots appeared larger, to minimize effects of spot size and subtraction of the intensity of the

spots printed with the printing buffer as background correction; these buffer subtracted values

are the “absolute” values given in the corresponding worksheet of the Supporting Data 3 Excel

workbook file. For each experimental spot, the absolute value was normalized to the highest

signal for the respective lectin. For example, the very first absolute binding intensity value for

lectin BSL for glycan “GA000005” listed in the worksheet is 77 (slide 1, spot 1), and this value

was normalized to the average of the best BSL binder “GA000175” spot intensities on the

same slide (77/(0.5*(47434+44265))=0.001679). All such resulting “relative” intensities are

given in the corresponding worksheet of the Supporting Data 3 Excel workbook file. The

reported value is generally the mean of four spots from two independent experiments unless

visibly unfit spots had to be removed from the analysis.

Monoclonal antibodies

Monoclonal rat antibody LM10 against oligoxylans (cell culture supernatant diluted 1:20,

PlantProbes, Leeds, UK)(1), murine monoclonal antibodies (concentration: 1 µg/mL, purity

>95% estimated by SDS-PAGE) 1A5(2), 1A10(3), and H16 (4) diluted in 3% BSA-PBS were

applied to blocked (1% BSA in 50 mM Hepes and 5 mM calcium chloride, pH 7.0, 1h at room

temperature) slides in 16 well format. Slides were incubated for 1 h at RT and washed three

times with PBS containing 0.1% Tween-20 (PBS-T). Secondary antibodies were diluted in 3%

BSA-PBS and incubated on the slide for 30 min. The following secondary antibodies were

used: goat anti-mouse IgG H+L AlexaFluor 635 (Life Technologies, Product-ID: A31574)

1:400; goat anti-rat IgG H+L AlexaFluor 555 (Life Technologies; Product-ID: A21434) 1:200.

Slides were washed once with PBS-T, twice with PBS, rinsed with water after removal of the

multiwell grid and dried by centrifugation. Intensities were read out using a GenePix 4300A

microarray scanner (Molecular Devices). Positive binding was evaluated from visual inspection

of the scanned glycan array images (2–5).

Sera from healthy human individuals

Sera for human studies were obtained as a gift from Sphingotec GmbH, Henningsdorf,

Germany. Sera from 15 apparently healthy human individuals were diluted 1:100 in 3% BSA-

PBS containing 0.1% Tween-20 and incubated at 37 °C for 20 min to dissolve lipid particles(6).

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Following centrifugation at 11000 rpm for 1 min, 100 µL of supernatant per serum sample was

applied to blocked slides (1% BSA-PBS, 30 min room temperature) with attached 16 well

incubation grid and incubated for 1.5 h at RT. Wells were washed three times for 5 min with

PBS-T. Secondary antibody solution (goat anti-human IgG-Fc AlexaFluor 488 (Dianova,

Product-ID: 109-545-098) diluted 1:400 in 3% BSA-PBS containing 0.1% Tween-20 was

applied to the wells. After incubation for 30 min at RT, slides were washed twice for 5 min with

PBS-T after which the multiwell gasket was removed and the slides washed in a Petri dish for

5 min with PBS, rinsed with water and dried by centrifugation. Slides were scanned using a

GenePix 4300A microarray scanner (Molecular Devices) at a detector voltage that prohibits

detector signal saturation at 10 µm resolution that is adequate for the size of the spots. Binding

analysis: binding was considered positive when a signal-to-noise ratio (SNR) of 7 was reached

for at least three of four spots from two independent experiments when the circle diameter was

manually adjusted to the spot size. Intensity evaluation: intensities were evaluated using

GenePix Pro 7.2 (Molecular Devices) with a spot diameter of 70 μm at the position of greatest

intensity, even if spots appeared larger, to minimize effects of spot size and subtraction of the

intensity of the spots printed with the printing buffer as background correction; these buffer

subtracted values are the “absolute” values given in the corresponding worksheet of the

Supporting Data 2 Excel workbook file. For both experimental repeats, the values were

normalized to the average intensity of all spots on the array. For example, the absolute value

for serum 1, slide 1, spot 1 is 2.5, which was divided by the average signal over all spots from

the first experiment (1658.39) to give the relative intensity of 1,51E-03. All such resulting

“relative” intensities are given in the corresponding worksheet of the Supporting Data 2 Excel

workbook file. The reported value is the mean of four spots from two glycan array experiments.

DC-SIGN

Generation and Expression of DC-SIGN-D

DC-SIGN-D was generated as a fusion protein consisting of the ECD of DC-SIGN and the Fc

part of human IgG1. Cloning and production was performed as described previously (7). In

brief, the sequence encoding the CRD and the neck domain of DC-SIGN (amino acids 61-404)

was amplified by RT-PCR and ligated in-frame into the expression vector pFUSE hIgG1-Fc2

(Invivogen, San Diego, CA, USA). The resulting construct was expressed in CHO-S cells using

the Freestyle™ MAX CHO Expression System (Life Technologies, Carlsbad, CA, USA).

Secreted protein was purified from the supernatant using a HiTrap™ Protein G HP column

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(GE Healthcare, Freiburg, Germany). Identity and homogeneity were confirmed by SDS-PAGE

and Western Blot analysis.

Expression of DC-SIGN-T

DC-SIGN-T (tetrameric ECD) comprising residues 66–404 was overexpressed in E. coli as

inclusion bodies, then refolded and purified to homogeneity as determined by SDS-PAGE and

size-exclusion chromatography in a functional form by an automated multistep purification

protocol using a high-throughput Akta Xpress System. This automated purification process,

relying on a mannan-agarose column, followed the report of Tabarani et al 2009(8).

Glycan arrays

Slides were blocked with 1% BSA (w/v) in 50 mM Hepes and 5 mM calcium chloride, pH 7.0

(BDC-buffer) for 1 h at rt, washed twice with water and dried by centrifugation (300 x g, 5 min).

A 16 well incubation grid was attached to the microarray slide. Recombinant DC-SIGN

constructs were diluted to 25 ng/µL, 5 ng/µL, and 1 ng/µL in BDC buffer. EDTA (final

concentration 10 mM) was added to a 25 ng/µL sample of each DC-SIGN construct.

Recombinant human IgG1-Fc was diluted to 25 ng/µL. All samples were incubated for 15 min

at 37°C to allow for calcium to dissociate in the negative controls. The samples were applied

to the glycan array slides (60 µL per well) and incubated at 4 °C for 18 h at high humidity and

washed three times with 0.1% Tween-20 (v/v) in 50 mM Hepes and 5 mM CaCl2, pH 7.0 (WDC-

buffer). To detect DC-SIGN-T, microarray wells were incubated with primary murine anti-

human CD209 (DC-SIGN) (Clone 9E9A8, Biolegend, Product-ID 330102) antibody diluted 1:50

in BDC-buffer for 30 min at RT, washed three times with WDC-buffer, and incubated for 30 min

with goat anti-mouse IgG Alexa Fluor 635 (Life Technologies, Product-ID: A31574) diluted

1:400. To detect DC-SIGN-D and human IgG1-Fc, wells were incubated with goat anti-human

IgG Alexa Fluor 647 (Life Technologies, Product-ID A21445) diluted 1:400 in BDC-buffer for 1

h at RT. Wells were washed once with WDC-buffer, twice with 10 mM Hepes, 1 mM calcium

chloride, rinsed with deionized water and dried by centrifugation (300 x g, 5 min). Read-out of

fluorescence intensity signals was performed with a GenePix 4300A microarray scanner

(Molecular Devices) at a detector voltage that prohibits detector signal saturation at 10 µm

resolution that is adequate for the size of the spots and image analysis was performed with the

GenePix Pro 7 software (Molecular Devices). Intensity evaluation: intensities were evaluated

with a spot diameter of 70 μm at the position of greatest intensity, even if spots appeared

larger, to minimize effects of spot size and subtraction of the intensity of the spots printed with

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the printing buffer as background correction; these buffer subtracted values are the “absolute”

values given in the two corresponding worksheets (DC-SIGN-T and DC-SIGN-D) of the

Supporting Data 4 Excel workbook file. These values were normalized to best binder Ley

antigen 157 at 25 ng/µL. One example of how the “relative” intensity values were obtained

(given in the two corresponding worksheets (DC-SIGN-T and DC-SIGN-D) of the Supporting

Data 4 Excel workbook file) is the first value for DC-SIGN-T (-132.5), which was then divided

by the average of the two spot intensities for best binder Ley antigen 157 at 25 ng/µL

(“GA000157”) as follows: -132.5/(0.5*(38408.5+40271.5))=-0.00337. All such resulting

“relative” intensities are given in the corresponding worksheet of the Supporting Data 2 Excel

workbook file. All such resulting “relative” intensities are given in the two corresponding

worksheets (DC-SIGN-T and DC-SIGN-D) of the Supporting Data 4 Excel workbook file.

Reported intensities are the mean of four spots from two independent glycan array experiments

normalized to the intensity of Ley antigen 157 at 25 ng/µL.

Surface Plasmon Resonance of Immobilized DC-SIGN and Immobilized Di-Hep

Binding avidity of DC-SIGN-T towards disaccharide 178 was measured at 25 °C using a

Biacore T100 instrument (GE Healthcare). DC-SIGN-T samples were cooled down to 10 °C

before injection into flow cells. For immobilization of oligosaccharides a C1 sensor chip surface

was modified with O-(2-aminoethyl)-O′-(2-carboxyethyl)-polyethylene glycol hydrochloride

(HCl·H2N-PEG-COOH) (Sigma Aldrich, molecular weight = 5,000). Sample and reference flow

cells were functionalized using 1 mg/mL HCl·H2N-PEG-COOH dissolved in 50 mM sodium

phosphate buffer, pH 8.5 at a flow rate of 10 μL/min and 420 s contact time using the Amine

Coupling Kit (GE Healthcare) according to the manufacturer’s recommendations, yielding

about 50 response units (RU). In the sample cell disaccharide 178 was immobilized at 1 mM

in 50 mM sodium phosphate buffer, pH 8.5 at a flow rate of 10 μL/min and 420 s contact time

using the Amine Coupling Kit (GE Healthcare), yielding about 140 RU. As a reference 1 mM

mono-Rha 167 was used under the same conditions, yielding about 9 RU. DC-buffer was used

as running buffer.

Binding runs were performed with DC-SIGN construct dilutions at indicated concentrations at

a flow rate of 30 μL/min for 60 s of contact time and 180 s of dissociation time. Flow cells were

regenerated twice with 2 M guanidine-hydrochloride for 30 s, followed by 2 M guanidine-

hydrochloride for 20 s and 300 s stabilization period. The binding signals were calculated as

the difference of RUs between ligand and reference flow cells and monitored as a function of

time represented as sensorgrams. Kinetic evaluation of binding responses was performed with

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the Biacore T100 Evaluation software (GE Healthcare), using reference-subtracted

sensorgrams. Apparent Kd values were determined with a steady-state affinity model.

Burkholderia lectins

Proteins BC2L-A and BC2L-C-ct were produced in E. coli BL21(DE3) as previously described

(9, 10). Transformed cells were cultured in LB medium (Duchefa Biochemie) containing

ampicillin (100 µg/mL) at 37 °C. When the culture reached an OD600 of 0.5, protein

overexpression was induced by isopropyl 1-thio-β-D-galactopyranoside (IPTG, Duchefa

Biochemie) added to a final concentration of 0.5 mM. Cells were incubated at 27 °C for 4 hours

(BC2L-A) or at 30 °C for 3 h (BC2L-C-ct), harvested by centrifugation and resuspended in

equilibration buffer (20 mM Tris/HCl, 100 mM NaCl, pH 7.4). Cells were then lysed by

sonication and cytosolic fractions containing soluble proteins were separated by centrifugation.

Lectins were purified by affinity chromatography on a D-mannose-agarose (Sigma-Aldrich)

column using an FPLC system (ÄKTA, GE Healthcare) and eluted with 20 mM Tris/HCl, 150

mM NaCl, 10 mM EDTA, pH 7.4. No impurities were detected using SDS-PAGE. Purified

proteins were dialysed against 10 mM Tris/HCl, 50 mM NaCl, 1 mM CaCl2 for 2 days, then

against 50 mM NH4HCO3 for 2 days, freeze-dried and stored at -20 °C

Freeze-dried lectins were dissolved in 10 mM HEPES, 150 mM NaCl, 0.005% TWEEN 20, pH

7.4 and incubated overnight at 4°C. Lectins were filtered and then transferred into conjugation

buffer (50 mM borate buffer, pH 8.5) using Zeba Spin Desalting Columns (Thermo Fisher

Scientific). Lectins in conjugation buffer were mixed in a 1:20 molar ratio with FITC (Thermo

Fisher Scientific) which was freshly dissolved in N,N-dimethylformamide to a concentration of

5 mg/mL. The solution was incubated with mixing at 400 rpm for 2 hours in the dark at RT.

Excess FITC was removed by subsequent buffer exchange (Zeba Spin Desalting Columns)

and lectins were transferred to buffer 100 mM Tris/HCl, 0.5 mM CaCl2, pH 7.4 during this

procedure. Lectins were concentrated by Vivaspin500 centrifugal concentrators (Sartorius

Stedim Biotech SA) to concentrations 0.8 mg/mL of BC2L-A and 1 mg/mL of BC2L-C-ct.

Activity of labelled lectins was tested by yeast agglutination experiment. 10 μL of BC2L-A (7.7

mg/ml) or BC2L-C-ct (10.5 mg/mL) was mixed with 10 μL of 5% yeast cell suspension and

incubated for 10 minutes at room temperature. Then the mixture was applied to a glass slide

and observed under fluorescent microscope (Olympus Corporation). Both lectins were active

because a well-visible agglutination was observed in the samples. Also, fluorescence of

agglutinates was visible in both samples. 10 μL of the buffer (100 mM Tris.Cl, 0.5 mM CaCl2,

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pH 7.42) instead of the lectin sample was used as a negative control and no agglutination was

observed in the sample.

Isothermal titration calorimetry using Auto-iTC200 (Malvern Instruments) was performed with

labelled lectins (dissolved in 100 mM Tris/HCl, 0.5 mM CaCl2, pH 7.4) to test the influence of

the label to their binding properties. 20 aliquots of 2 μL of methyl α-D-mannopyranoside

(dissolved in the same buffer) were automatically added at 4 minute interval to the protein

solution present in the calorimeter cell tempered to 25 ºC. Integrated heat effects were

analyzed using Microcal Origin 7 software (Malvern Instruments). Measurements were

performed in triplicates. Labelling had no significant influence on activity of the lectins because

obtained binding parameters corresponded to thermodynamic values of non-labelled lectins

(9, 10).

For glycan array incubation, lectins were diluted in 100 mM Tris/HCl pH 7.4 containing 2 mM

calcium chloride and 0.05% (v/v) Tween-20. The concentration series of the lectins differed

slightly resulting in the 1 mg/mL sample shown in Figure 6 to be 0.96 mg/mL for BC2L-A and

1.05 mg/mL for BC2L-C-ct. At this concentration, EDTA was added to a final concentration of

5 mM as negative binding control. Lectins were incubated for 15 min at 37 °C to allow for

calcium to dissociate in the negative controls and then applied to unblocked slides (60 µL per

well). Microarrays were incubated for 1 h at RT. Wells were washed twice for 1 min with 20

mM Tris/HCl pH 7.4. The incubation grid was removed; the slides washed for 30 s with water,

dried by centrifugation and scanned with a GenePix 4300A microarray scanner (Molecular

Devices) at a detector voltage that prohibits detector signal saturation at 10 µm resolution that

is adequate for the size of the spots. Intensity evaluation: intensities were quantified using

GenePix Pro 7.2 (Molecular Devices). To adjust for different spots sizes, spot intensities were

evaluated from 70 µm diameter circles in the center of the spots and the values for the printing

buffer spots from the same well were subtracted as background correction; these buffer

subtracted values are the “absolute” values given in the two corresponding worksheets (BC2L-

A and BC2L-C-ct) of the Supporting Data 1 Excel workbook file. For every slide, intensities

were normalized to the signal of the same lectin for arabinomannan hexasaccharide 257 at

962.5 µg/µL or 1050 µg/µL, closest to a 1 mg/mL concentration. One example calculation

follows for the normalization of the first absolute value given in the table for BC2L-A at 1925

µg/µL, slide 1, spot 1 (53.5): 53.5/(0.5+(46214+46150))=0.01158. All such resulting “relative”

intensities are given in the two corresponding worksheets (BC2L-A and BC2L-C-ct) of the

Supporting Data 1 Excel workbook file. The reported values are generally the mean of six spots

from three independent experiments unless intensities had to be removed because of aberrant

spot shapes. For avidity analysis, fluorescence intensities (generally the mean of two spots

per glycan, lectin and concentration for three concentration series) of glycans containing an

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aminopentanol linker were plotted against lectin concentration using SigmaPlot 13.

Background-corrected intensities (I) were fitted against the hyperbolic equation 𝐼 =

𝐼𝑚𝑎𝑥𝑐(𝑃𝑟𝑜𝑡𝑒𝑖𝑛)

𝐾𝑑+𝑐(𝑃𝑟𝑜𝑡𝑒𝑖𝑛) with Kd being the apparent dissociation constant.

Diversity scores

To quantify the diversity of the glycan libraries we used the glycan fingerprint method as

previously described (11). Briefly, glycans were converted into xml format and 512 bit

fingerprints were calculated form all possible sub-trees of a glycan structure up to a chain

length of seven. The resulting hashed bit strings were then compared using the modified

Tanimoto coefficient to yield a similarity score between zero and one. The diversity of each

library was calculated from the average pairwise distance of all members. This matrix was also

used to build the tree using a neighbor-joining algorithm(12) and FigTree (version 1.4.3,

http://tree.bio.ed.ac.uk/software/figtree/) for visualization.

http://tree.bio.ed.ac.uk/software/figtree/

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2. Supporting Tables

Table S1: Plant lectins used for validation of glycan printing. Colors in specificity column

correspond to the colors used in Figure 3 and SI Appendix Figure S1. Specificity assignment

is based on vendor data and review publications(13, 14, 15). Additional literature precedent is

indicated for specificities not stated in these general sources.

Lectin Conc. Specificity

Bandeiraea simplicifolia lectin I (BSL) 20 µg/mL Orange: Terminal α-Gal

Blue: Terminal α-GalNAc

Concanavalin A (ConA) 20 µg/mL Orange: Terminal α-Man

Blue: Terminal α-Glc

Dolichos biflorus agglutinin (DBA) 50 µg/mL Orange: Terminal α-GalNAc

Blue: Terminal β-GalNAc

Erythrina cristagalli lectin (ECL) 20 µg/mL Orange: Terminal β-Gal

Maackia amurensis lectin I (MALI) 20 µg/mL Orange: Terminal β-Gal(1,4)Glc/GlcNAc

Blue: Terminal α-Neu5Ac(2,3)β-Gal(1,4)Glc/GlcNAc

(Both cases except glycans with fucose linked to

GlcNAc) (16)

Peanut agglutinin (PNA) 20 µg/mL Orange: Terminal β-Gal

Ricinus communis agglutinin

(RCA120)

20 µg/mL Orange: Terminal β-Gal

Blue: Terminal α-Neu5Ac(2,6)β-Gal (18)

Soybean agglutinin (SBA) 20 µg/mL Orange: Terminal GalNAc

Blue: Terminal Gal

Sambucus nigra lectin (SNL) 50 µg/mL Orange: Terminal α-(2,6) sialic acid

Ulex europaeus agglutinin I (UEAI) 50 µg/mL Orange: Terminal α-Fuc

Wheat germ agglutinin (WGA) 20 µg/mL Orange: Terminal GlcNAc or sialic acid

Blue: Terminal GalNAc (19, 20)

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Table S2: Glycans immobilized on 140 compound array.

Glycan ID

Glycan Structure (Microbial origin if applicable)

Reference DOI

Lectin Detected by mono-

clonal antibody

Signal from n human

sera (out of 15)

Number saccharide

units

Printed in

Predicted Bound (of predicted)

Not Predicted

Bounc

Predicted Not Bound

5

Neu5Ac(a2-6)Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)Glc(b1-1)aminohexanol

Hanashima (2007)

10.1002/asia.200600424 RCA, SNL,

WGA RCA, SNL WGA >5 5 NaP 8.5

6

Neu5Ac(a2-3)Gal(b1-3)GlcNAc(b1-3)Gal(b1-4)Glc(b1-1)aminohexanol

Hanashima (2007)

10.1002/asia.200600424 WGA WGA >5 5 NaP 8.5

7

Fuc(a1-3)[Neu5Ac(a2-3)Gal(b1-4)]GlcNAc(b1-3)Gal(b1-4)Glc(b1-1)aminohexanol

Hanashima (2007b)

10.1021/ol0704946 UEA, WGA WGA UEA 3 6 NaP 8.5

8

Neu5Ac(a2-6)Gal(b1-4)Glc(b1-1)aminohexanol

Hanashima (2007)

10.1002/asia.200600424 RCA, SNL,

WGA RCA, SNL,

WGA 3 NaP 8.5

9

Neu5Ac(a2-3)Gal(b1-4)Glc(b1-1)aminohexanol

Hanashima (2007)

10.1002/asia.200600424 MAL I, WGA MAL I, WGA 2 3 NaP 8.5

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10

Neu5Ac(a2-6)Gal(b1-4)GlcNAc-6-sulfate(b1-1)aminohexanol

RCA, SNL,

WGA RCA, SNL,

WGA ECL 1 3 NaP 8.5

11

Gal(b1-4)Glc(b1-1)aminohexanol

ECL, MAL I, PNA, RCA,

SBA


SBA >5 2 NaP 8.5

12

Gal(b1-4)GlcNAc-6-sulfate(b1-1)aminohexanol


SBA

ECL, MAL I, RCA, SBA

WGA PNA >5 2 NaP 8.5

69

Araf(a1-5)Araf(a1-1)aminopentanol

(Mycobacterium tuberculosis)

Kandasamy (2013)

10.1039/c3cc00042g >5 2 NaP 8.5

70

Araf(a1-5)Araf(a1-3)[Araf(a1-5)Araf(a1-5)]Araf(a1-5)Araf(a1-1)aminopentanol


Kandasamy (2013)

10.1039/c3cc00042g >5 6 NaP 8.5

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71

Araf(a1-3)[Araf(a1-5)]Araf(a1-1)aminopentanol


Kandasamy (2013)

10.1039/c3cc00042g >5 3 NaP 8.5

72

Araf(a1-5)Araf(a1-5)Araf(a1-5)Araf(a1-5)Araf(a1-5)Araf(a1-5)aminopentanol


Kandasamy (2013)

10.1039/c3cc00042g >5 6 NaP 8.5

73

Col(a1-3)[Col(a1-6)]Glc(a1-4)Gal(a1-3)GlcNAc(b1-1)aminopentanol

(Escherichia coli O111)

Calin (2013) 10.1002/chem.20120439

4 >5 5 NaP 8.5

74

ManNAc(b1-3)FucNAc(a1-3)GalNAc(a1-4)Gal(a1-1)aminopentanol

(Streptococcus pneumoniae)

Pereira (2015) 10.1002/anie.201504847 BSL H16 >5 4 NaP 8.5

75

GalNAc(a1-4)Gal(a1-1)aminopentanol


Pereira (2015) 10.1002/anie.201504847 BSL, DBA,

SBA, WGA BSL, SBA,

WGA SNL DBA H16 >5 2 NaP 8.5

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76

GalNAc(b1-4)Gal(a1-1)aminopentanol

Pereira (2015) 10.1002/anie.201504847 DBA, SBA,

WGA DBA, SBA,

WGA BSL >5 2 NaP 8.5

77

FucNAc(a1-3)GalNAc(a1-4)Gal(a1-1)aminopentanol


Pereira (2015) 10.1002/anie.201504847 BSL, UEA >5 3 NaP 8.5

78

FucNAc(b1-3)GalNAc(a1-4)Gal(a1-1)aminopentanol

Pereira (2015) 10.1002/anie.201504847 BSL, SBA, UEA

H16 >5 3 NaP 8.5

80

GalNAc(b1-1)aminoethanol

Pereira (2015) 10.1002/anie.201504847 DBA, SBA,

WGA DBA, SBA,

WGA BSL, RCA,

SNL >5 1 NaP 8.5

81

FucNAc(a1-1)aminopentanol

Lisboa (2017) 10.1073/pnas.170687511

4 UEA H16 >5 1 NaP 8.5

82

Man(a1-2)Man(a1-2)[Gal(b1-4)]Man(a1-1)aminopentanol

(Leishmania donovani)

Anish (2013) 10.1021/cb400602k ConA, ECL, PNA, RCA,

SBA

ConA, ECL, PNA, RCA,

SBA >5 4 NaP 8.5

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83

Man(a1-2)Man(a1-2)Man(a1-1)aminopentanol


ConA ConA >5 3 NaP 8.5

84

Gal(b1-4)Man(a1-1)aminopentanol

(Leishmania chagasi)

Anish (2013) 10.1021/cb400602k ECL, PNA, RCA, SBA

ECL, PNA, RCA, SBA

ConA >5 2 NaP 8.5

85

Man(a1-2)Man(a1-1)aminopentanol

ConA ConA 2 2 NaP 8.5

90

Glc(b1-1)aminoethanol

Parameswarappa (2016)

10.1016/j.chembiol.2016.09.016

>5 1 NaP 8.5

91

GlcNAc(a1-2)Hep(a1-3)Hep(a1-5)Kdo(a2-1)aminopentanol

(Neisseria meningitidis)

Yang (2012) 10.1039/c1sc00804h;

10.1021/ja401164s WGA WGA 1A5 >5 4 NaP 8.5

92

Hep(a1-3)Hep(a1-5)Kdo(a2-1)aminopentanol


Yang (2013) 10.1021/ja401164s 1A5 3 NaP 8.5

S16

93

Hep(a1-3)Hep(a1-5)[L-Ara4N(b1-8)]Kdo(a2-1)aminopentanol

(Proteus spec.)

Yang (2013) 10.1021/ja401164s 1A5 1 4 NaP 8.5

94

Hep(a1-7)Hep(a1-3)Hep(a1-5)Kdo(a2-1)aminopentanol

(Yersinia pestis)

Yang (2013) 10.1021/ja401164s 1A5 4 NaP 8.5

95

Hep(a1-2)Hep(a1-3)Hep(a1-5)Kdo(a2-1)aminopentanol

(Haemophilus influenzae)

Yang (2013) 10.1021/ja401164s 1A5 4 NaP 8.5

96

Hep(a1-5)Kdo(a2-1)aminopentanol

Yang (2013) 10.1021/ja401164s BSL 1A5 2 NaP 8.5

97

Hep(a1-7)Hep(a1-3)Hep(a1-1)aminopentanol

(Yersinia pestis)

Anish (2013b) 10.1002/anie.201301633 3 NaP 8.5

S17

98

Kdo(a2-8)Kdo(a2-4)Kdo(a2-1)aminopentanol

(Chlamydia spec.)

1A5 >5 3 NaP 8.5

99

Kdo(a2-1)aminopentanol

Yang (2013) 10.1021/ja401164s 1A5 >5 1 NaP 8.5

100

Hep(a1-1)aminopentanol

Yang (2013) 10.1021/ja401164s 3 1 NaP 8.5

101

Glc(b1-1)aminopentanol

Leonori (2013) 10.3762/bjoc.9.38 >5 1 NaP 8.5

102

D-FucNAc(b1-1)aminopentanol

Leonori (2013) 10.3762/bjoc.9.38 BSL, DBA, RCA, SNL,

WGA >5 1 NaP 8.5

103

FucNAc(b1-1)aminopentanol

Leonori (2013) 10.3762/bjoc.9.38 UEA H16 >5 1 NaP 8.5

104

Glc(b1-3)D-FucNAc(b1-1)aminopentanol

(Pseudomonas aeruginosa)

Leonori (2013) 10.3762/bjoc.9.38 >5 2 NaP 8.5

S18

105

Glc(b1-3)FucNAc(b1-1)aminopentanol

(Pseudomonas aeruginosa)

Leonori (2013) 10.3762/bjoc.9.38 >5 2 NaP 8.5

153

Gal(b1-3)GalNAc(a1-1)aminopentanol

ECL, PNA, RCA, SBA

PNA, RCA, SBA

ECL >5 2 NaP 8.5

154

Fuc(a1-3)[Gal(b1-4)]GlcNAc(b1-1)aminopentanol

Schlegel (2011) 10.1002/cbic.201100511 ECL, PNA, RCA, SBA,

UEA

BSL ECL, PNA, RCA, SBA,

UEA 2 NaP 8.5

155

Neu5Ac(a2-6)GalNAc(a1-1)aminopentanol

SNL, WGA WGA BSL, PNA, RCA, SBA

SNL 4 2 NaP 8.5

156

Gal(b1-4)[Gal(b1-4)Glc(b1-6)]GlcNAc(b1-1)aminopentanol



SBA


SBA WGA >5 4 NaP 8.5

S19

157

Fuc(a1-3)[Fuc(a1-2)Gal(b1-4)]GlcNAc(b1-1)aminopentanol

UEA UEA BSL 4 NaP 8.5

158

Gal(b1-3)[Fuc(a1-4)]GlcNAc(b1-1)aminopentanol

ECL, PNA, RCA, SBA,

UEA UEA

ECL, PNA, RCA, SBA

1 3 NaP 8.5

159

Fuc(a1-2)Gal(b1-3)[Fuc(a1-4)]GlcNAc(b1-1)aminopentanol

UEA UEA BSL 2 3 NaP 8.5

160

Gal-2,3-Pyruvate(a1-1)aminopentanol (mixture of R/S pyruvate)


Pereira (2015) 10.1002/anie.201504847 BSL >5 1 NaP 8.5

161

Gal(a1-3)Gal(b1-4)Glc(b1-1)aminopentanol

Hofmann (2015) 10.1038/nature15388;

10.1021/acs.joc.6b00554 BSL, SBA BSL, SBA RCA >5 3 NaP 8.5

162

Gal(a1-3)Gal(b1-4)GlcNAc(b1-1)aminopentanol

Hahm (2016) 10.1021/acs.joc.6b00554 BSL, SBA BSL, SBA ECL, RCA,

WGA >5 3 NaP 8.5

S20

163

Gal(a1-3)Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)Glc(b1-1)aminopentanol

Hahm (2016) 10.1021/acs.joc.6b00554 BSL, SBA BSL, SBA RCA, WGA >5 5 NaP 8.5

164

Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)Glc(b1-1)aminopentanol


Hahm (2016) 10.1021/acs.joc.6b00554 ECL, MAL I, PNA, RCA,

SBA


WGA PNA >5 4 NaP 8.5

165

Fuc(a1-2)Gal(b1-3)GlcNAc(b1-3)Gal(b1-4)Glc(b1-1)aminopentanol

Hahm (2016) 10.1021/acs.joc.6b00554 UEA UEA >5 5 NaP 8.5

166


Hahm (2016) 10.1021/acs.joc.6b00554 ECL, PNA, RCA, SBA

PNA, RCA, SBA

ECL >5 4 NaP 8.5

167

Rha(a1-1)aminopentanol

Martin (2013) 10.1021/ja401410y 1A10 >5 1 NaP 8.5

168

Rha(a1-3)Glc(b1-1)aminopentanol

Martin (2013) 10.1021/ja401410y 1A10 >5 2 NaP 8.5

169

Glc(a1-2)Glc(a1-1)aminopentanol

(Clostridium difficile)

Martin (2013) 10.1021/ja401410y ConA ConA >5 2 NaP 8.5

S21

170

Glc(b1-4)Glc(a1-2)Glc(a1-1)aminopentanol


Martin (2013) 10.1021/ja401410y ConA >5 3 NaP 8.5

171

Rha(a1-3)Glc(b1-4)Glc(a1-1)aminopentanol


Martin (2013) 10.1021/ja401410y 1A10 >5 3 NaP 8.5

172

Gal(b1-3)GalNAc(b1-3)Gal(a1-4)Gal(b1-4)Glc(b1-1)aminopentanol

ECL, PNA, RCA, SBA

PNA, RCA, SBA

BSL ECL >5 5 NaP 8.5

173

Neu5Ac(a2-8)Neu5Ac(a2-3)[GalNAc(b1-4)]Gal(b1-4)Glc(b1-1)aminopentanol

DBA, SBA,

WGA DBA, SBA WGA 5 NaP 8.5

174

Gal(a1-4)Gal(b1-4)Glc(b1-1)aminopentanol

Hofmann (2015) 10.1038/nature15388 BSL, SBA BSL, SBA RCA >5 3 NaP 8.5

175

GalNAc(a1-1)AminoLinker2

Schlegel (2011) 10.1002/cbic.201100511 BSL, DBA, SBA, WGA

BSL, DBA, SBA, WGA

2 1 NaP 8.5

S22

176

Fuc(a1-3)[Gal(b1-4)]GlcNAc(b1-1)AminoLinker2

Schlegel (2011) 10.1002/cbic.201100511 ECL, PNA, RCA, SBA,

UEA

BSL ECL, PNA, RCA, SBA,

UEA 3 NaP 8.5

177

GlcNAc(a1-2)Hep(a1-3)Hep(a1-1)aminopentanol


Reinhardt (2015) 10.1016/j.chembiol.2014.

11.016 WGA WGA >5 3 NaP 8.5

178

Hep(a1-3)Hep(a1-1)aminopentanol

Reinhardt (2015) 10.1016/j.chembiol.2014.

11.016 1 2 NaP 8.5

179

Gal(b1-4)Glc(b1-1)aminopentanol


SBA


SBA >5 2 NaP 8.5

180

GalNAc(b1-4)Gal(b1-4)Glc(b1-1)aminopentanol

DBA, SBA,

WGA DBA, SBA WGA >5 3 NaP 8.5

181

Neu5Ac(a2-3)Gal(b1-4)Glc(b1-1)aminopentanol

Fair (2015) 10.1039/c5cc01368b MAL I, WGA MAL I, WGA 2 3 NaP 8.5

S23

182

GalNAc-4-sulfate(b1-1)aminopentanol

Kandasamy (2014)

10.1039/c3cc48860h 1 1 NaP 8.5

183

IdoA-2,4-disulfate(a1-1)aminopentanol

Nonaka (2014) 10.1039/c3cc48860h;

ConA, DBA, ECL, PNA, RCA, SNL,

UEA

>5 1 NaP 8.5

184

IdoA(a1-3)GalNAc-4-sulfate(b1-1)aminopentanol

Kandasamy (2014)

10.1039/c3cc48860h 2 NaP 8.5

185

IdoA-2-sulfate(a1-3)GalNAc-4-sulfate(b1-1)aminopentanol

Kandasamy (2014)

10.1039/c3cc48860h ConA, SNL 2 NaP 8.5

186

IdoA(a1-3)GalNAc(b1-1)aminopentanol

Kandasamy (2014)

10.1039/c3cc48860h 1 2 NaP 8.5

187

GlcA(b1-4)Glc(b1-3)GlcA(b1-4)Glc(b1-1)aminoethanol



10.1016/j.chembiol.2016.09.016

>5 4 NaP 8.5

S24

188

Glc(b1-3)GlcA(b1-4)Glc(b1-1)aminoethanol



10.1016/j.chembiol.2016.09.016

>5 3 NaP 8.5

189

GalNAc(a1-1)Thr-Linker

BSL, DBA, SBA, WGA

BSL, DBA, SBA, WGA

SNL 5 1 NaP 8.5

190

Glc(b1-3)Glc(b1-3)[Glc(b1-6)]Glc(b1-3)Glc(b1-1)aminopentanol

(Candida spec.)

Weishaupt (2017)

10.1039/C7CC00520B >5 5 NaP 8.5

191

Glc(b1-3)Glc(b1-3)[Glc(b1-6)]Glc(b1-3)Glc(b1-3)Glc(b1-3)Glc(b1-3)Glc(b1-3)Glc(b1-1)aminopentanol

(Candida spec.)

Weishaupt (2017)

10.1039/C7CC00520B >5 9 NaP 8.5

192

Glc(b1-3)Glc(b1-3)[Glc(b1-6)]Glc(b1-3)Glc(b1-3)Glc(b1-3)Glc(b1-3)Glc(b1-3)Glc(b1-3)Glc(b1-3)Glc(b1-3)Glc(b1-3)Glc(b1-1)aminopentanol

(Candida spec.)

Weishaupt (2017)

10.1039/C7CC00520B >5 13 NaP 8.5

193

Glc(b1-3)Glc(b1-3)Glc(b1-3)Glc(b1-3)Glc(b1-3)Glc(b1-3)Glc(b1-3)Glc(b1-3)Glc(b1-3)Glc(b1-3)Glc(b1-3)Glc(b1-1)aminopentanol

(Candida spec.)

Weishaupt (2013)

10.1002/chem.201204518

>5 12 NaP 8.5

S25

194

L-PneNAc(a1-2)GlcA(b1-3)FucNAc(a1-3)D-FucNAc(b1-1)aminopentanol


Lisboa (2017) 10.1073/pnas.170687511

4 H16 >5 4 NaP 8.5

195

Mixture of: D-6d-xylHexpNAc-4-ulo(b1-1)aminopentanol (Sugp(b1-1)aminopentanol) and D-FucNAc(b1-1)aminopentanol


Lisboa (2017) 10.1073/pnas.170687511

4

BSL, DBA, RCA, SNL,

WGA 3 1 NaP 8.5

196

Mixture of: FucNAc(a1-3)D-6d-xylHexpNAc-4-ulo(b1-1)aminopentanol and FucNAc(a1-3)D-FucNAc(b1-1)aminopentanol


Lisboa (2017) 10.1073/pnas.170687511

4 H16 >5 2 NaP 8.5

197

FucNAc(a1-3)D-FucNAc(b1-1)aminopentanol


Lisboa (2017) 10.1073/pnas.170687511

4 H16 >5 2 NaP 8.5

198

GlcA(b1-4)FucNAc(a1-1)aminopentanol


Lisboa (2017) 10.1073/pnas.170687511

4 H16 >5 2 NaP 8.5

S26

199

Glc(b1-3)FucNAc(a1-1)aminopentanol


Lisboa (2017) 10.1073/pnas.170687511

4 H16 >5 2 NaP 8.5

200

L-PneNAc(a1-2)GlcA(b1-1)aminopentanol


Lisboa (2017) 10.1073/pnas.170687511

4 5 2 NaP 8.5

201

L-PneNAc(a1-1)aminopentanol


Lisboa (2017) 10.1073/pnas.170687511

4 H16 5 1 NaP 8.5

202

L-PneNAc(b1-1)aminopentanol


Lisboa (2017) 10.1073/pnas.170687511

4 >5 1 NaP 8.5

203

Gal(b1-4)[Glc(b1-6)]GlcNAc(b1-3)Gal(b1-1)aminopentanol




WGA >5 4 NaP 8.5

204

Glc(a1-4)Gal(a1-4)GlcA(b1-4)Glc(b1-1)aminoethanol


Schumann (2017) 10.1126/scitranslmed.aaf

5347 ConA ConA >5 4 NaP 8.5

S27

205

Glc(a1-4)Gal(a1-1)aminoethanol




206

GlcA(b1-4)Glc(b1-4)Glc(a1-4)Gal(a1-1)aminoethanol



5347 >5 4 NaP 8.5

207

Glc(a1-4)Gal(a1-4)GlcA(b1-4)Glc(b1-1)aminopentanol




208

Gal(a1-4)GlcA(b1-4)Glc(b1-4)Glc(a1-1)aminopentanol



5347 BSL, SBA BSL SBA >5 4 NaP 8.5

209

GlcA(b1-4)Glc(b1-4)Glc(a1-4)Gal(a1-1)aminopentanol



5347 >5 4 NaP 8.5

210

Glc(b1-4)Glc(a1-4)Gal(a1-4)GlcA(b1-1)aminopentanol



5347 >5 4 NaP 8.5

S28

211

Xyl(b1-4)Xyl(b1-1)aminopentanol

Schmidt (2015) 10.1002/chem.20150006

5 LM10 3 2 NaP 8.5

212

Xyl(b1-4)Xyl(b1-4)Xyl(b1-4)Xyl(b1-1)aminopentanol

Schmidt (2015) 10.1002/chem.20150006

5 LM10 2 4 NaP 8.5

213 Xyl(b1-4)Xyl(b1-4)Xyl(b1-4)Xyl(b1-4)Xyl(b1-4)Xyl(b1-1)aminopentanol

Schmidt (2015) 10.1002/chem.20150006

5 LM10 2 6 NaP 8.5

214 Xyl(b1-4)Xyl(b1-4)Xyl(b1-4)Xyl(b1-4)Xyl(b1-4)Xyl(b1-4)Xyl(b1-4)Xyl(b1-1)aminopentanol

Schmidt (2015) 10.1002/chem.20150006

5 LM10 2 8 NaP 8.5

215

Glc(b1-4)Glc(b1-4)Glc(b1-4)Glc(b1-1)aminopentanol

PNA >5 4 NaP 8.5

216

Glc(b1-3)GlcA(b1-4)Glc(b1-1)aminopentanol


Weishaupt (2016)

10.3762/bjoc.12.139 >5 3 NaP 8.5

217

GlcA(b1-4)Glc(b1-1)aminoethanol



10.1016/j.chembiol.2016.09.016

>5 2 NaP 8.5

S29

218

Glc(b1-3)GlcA(b1-1)aminoethanol



10.1016/j.chembiol.2016.09.016

>5 2 NaP 8.5

219

ManNAc(b1-3)FucNAc(a1-3)GalNAc(a1-4)Gal-2,3-pyruvate(a1-1)aminopentanol


Pereira (2015) 10.1002/anie.201504847 H16 >5 4 NaP 8.5

220

GlcA(b1-1)aminoethanol


10.1016/j.chembiol.2016.09.016

1 NaP 8.5

225

Glc(a1-4)GalNAc(b1-4)Man(a1-1)aminopentanol

(Toxoplasma gondii)

Goetze (2014) 10.1002/anie.201406706 ConA ConA >5 3 NaP 8.5

226

Glc(a1-4)GalNAc(b1-4)[Man(a1-2)Man(a1-6)]Man(a1-1)aminopentanol

(Toxoplasma gondii)


S30

227

Glc(a1-4)GalNAc(b1-4)[Man-6-PEtN(a1-2)Man(a1-6)]Man(a1-1)aminopentanol

(Toxoplasma gondii)


229

GalNAc(b1-4)Man(a1-1)aminopentanol

DBA, SBA,

WGA DBA, SBA,

WGA BSL, ECL, RCA, SNL

>5 2 NaP 8.5

230

GalNAc(b1-4)[Man-6-PEtN(a1-2)Man(a1-6)]Man(a1-1)aminopentanol

DBA, SBA,

WGA DBA, SBA,

WGA ConA, ECL 4 4 NaP 8.5

231

GalNAc(b1-4)[Man(a1-2)Man(a1-6)]Man(a1-1)aminopentanol

ConA, DBA, SBA, WGA

ConA, DBA, SBA, WGA

ECL, RCA 4 4 NaP 8.5

S31

232

GalNAc(b1-4)[Man-6-PEtN(a1-2)Man(a1-6)]Man-2-PEtN(a1-1)aminopentanol

DBA, SBA,

WGA DBA, SBA ConA WGA 2 4 NaP 8.5

233

GalNAc(b1-4)Man(a1-1)aminododecanol

DBA, SBA,

WGA DBA, SBA,

WGA ECL >5 2 NaP 8.5

234

GalNAc(b1-4)Man(a1-1)p-aminocyclohexanol

DBA, SBA,

WGA DBA, SBA,

WGA ConA, ECL >5 2 NaP 8.5

235

GlcA(b1-4)Glc(b1-3)GlcA(b1-1)aminoethanol



10.1016/j.chembiol.2016.09.016

>5 3 NaP 8.5

236

GlcA(b1-4)Glc(b1-3)Glc(b1-4)Glc(b1-1)aminoethanol and/or Glc(b1-4)Glc(b1-3)GlcA(b1-4)Glc(b1-1)aminoethanol

>5 4 NaP 8.5

237

Man(a1-1)aminopentanol

ConA ConA 1 NaP 8.5

S32

238

GlcNAc-6-P-phosphoaminopentanol(a1-3)GlcNAc-6-P-phosphoaminopentanol(a1-2)glyceric acid


Martin (2013b) 10.1039/c3cc43545h >5 2 NaP 8.5

239

GlcA(a1-3)Gal(a1-3)ManNAc(b1-4)Glc(b1-4)Glc(a1-1)aminopentanol


>5 5 NaP 8.5

240

GlcA(a1-3)Gal(a1-3)ManNAc-6-acetate(b1-4)Glc(b1-4)Glc(a1-1)aminopentanol


5 5 NaP 8.5, PBS

241

GlcA(a1-3)Gal(a1-1)aminopentanol


>5 2 NaP 8.5

242

Glc(b1-4)Glc(a1-1)aminopentanol

PNA >5 2 NaP 8.5

243

ManNAc(b1-4)Glc(b1-4)Glc(a1-1)aminopentanol


>5 3 NaP 8.5

S33

244

GalNAc(b1-3)GalNAc(b1-1)aminopentanol

DBA, SBA,

WGA SBA SNL DBA, WGA 3 2 NaP 8.5

245

Glc(b1-4)Gal(b1-4)Glc(b1-1)aminopentanol

Hofmann (2015) 10.1038/nature15388 RCA, SBA >5 3 NaP 8.5

247

Rha(a1-3)[Rha(a1-3)Glc(b1-4)]Glc(a1-2)Glc(a1-1)aminopentanol


Martin (2013) 10.1021/ja401410y ConA 1A10 >5 5 NaP 8.5

248

GlcNAc(a1-3)GlcNAc-6-P-phosphoaminopentanol(a1-2)glyceric acid


Martin (2013b) 10.1039/c3cc43545h WGA WGA >5 2 NaP 8.5

249

GlcNAc(a1-3)GlcNAc[(a1-2)glyceric acid](6-P-6)GlcNAc(a1-3)GlcNAc-6-P-phosphoaminopentanol(a1-2)glyceric acid


Martin (2013b) 10.1039/c3cc43545h WGA WGA >5 4 NaP 8.5

S34

250

Man(a1-2)Man(a1-2)[Gal(b1-4)]Man(a1-1)aminoethanol


Anish (2013) 10.1021/cb400602k ConA, ECL, PNA, RCA,

SBA

ConA, ECL, PNA, RCA

SBA >5 4 NaP 8.5

251

Glc(b1-3)Gal(b1-4)Man(a1-1)aminopentanol

(Leishmania chagasi)

Anish (2013) 10.1021/cb400602k 3 3 NaP 8.5

252

Rha(a1-2)Rha(a1-2)Rha(a1-1)aminopentanol

(Klebsiella pneumoniae)

Seeberger (2017) 10.1002/anie.201700964 1A10 >5 3 NaP 8.5

253

GalNAc-2,3-Oxazolidinone(a1-4)GalNAc-2,3-Oxazolidinone(a1-1)aminopentanol

BSL, SBA, SNL, WGA

>5 2 NaP 8.5

S35

254

Glc(a1-2)Glc(a1-3)[FucNAc(a1-3)GalNAc(b1-4)]ManNAcA(b1-1)aminopentanol


Seeberger (2017b)

10.3762/bjoc.13.19 ConA ConA H16 >5 5 NaP 8.5

255

Glc(a1-2)Glc(a1-3)[Gal(a1-3)FucNAc(a1-3)GalNAc(b1-4)]ManNAcA(b1-1)aminopentanol


Seeberger (2017b)

10.3762/bjoc.13.19 BSL, ConA,

SBA BSL ConA, SBA H16 >5 6 NaP 8.5

256

Araf(a1-3)[Araf(a1-5)]Araf(a1-5)Araf(a1-1)aminopentanol


>5 4 NaP 8.5

257

Man(a1-5)Araf(a1-3)[Man(a1-5)Araf(a1-5)]Araf(a1-5)Araf(a1-1)aminopentanol


Hahm (2017) 10.1073/pnas.170014111


S36

258

GalNAc-3,4-diacetate(a1-4)GalNAc-3-acetate(a1-4)GalNAc-3-acetate(a1-4)GalNAc-3-acetate(a1-1)aminopentanol

DBA >5 4 PBS

Abbreviations: NaP 8.5 (50 mM sodium phosphate buffer, pH 8.5), PBS (phosphate buffered saline); For lectins see Supporting Table 1

S37

Monosaccharide representations:

S38

All synthetic glycans meet the standards of purity set by the Jounral of Organic Chemistry which means that 1H-, 13C-NMR as well as high resolution mass spec analyses were performed. The following publications describe the synthesis of the majority of glycans as referred to in the table.

References Anish (2013) ACS Chem. Biol. 8 2412−2422 (2013) 10.1021/cb400602k

Anish (2013b) Angew. Chem. Int. Ed. 52 9524 –9528 (2013) 10.1002/anie.201301633 Calin (2013) Chem. Eur. J. 19 3995 – 4002 (2013) 10.1002/chem.201204394 Fair (2015) Chem. Commun. 51 6183-6185 (2015) 10.1039/c5cc01368b Goetze (2014) Angew. Chem. Int. Ed. 53 13701 –13705 (2014) 10.1002/anie.201406706 Hahm (2016) J. Org. Chem. 81 5866–5877 (2016) 10.1021/acs.joc.6b00554 Hahm (2017) Proc .Natl. Acad. Sci. USA 114 E3385–E3389 (2017) 10.1073/pnas.1700141114 Hanashima (2007) Chem. Asian J. 2 1447 – 1459 (2007) 10.1002/asia.200600424 Hanashima (2007b) Org. Lett. 9 1777–1779 (2007) 10.1021/ol0704946 Hofmann (2015) Nature 526 241–244 (2015) 10.1038/nature15388 Kandasamy (2013) Chem. Commun. 49 4453-445 (2013) 10.1039/c3cc00042g Kandasamy (2014) Chem. Commun. 50 1875-1877 (2014) 10.1039/c3cc48860h Leonori (2013) Beilstein J. Org. Chem. 9 332–341 (2013) 10.3762/bjoc.9.38 Lisboa (2017) Proc .Natl. Acad. Sci. USA 114 11063-11068 (2017) 10.1073/pnas.1706875114 Martin (2013) J. Am. Chem. Soc. 135 9713−9722 (2013) 10.1021/ja401410y Martin (2013b) Chem. Commun. 49 7159-7161 (2013) 10.1039/c3cc43545h Nonaka (2014) Proc .Natl. Acad. Sci. USA 111 8173-8178 (2014) 10.1073/pnas.1319870111 Parameswarappa (2016) Cell Chem. Biol. 23 1407-1416 10.1016/j.chembiol.2016.09.016 Pereira (2015) Angew. Chem. Int. Ed. 54 10016 –10019 (2015) 10.1002/anie.201504847 Reinhardt (2015) Chem. Biol. 22 38–49 (2015) 10.1016/j.chembiol.2014.11.016 Schlegel (2011) ChemBioChem 12 2791 – 2800 (2011) 10.1002/cbic.201100511 Schmidt (2015) Chem. Eur. J. 21 5709 – 5713 (2015) 10.1002/chem.201500065 Schumann (2017) Sci. Transl. Med. Advance Online Publication 10.1126/scitranslmed.aaf5347 Seeberger (2017) Angew. Chem. Int. Ed. 56 13973 –13978 (2017) 10.1002/anie.201700964 Seeberger (2017b) Beilstein J. Org. Chem. 13 164–173 (2017) 10.3762/bjoc.13.19 Weishaupt (2013) Chem. Eur. J. 19 12497 – 12503 (2013) 10.1002/chem.201204518 Weishaupt (2016) Beilstein J. Org. Chem. 12 1440-1446 (2016) 10.3762/bjoc.12.139 Weishaupt (2017) Chem. Commun. Advance Online Publication 10.1039/C7CC00520B Yang (2012) Chem. Sci. 3 896-899 (2012) 10.1039/c1sc00804h Yang (2013) J. Am. Chem. Soc. 135 6262–6271 (2013) 10.1021/ja401164s

S39

Table S3: Additional published glycans in the library

Glycan ID

Glycan Structure (Microbial origin if applicable)

DOI Paper Saccharides

number

3

Man(a1-2)[Man(a1-6)Man(a1-6)]Man(a1-6)Man(a1-6)Man(a1-1)thiopentanol


10.1021/jo061233x 6

4

Araf(a1-5)Araf(a1-3)[Araf(a1-5)]Araf(a1-5)Araf(a1-5)Araf(a1-1)thiohexanol


10.1021/jo061233x 6

14

Fuc(a1-2)Gal(b1-3)GalNAc(b1-3)Gal(a1-4)Gal(b1-4)Glc(b1-1)thiopentanol

10.1021/ja069218x 6

16

GlcNAc(b1-1)thiopentanol

10.1002/cbic.201000020 1

17

Man(a1-2)Man(a1-1)thiopentanol

10.1002/cbic.201000020 2

19

Gal(b1-4)GlcNAc(b1-2)Man(a1-1)thiopentanol

10.1002/cbic.201000020 3

S40

21

Man(a1-6)Man(a1-4)GlcN(a1-6)Ino(1-P)phosphothiohexanol

(Plasmodium falciparum)

10.1038/nchembio.75 4

22

Man(a1-2)Man(a1-6)Man(a1-4)GlcN(a1-6)Ino(1-P)phosphothiohexanol

(Plasmodium falciparum)

10.1038/nchembio.75 5

23

Gal(b1-4)Glc(b1-1)thiopentanol

10.1002/cbic.201000020 2

24

Gal(a1-3)[Fuc(a1-2)]Gal(b1-4)GlcNAc(b1-1)thiopentanol

10.1002/cbic.201000020 4

25

Gal(b1-4)GlcNAc(b1-1)thiopentanol

10.1002/cbic.201000020 2

26

Gal(b1-2)Gal(b1-1)thiopentanol

10.1002/cbic.201000020 2

27

Gal(b1-3)Gal(b1-4)Glc(b1-1)thiopentanol

10.1002/cbic.201000020 3

28

Fuc(a1-2)Gal(b1-4)GlcNAc(b1-1)thiopentanol

10.1002/cbic.201000020 3

S41

29

Gal(b1-3)GlcNAc(b1-1)thiopentanol

10.1002/cbic.201000020 2

30

Gal-3-sulfate(b1-1)ThiolinkerA

10.1002/cbic.201000020 1

31

Gal-6-sulfate(b1-1-)ThiolinkerA

10.1002/cbic.201000020 1

32

GalNAc(b1-4)GlcNAc(b1-1)thiopentanol

10.1002/cbic.201000020 2

33

Gal(b1-3)GalNAc(a1-1)thiopentanol

10.1002/cbic.201000020 2

51

Gal(a1-1)PEG-Thiol

10.1021/bi8022703 1

52

Anthrose(b1-3)Rha(a1-3)Rha(a1-2)Rha(a1-1)thiopentanol

(Bacillus anthracis)

10.1111/j.1365-2672.2008.04129.x

4

53

Rha(a1-2)Rha(a1-1)thiopentanol

10.1111/j.1365-2672.2008.04129.x

2

S42

54

Rha(a1-1)thiopentanol

10.1111/j.1365-2672.2008.04129.x

1

55

Anthrose(b1-3)Rha(a1-3)Rha(a1-1)thiopentanol


10.1111/j.1365-2672.2008.04129.x

4

56

Rha(a1-3)Rha(a1-1)thiopentanol

10.1111/j.1365-2672.2008.04129.x

2

57

2-methyl-4-N-acetyl-4,6-dideoxyglucose(b1-3)Rha(a1-3)Rha(a1-2)Rha(a1-1)thiopentanol

10.1111/j.1365-2672.2008.04129.x

4

58

Anthrose(b1-3)Rha(a1-1)thiopentanol


10.1111/j.1365-2672.2008.04129.x

2

S43

59

Anthrose(b1-1)thiopentanol


10.1111/j.1365-2672.2008.04129.x

1

60

2-Demethyl-Anthrose(b1-1)thiopentanol

10.1111/j.1365-2672.2008.04129.x

1

61

2-Demethyl-Anthrose(b1-3)Rha(a1-1)thiopentanol

10.1111/j.1365-2672.2008.04129.x

2

S44

62

2-Demethyl-Anthrose(b1-3)Rha(a1-3)Rha(a1-2)Rha(a1-1)thiopentanol

10.1111/j.1365-2672.2008.04129.x

4

63

2-methyl-4-N-tert-butanoyl-4,6-dideoxyglucose(b1-3)Rha(a1-3)Rha(a1-2)Rha(a1-1)thiopentanol

10.1111/j.1365-2672.2008.04129.x

4

64 2-methyl-4-N-isobutanoyl-4,6-dideoxyglucose(b1-3)Rha(a1-3)Rha(a1-2)Rha(a1-1)thiopentanol

10.1111/j.1365-2672.2008.04129.x

4

S45

65

2-methyl-4-N-(3R-3-hydroxybutanoyl)l-4,6-dideoxyglucose(b1-3)Rha(a1-3)Rha(a1-2)Rha(a1-1)thiopentanol

10.1111/j.1365-2672.2008.04129.x

4

S46

66

2-methyl-4-N-(3S-3-hydroxybutanoyl)l-4,6-dideoxyglucose(b1-3)Rha(a1-3)Rha(a1-2)Rha(a1-1)thiopentanol

10.1111/j.1365-2672.2008.04129.x

4

67

Man(a1-2)[Araf(a1-5)Araf(a1-3)[Araf(a1-5)]Araf(a1-5)Araf(a1-5)Araf(a1-2)[Man(a1-6)]Man(a1-6)]Man(a1-6)Man(a1-1)thiohexanol

l(Mycobacterium tuberculosis)

10.1021/jo061233x 13

68

Araf(a1-5)Araf(a1-1)thiohexanol


10.1021/jo061233x 2

S47

106

GlcN-6,N-disulfate(a1-4)IdoA-2-sulfate(a1-4)GlcN-6,N-disulfate(a1-4)IdoA-2-sulfate(a1-4)GlcN-6,N-disulfate(a1-4)IdoA-2-sulfate(a1-1)AminoLinker1

10.1002/chem.200601103 6

107

GlcNAc-6-sulfate(a1-4)IdoA-2-sulfate(a1-4)GlcNAc-6-sulfate(a1-4)IdoA-2-sulfate(a1-4)GlcNAc-6-sulfate(a1-4)IdoA-2-sulfate(a1-1)aminopentanol

10.1002/chem.200601103 6

108

GlcN-N-sulfate(a1-4)IdoA(a1-4)GlcN-N-sulfate(a1-4)IdoA(a1-4)GlcN-N-sulfate(a1-4)IdoA(a1-1)aminopentanol

10.1002/chem.200601103 6

109

GlcNAc(a1-4)IdoA(a1-4)GlcNAc(a1-4)IdoA(a1-4)GlcNAc(a1-4)IdoA(a1-1)aminopentanol

10.1002/chem.200601103 6

110

IdoA-2-sulfate(a1-4)GlcN-N-sulfate(a1-4)IdoA-2-sulfate(a1-4)GlcN-N-sulfate(a1-4)IdoA-2-sulfate(a1-4)GlcN-N-sulfate(a1-1)aminopentanol

10.1002/chem.200601103 6

111 IdoA-2-sulfate(a1-4)GlcNAc(a1-4)IdoA-2-sulfate(a1-4)GlcNAc(a1-4)IdoA-2-sulfate(a1-4)GlcNAc(a1-1)aminopentanol

10.1002/chem.200601103 6

S48

112

GlcN-6,N-disulfate(a1-4)IdoA-2-sulfate(a1-4)GlcN-6,N-disulfate(a1-4)IdoA-2-sulfate(a1-1)AminoLinker1

10.1002/chem.200601103 4

113

IdoA(a1-4)GlcN-N-sulfate(a1-4)IdoA(a1-4)GlcN-N-sulfate(a1-1)aminopentanol

10.1002/chem.200601103 4

114

IdoA(a1-4)GlcN(a1-4)GlcA(b1-3)GlcNAc(a1-1)AminoLinker1

10.1002/chem.200601103 4

115

GlcN-6,N-disulfate(a1-4)IdoA-2-sulfate(a1-1)AminoLinker1

10.1002/chem.200601103 2

116

IdoA-2,4-disulfate(a1-1)AminoLinker1

10.1002/chem.200601103 1

S49

117

GlcN-6,N-disulfate(a1-1)AminoLinker1

10.1002/chem.200601103 1

221

Glc(a1-4)GalNAc(b1-4)[Man-6-PEtN(a1-2)Man(a1-6)]Man(a1-4)GlcN(a1-6)Ino(1-P)phosphothiohexanol

(Toxoplasma gondii)

10.1002/anie.201406706 7

222

GalNAc(b1-4)[Man-6-PEtN(a1-2)Man(a1-6)]Man(a1-4)GlcN(a1-6)Ino(1-P)phosphothiohexanol

(Toxoplasma gondii)

10.1002/anie.201406706 6

223

GalNAc(b1-4)[Man-6-PEtN(a1-2)Man(a1-6)]Man-2-PEtN(a1-4)GlcN(a1-6)Ino(1-P)phosphothiohexanol

10.1002/anie.201406706 6

S50

224

GalNAc(b1-4)[Man(a1-2)Man(a1-6)]Man(a1-4)GlcN(a1-6)Ino(1-P)phosphothiohexanol

(Toxoplasma gondii)

10.1002/anie.201406706 6

228

GlcN(a1-6)Ino(1-P)phosphothiohexanol

10.1002/anie.201406706 2

259

Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P)phosphoaminopentanol


10.1039/C7SC04521B 8

260

Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P)phosphoaminopentanol


10.1039/C7SC04521B 12

261

Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P)phosphoaminopentanol


10.1039/C7SC04521B 16

S51

262

Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P-3)Ribf(b1-1)ribitol(5-P)phosphoaminopentanol


10.1039/C7SC04521B 20

264

Gal(b1-3)Gal(b1-4)Glc(b1-1)aminopentanol

10.1038/nature15388 3

265

Glc(b1-3)Gal(b1-4)Glc(b1-1)aminopentanol

10.1038/nature15388 3

267

Araf(a1-5)Araf(a1-5)Araf(a1-6)Man(a1-6)Man(a1-6)Man(a1-1)aminopentanol


10.1073/pnas.1700141114 6

268

Gal(b1-3)[Rha(a1-4)]GalA(a1-2)Rha(a1-2)Rha(a1-2)Rha(a1-1)aminopentanol


10.1002/anie.201700964 6

269

Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)GlcNAc(b1-1)aminopentanol

10.1016/j.chempr.2016.12.004 4

S52

270

Gal(b1-4)GlcNAc(b1-3)[Gal(b1-4)GlcNAc(b1-6)]Gal(b1-4)GlcNAc(b1-1)aminopentanol

10.1038/nature15388 6

271

Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)GlcNAc(b1-1)aminopentanol

10.1038/nature15388 6

272

Gal-6-sulfate(b1-4)GlcNAc(b1-3)Gal-6-sulfate(b1-4)GlcNAc(b1-1)aminopentanol

10.1016/j.chempr.2016.12.004 4

273

Gal-3,6-disulfate(b1-4)GlcNAc(b1-3)Gal-6-sulfate(b1-4)GlcNAc(b1-1)aminopentanol

10.1016/j.chempr.2016.12.004 4

274

Gal(b1-4)GlcNAc-6-sulfate(b1-3)Gal(b1-4)GlcNAc-6-sulfate(b1-1)aminopentanol

10.1016/j.chempr.2016.12.004 4

275

Gal-6-sulfate(b1-4)GlcNAc-6-sulfate(b1-3)Gal-6-sulfate(b1-4)GlcNAc-6-sulfate(b1-1)aminopentanol

10.1016/j.chempr.2016.12.004 4

S53

276

L-Araf(a1-3)[Xyl(b1-4)Xyl(b1-4)]Xyl(b1-4)Xyl(b1-4)Xyl(b1-4)Xyl(b1-1)aminopentanol

10.1002/chem.201500065 7

277

L-Araf(a1-3)[L-Araf(a1-3)[Xyl(b1-4)]Xyl(b1-4)Xyl(b1-4)Xyl(b1-4)]Xyl(b1-4)Xyl(b1-1)aminopentanol

10.1002/chem.201500065 8

278

L-Araf(a1-3)[Xyl(b1-4)]Xyl(b1-4)Xyl(b1-1)aminopentanol

10.1002/chem.201500065 4

279

L-Araf(a1-3)Xyl(b1-4)Xyl(b1-4)Xyl(b1-1)aminopentanol

10.1002/chem.201500065 4

S54

280

Xyl(b1-2)L-Araf(a1-3)[Xyl(b1-4)]Xyl(b1-4)Xyl(b1-1)aminopentanol

10.1002/chem.201500065 5

281

L-Araf(a1-3)[L-Araf(a1-3)[Xyl(b1-4)Xyl(b1-4)]Xyl(b1-4)]Xyl(b1-4)Xyl(b1-1)aminopentanol

10.1002/chem.201500065 7

282

L-Araf(a1-3)[L-Araf(a1-3)[Xyl(b1-4)]Xyl(b1-4)Xyl(b1-4)]Xyl(b1-4)Xyl(b1-1)aminopentanol

10.1002/chem.201500065 7

S55

285

Gal(b1-3)[Rha(a1-4)]GalA(a1-1)aminopentanol


10.1002/anie.201700964 3

286

Gal(b1-3)GalA(a1-1)aminopentanol


10.1002/anie.201700964 2

287

Gal(b1-3)GalA(b1-1)aminopentanol


10.1002/anie.201700964 2

288

Rha(a1-3)Gal(b1-1)aminopentanol


10.1002/anie.201700964 2

289

Rha(a1-4)GalA(a1-1)aminopentanol


10.1002/anie.201700964 2

290

Glc(b1-3)Rha(a1-3)Rha(a1-1)aminopentanol


10.1021/jacs.7b07836 3

291

GlcA(a1-6)Glc(a1-2)Rha(a1-3)Rha(a1-1)aminopentanol


10.1021/jacs.7b07836 4

S56

292

Rha(a1-3)Rha(a1-1)aminopentanol

10.1021/jacs.7b07836 2

293

Rha(b1-4)Glc(b1-3)Rha(a1-3)Rha(a1-1)aminopentanol


10.1021/jacs.7b07836 4

294

Rha(b1-4)Glc(b1-1)aminopentanol


10.1021/jacs.7b07836 2

295

GlcA(a1-6)Glc(a1-1)aminopentanol


10.1021/jacs.7b07836 2

296

GlcA(a1-6)Glc(a1-2)[Rha(b1-4)Glc(b1-3)]Rha(a1-3)Rha(a1-1)aminopentanol


10.1021/jacs.7b07836 6

297

Fuc(a1-2)Gal(b1-3)GalNAc(b1-3)Gal(a1-4)Gal(b1-4)Glc(b1-1)aminopentanol

10.1038/ncomms12482 6

299

Glc(a1-3)Glc(a1-3)Glc(a1-3)Glc(a1-6)Glc(a1-1)aminopentanol

10.1038/ncomms12482 5

S57

300


10.1038/ncomms12482 5

301


10.1038/ncomms12482 5

302


10.1038/ncomms12482 5

303

Glc(a1-6)Glc(a1-4)Glc(a1-4)Glc(a1-1)aminopentanol

10.1038/ncomms12482 4

304

Glc(a1-3)Glc(a1-3)Glc(a1-3)Glc(a1-1)aminopentanol

10.1038/ncomms12482 4

305

Gal(b1-3)GalNAc(b1-4)Gal(b1-4)Glc(b1-1)aminopentanol

10.1039/C5CC01368B 4

306


10.1039/C5CC01368B 4

S58

307


10.1039/C5CC01368B 4

308

Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)Glc(b1-1)aminopentanol

10.1039/C5CC01368B 6

309

Neu5Ac(a2-3)Gal(b1-3)GalNAc(b1-4)Gal(b1-4)Glc(b1-1)aminopentanol

10.1039/C5CC01368B 5

310

Neu5Ac(a2-3)Gal(b1-3)GlcNac(b1-3)Gal(b1-4)Glc(b1-1)aminopentanol

10.1039/C5CC01368B 5

311

Neu5Ac(a2-3)Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)Glc(b1-1)aminopentanol

10.1039/C5CC01368B 5

312

Neu5Ac(a2-3)Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)GlcNAc(b1-3)Gal(b1-4)Glc(b1-1)aminopentanol

10.1039/C5CC01368B 7

S59

314

LegA5Ac(b2-1)thioethanol

(Legionella pneumophila)

10.1021/jacs.5b00455 1

315

LegA5Ac7Ac(b2-1)thioethanol

(Legionella pneumophila)

10.1021/jacs.5b00455 1

320

GalA(a1-1)thiohexanol

10.1039/C3SC53362J 1

321

Gal-4,6-pyruvate(b1-1)thiohexanol

10.1039/C3SC53362J 1

322

Galf(b1-3)[AAT(a1-4)]GalNAc(a1-3)Gal-4,6-pyruvate(b1-1)thiohexanol

(Bacteroides fragilis)

10.1039/C3SC53362J 4

323

AAT(a1-4)GalA(a1-3)GalA(a1-1)thioethanol


10.1039/C3SC53362J 3

S60

325

Glc(a1-4)Gal(a1-4)GlcA(b1-4)Glc(b1-4)Glc(a1-4)Gal(a1-1)aminoethanol


10.1126/scitranslmed.aaf5347 6

326

Gal(a1-4)GlcA(b1-4)Glc(b1-4)Glc(a1-4)Gal(a1-1)aminoethanol



327

Glc(b1-4)Glc(b1-4)Glc(a1-4)Gal(a1-1)aminoethanol



328

Glc(b1-4)Glc(a1-4)Gal(a1-1)aminoethanol



329

Glc(b1-4)Glc(b1-4)Glc(b1-1)aminopentanol

10.1039/C5OB02226F 3

330

Glc(b1-4)Glc(b1-4)Glc(b1-4)Glc(b1-4)Glc(b1-1)aminopentanol

10.1039/C5OB02226F 5

S61

331

Glc(b1-4)[Xyl(a1-6)]Glc(b1-4)Glc(b1-1)aminopentanol

10.1039/C5OB02226F 4

332

Glc(b1-4)[Xyl(a1-6)]Glc(b1-4)[Xyl(a1-6)]Glc(b1-4)Glc(b1-1)aminopentanol

10.1039/C5OB02226F 6

333

Glc(b1-4)[Xyl(a1-6)]Glc(b1-4)[Xyl(a1-6)]Glc(b1-4)[Xyl(a1-6)]Glc(b1-4)Glc(b1-1)aminopentanol

10.1039/C5OB02226F 8

S62

334

Glc(b1-4)[Xyl(a1-6)]Glc(b1-4)Glc(b1-4)[Xyl(a1-6)]Glc(b1-4)Glc(b1-1)aminopentanol

10.1039/C5OB02226F 7

335

Xyl(a1-6)Glc(b1-4)[Xyl(a1-6)]Glc(b1-4)Glc(b1-4)Glc(b1-1)aminopentanol

10.1039/C5OB02226F 6

336

Gal(b1-3)Gal(b1-3)Gal(b1-1)aminopentanol

10.1021/acs.orglett.5b02185 3

337

Gal(b1-3)Gal(b1-3)Gal(b1-3)Gal(b1-1)aminopentanol

10.1021/acs.orglett.5b02185 4

338

Gal(b1-6)Gal(b1-6)Gal(b1-1)aminopentanol

10.1021/acs.orglett.5b02185 3

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339

Gal(b1-3)[Gal(b1-6)]Gal(b1-3)Gal(b1-1)aminopentanol

10.1021/acs.orglett.5b02185 4

340

Gal(b1-3)[Gal(b1-6)Gal(b1-6)]Gal(b1-3)Gal(b1-1)aminopentanol

10.1021/acs.orglett.5b02185 5

341

Gal(b1-3)[Gal(b1-6)Gal(b1-6)Gal(b1-6)]Gal(b1-3)Gal(b1-1)aminopentanol

10.1021/acs.orglett.5b02185 6

342

Gal(b1-3)Gal(b1-3)[Gal(b1-6)Gal(b1-6)]Gal(b1-3)Gal(b1-1)aminopentanol

10.1021/acs.orglett.5b02185 6

343

L-Araf(a1-3)Gal(b1-3)Gal(b1-3)Gal(b1-1)aminopentanol

10.1021/acs.orglett.5b02185 4

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344

Gal(b1-3)[L-Araf(a1-6)]Gal(b1-3)Gal(b1-1)aminopentanol

10.1021/acs.orglett.5b02185 4

345

L-Araf(a1-3)[Gal(b1-6)]Gal(b1-3)Gal(b1-1)aminopentanol

10.1021/acs.orglett.5b02185 4

346

Gal(b1-6)[L-Araf(a1-3)]Gal(b1-6)[Gal(b1-3)]Gal(b1-3)Gal(b1-1)aminopentanol

10.1021/acs.orglett.5b02185 6

347

Gal(b1-6)[L-Araf(a1-3)]Gal(b1-6)Gal(b1-1)aminopentanol

10.1021/acs.orglett.5b02185 4

S65

348

L-Araf(a1-3)Gal(b1-6)[Gal(b1-3)]Gal(b1-3)Gal(b1-1)aminopentanol

10.1021/acs.orglett.5b02185 5

349

Gal(b1-6)[L-Araf(a1-3)]Gal(b1-6)Gal(b1-6)[Gal(b1-3)]Gal(b1-3)Gal(b1-1)aminopentanol

10.1021/acs.orglett.5b02185 7

350

Glc(b1-4)FucNAc(a1-3)D-FucNAc(b1-1)aminopentanol


10.1073/pnas.1706875114 3

351

GlcA(b1-3)[Glc(b1-4)]FucNAc(a1-1)aminopentanol


10.1073/pnas.1706875114 3

S66

352

GlcA(b1-3)[Glc(b1-4)]FucNAc(a1-3)D-FucNAc(b1-1)aminopentanol


10.1073/pnas.1706875114 4

353

L-PneNAc(a1-2)GlcA(b1-3)[Glc(b1-4)]FucNAc(a1-3)D-FucNAc(b1-1)aminopentanol


10.1073/pnas.1706875114 5

354

L-PneNAc(a1-2)GlcA(b1-3)[Glc(b1-4)]FucNAc(a1-3)QuiNAc(b1-1)aminopentanol


10.1073/pnas.1706875114 5

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Table S4: Summary of linkers of glycans in the library

Thioethanol

Thiopentanol

Thiohexanol

Phosphothiohexanol

PEG-Thiol

ThiolinkerA

Aminoethanol

Aminopentanol

Aminohexanol

Aminododecanol

p-Aminocyclohexanol

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Aminolinker1

Aminolinker2

Thr/Threonine

Phosphoethanolamine

Phosphoaminopentanol

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Table S5: Estimated Kd values and avidity ranking for Burkholderia lectins towards aminopentanol-linked mannosides and heptosides

BC2L-A Rankd, BC2L-A BC2L-C-ct Rankd, BC2L-C-ct

Glycan Kd a Kd

Intensity

1 mg/mL

Intensity

0.4 mg/mL Kd

a Kd Intensity

1 mg/mL

Intensity

0.4 mg/mL

082 7.69 (0.280) ± 4.91 (0.178) 7 5 5 112.9 (4.53) ± 8.30 (0.33) 7 4 5

083 4.58 (0.167) ± 1.77 (0.0644) 3 4 4 97.2 (3.90) ±11.10 (0.45) 4 5 4

085 3.89 (0.141) ± 0.622 (0.0226) 2 2 2 64.53 (2.59) ± 15.59 (0.63) 1 3 2

226 5.90 (0.215) ± 2.66 (0.0970) 6 7 7 118.89 (4.77) ± 15.91 (0.64) 8 7 7

231 4.77 (0.173) ± 2.36 (0.0860) 4 6 6 110.02 (4.42) ± 13.46 (0.54) 6 6 6

237 3.66 (0.133) ± 1.30 (0.0474) 1 3 3 67.77 (2.72) ± 15.83 (0.64) 2 2 1

257 5.89 (0.214) ± 2.19 (0.0800) 5 1 1 82.47 (3.31) ± 18.58 (0.75) 3 1 3

092 n.d. b 12 12 >1050 (>42) c 14 13 14

093 n.d. 13 13 >1050 (>42) 15 16 16

094 382.9 (13.9) ± 112.6 (4.09) 11 11 11 301 (12.08) ± 116 (4.68) 11 11 11

095 n.d. 14 13 >1050 (>42) 13 14 13

096 n.d. 16 15 686 (27.54) ± 246 (9.9) 12 12 12

097 87.12 (3.17) ± 55.9 (2.03) 8 10 10 128 (5.12) ± 13.0 (0.52) 9 10 10

100 124.0 (4.51) ± 37.9 (1.38) 9 8 8 107 (4.31) ± 27 (1.1) 5 8 9

178 147.9 (5.48) ± 72.9 (2.65) 10 9 9 130 (5.22) ± 16.49 (0.66) 10 9 8

158 n.d. 15 16 >1050 (>42) 16 15 15

a Mean of three Kd values calculated from three glycan experiments. Format for Kd values: µg/mL (µM) ± SD from three experiments; molar Kd is based on the

concentration of the dimer with molecular weights of 27.5 kDa for BC2L-A and 24.9 kDa for BC2L-C-ct)..

b n.d. Signal intensities too low to determine dissociation constant (highest fluorescence intensities less than 5% of saturation intensity for 257)

c The value given for BC2L-C-ct is >1050 µg/mL (>42 µM) if not at least two data points of the concentration series were above the calculated Kd

d Ranking was performed from lowest to highest Kd values and from highest to lowest fluorescence intensities

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3. Supporting Figures

Figure S1: Cluster analysis of compounds of the MPS and the Imperial College London

Glycosciences Laboratory libraries (11). Each line terminus represents one glycan with MPS

glycans colored in orange, Imperial College glycans in blue, and structures contained in both

sets as well as inner branches leading to structures of both sets colored in green.

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Figure S2: Binding of eleven plant lectins to a glycan array printed with 140 synthetic

compounds. Values are the mean ± SD of four spots from two glycan array experiments

relative to the highest intensity glycan for each lectin. Glycans are arranged with expected

binders for each lectin to the left and color-coded according to SI Appendix Table S1 (see

Figure S3 for identical glycan arrangement for all lectins). Structures or class of strong

additional glycan ligands are indicated within the graphs. For several lectins, F1 represents

mono-D-FucNAc 102, F2 the mixture of mono-D-FucNAc with the oxidized form Sugp (glycan

195). For WGA, glycans marked with ‘a’ contain an internal GlcNAc residue that may mediate

binding (21). The complete information on which glycan was bound by which lectin can be

found in the list of glycans (SI Appendix Table S2).

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Figure S3: Comparison of lectin binding profiles for eleven plant lectins to a glycan array printed

with 140 synthetic compounds. Values are the mean ± SD of four spots from two glycan array

experiments relative to the highest intensity glycan for each lectin. Glycans are arranged

identically in each panel, see Figure S2 for a comparison between observed and predicted

binding. All intensity values are listed in the Supporting Data files.

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Figure S4: Binding of antibodies from the sera of 15 healthy individuals to the complete set of 140 immobilized glycans. Data are presented as

boxplots with each value being the mean of four spots from two glycan experiments normalized to the mean binding signal of each experiment. The

black bar represents the median, the red bar the mean fluorescence intensity of the 15 sera.

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Figure S5: Binding of Fc-Fusion protein DC-SIGN-D to selected compounds on the 140

compound glycan array. All signals are normalized to the intensity of LeY tetrasaccharide 157

at a protein concentration of 25 µg/mL. Error bars represent SD of four spots from two glycan

array experiments.

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Figure S6: Complete binding profile of DC-SIGN-T on 140 glycan array at 25 µg/mL. Each

value is the mean of four spots from two glycan array experiments normalized to the intensity

of LeY tetrasaccharide 157 at 25 µg/mL. Only structures of glycans that are not included in

main text Figure 5 are shown. Tetrasaccharide 250 is identical to 82 except that it bears an

aminoethanol instead of an aminopentanol linker.

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Figure S7: Complete binding profile of DC-SIGN-T on 140 glycan array at 5 µg/mL. Each value

is the mean of four spots from two glycan array experiments normalized to the intensity of LeY

tetrasaccharide 157 at 25 µg/mL.

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Figure S8: Complete binding profile of DC-SIGN-T on 140 glycan array at 1 µg/mL. Each value

is the mean of four spots from two glycan array experiments normalized to the intensity of LeY

tetrasaccharide 157 at 25 µg/mL.

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Figure S9: Complete binding profile of DC-SIGN-D on 140 glycan array at 25 µg/mL. Each


of LeY tetrasaccharide 157 at 25 µg/mL. Only structures of glycans that are not included in SI

Appendix Figure S3 are shown. Tetrasaccharide 250 is identical to 82 except that it bears an

aminoethanol instead of an aminopentanol linker.

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of LeY tetrasaccharide 157 at 25 µg/mL.

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of LeY tetrasaccharide 157 at 25 µg/mL.

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Figure S12: Interaction of DC-SIGN-T with immobilized LPS inner core diheptoside 178.

Sensorgrams (a) and the resulting saturation plot (b) are shown. (c) Resulting apparent (app)

Kd and Rmax values are indicated. Kd is the mean ± SD of two independent measurements.

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Figure S13: Binding of BC2L-A and BC2L-C-ct at 1 mg/mL to the 140 compound glycan array. Each value in the barchart is the mean ± SD of six

spots from three glycan experiments with the insets showing representative field images. Terminal mannosides and heptosides are labeled with M

and H, respectively. M* denotes terminal mannosides in which this saccharide is modified with a phosphoethanolamine.

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References 1. McCartney L, Marcus SE, Knox JP (2005) Monoclonal antibodies to plant cell wall xylans

and arabinoxylans. J. Histochem. Cytochem. 53:543–546.

2. Reinhardt A et al. (2015) Antigenic potential of a highly conserved Neisseria meningitidis lipopolysaccharide inner core structure defined by chemical synthesis. Chem. Biol. 22:38–49.

3. Broecker F et al. (2016) Multivalent display of minimal Clostridium difficile glycan epitopes mimics antigenic properties of larger glycans. Nat. Commun. 7:11224.

4. Geissner A, Pereira CL, Leddermann M, Anish C, Seeberger PH (2016) Deciphering Antigenic Determinants of Streptococcus pneumoniae Serotype 4 Capsular Polysaccharide using Synthetic Oligosaccharides. ACS Chem. Biol.

5. Schmidt D, Schuhmacher F, Geissner A, Seeberger PH, Pfrengle F (2015) Automated synthesis of arabinoxylan-oligosaccharides enables characterization of antibodies that recognize plant cell wall glycans. Chemistry 21:5709–5713.

6. Jacob F et al. (2012) Serum antiglycan antibody detection of nonmucinous ovarian cancers by using a printed glycan array. Int. J. Cancer 130:138–146.

7. Johannssen T, Lepenies B (2015) Identification and Characterization of Carbohydrate-Based Adjuvants. Methods Mol. Biol. 1331:173–187.

8. Tabarani G et al. (2009) DC-SIGN Neck Domain Is a pH-sensor Controlling Oligomerization: SAXS AND HYDRODYNAMIC STUDIES OF EXTRACELLULAR DOMAIN. J. Biol. Chem. 284:21229–21240.

9. Lameignere E et al. (2008) Structural basis for mannose recognition by a lectin from opportunistic bacteria Burkholderia cenocepacia. Biochem. J. 411:307.

10. Šulák O et al. (2011) Burkholderia cenocepacia BC2L-C Is a Super Lectin with Dual Specificity and Proinflammatory Activity. PLoS Pathog. 7:e1002238.

11. Rademacher C, Paulson JC (2012) Glycan Fingerprints: Calculating Diversity in Glycan Libraries. ACS Chem. Biol. 7:829–834.

12. Gascuel O (1997) BIONJ: an improved version of the NJ algorithm based on a simple model of sequence data. Mol. Biol. Evol. 14:685–695.

13. Rüdiger H, Gabius HJ (2001) Plant lectins: occurrence, biochemistry, functions and applications. Glycoconj. J. 18:589–613.

14. Kobayashi Y, Tateno H, Ogawa H, Yamamoto K, Hirabayashi J (2014) in Lectins, ed Hirabayashi J. (Springer New York, New York, NY), pp 555–577.

15. Cummings RD, Darvill AG, Etzler ME, Hahn MG (2015) in Essentials of Glycobiology, eds Varki A et al., pp 611–625.

17. Geisler C, Jarvis DL (2011) Effective glycoanalysis with Maackia amurensis lectins requires a clear understanding of their binding specificities. Glycobiology 21:988–993.

18. Song X et al. (2011) A sialylated glycan microarray reveals novel interactions of modified sialic acids with proteins and viruses. J. Biol. Chem. 286:31610–31622.

19. Peters BP, Ebisu S, Goldstein IJ, Flashner M (1979) Interaction of wheat germ agglutinin with sialic acid. Biochemistry 18:5505–5511.

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20. Wu AM, Wu JH, Song S, Tsai M, Herp A (1998) Studies on the binding of wheat germ agglutinin (Triticum vulgaris) to O-glycans. FEBS Lett. 440:315–319.

21. Gallagher JT, Morris A, Dexter TM (1985) Identification of two binding sites for wheat-germ agglutinin on polylactosamine-type oligosaccharides. Biochem. J. 231:115–122.

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