Sialoside analogue arrays for rapid identification of high affinity siglec ligands

Article abstract of DOI:10.1021/ja801052g

The siglec family of sialic acid binding proteins participates in diverse cell surface biology that includes regulation of immune cell signaling and the interaction of neuronal cells with glial cells. The weak intrinsic affinity of the natural sialoside ligands has hampered the development of synthetic ligand based probes needed to elucidate their roles in siglec function. In this report, we describe a glycan microarray comprising a library of 9-acyl-substituted sialic acids incorporated into sialosides containing the Neu5Acα2-3Gal and Neu5Acα2-6Gal linkages commonly recognized by the siglecs. The array is demonstrated to exhibit utility for detecting 9-acyl substituents that increase the affinity of siglecs for their ligands. Substituents that increase affinity are anticipated to be useful for the design of high affinity ligand based probes of siglec function. Copyright

Full text of DOI:10.1021/ja801052g

Published on Web 05/02/2008
Sialoside Analogue Arrays for Rapid Identification of High Affinity Siglec
Ligands
Ola Blixt,† Shoufa Han,‡ Liang Liao, Ying Zeng, Julia Hoffmann, Satoshi Futakawa, and
James C. Paulson*
Departments of Chemical Physiology and Molecular Biology, The Scripps Research Institute, 10550 North Torrey
Pines Road, La Jolla, California 92037
Received February 11, 2008; E-mail: jpaulson@scripps.edu
Scheme 1a
The siglec family of glycan binding proteins recognizes sialic
acid containing glycans of glycoproteins and glycolipids as ligands.
Most are expressed on various white blood cells that mediate
immune function, and one of them, myelin associated glycoprotein
(MAG), is expressed on glial cells and functions in myelin-axon
interactions.1 In general, siglecs bind with low intrinsic affinity
(0.1-1 mM) to their natural sialoside ligands, and while several
exhibit unique specificities, most siglecs bind to sialosides contain-
ing the common Neu5AcR2-6Gal and/or Neu5AcR2-3Gal
linkages.2–4
To investigate the roles of ligand binding in the functions of
siglecs, high affinity probes are desired to compete with natural
ligands and bind to siglecs on native cells. Several studies have
a Reagents and conditions: (a) ST3Gal and CMP-9-azido-9-deoxy-
documented the importance of sialic acid substituents (e.g., 9C and
5C positions) for modulating the affinity of siglec binding5–9 and
Neu5Ac; (b) GalT-GalE, UDP-Gal, hST6Gal I, CMP-9-azido-9-deoxy-
Neu5Ac; (c) PPh3, MeOH:TFA:H2O (4:5:1); (d) acylation, R2COCl (R2 )
the utility of high affinity sialoside ligands as biological probes of
1-34, see Table S1) (2-4 equiv), base (5 equiv); (e) I2 (5 equiv), MeOH:
H2O (9:1); (f) Pd/C, H2.
Siglec-2 (CD22).10,11 To facilitate development of such ligands for
other siglecs, we have implemented a strategy of making a sialoside
analogue library compatible with glycan microarray technology12
to screen for substituents that increase affinity.
The synthesis of sialosides with 9-azido-Neu5Ac opens a route
to the facile synthesis of 9-substituted sialic acids. Synthesis of
the key azido intermediates (C and D) with 9-azido-Neu5AcR2-3Gal
and 9-azido-Neu5AcR2-6Gal termini was accomplished by enzy-
matic transfer of 9-azido-Neu5Ac from CMP-9-azido-Neu5Ac13,14
to an oligosaccharide precursor containing a 4-pentenoyl or CBZ-
protected 2-amino ethyl aglycone (Scheme 1). Portions (1-5 mg)
of the azido intermediates were reduced to the amine with PPh3
and acylated in excess from a library of acylchlorides (see Table
S1 in Supporting Information). After quenching of excess acylating
reagents with water, glycans were deprotected15,16 to expose the
amine (E or F) for printing onto amino-reactive N-hydroxy
succinimide (NHS)-activated glass slides (Schott Nexterion, Slide-
H).12 In total, the library comprised 16 R2-3 and 28 R2-6
sialosides.
To determine if the increased affinity of siglecs toward acyl
substituents could be detected on a glycan array, we tested the
binding of CD22 (Siglec-2) to an array of selected analogues of
Figure 1. Binding of CD22-Fc chimera to sialoside analogues printed on
a glycan array. Sialosides were printed with 10 replicates of 10, 2-fold
dilutions from 100 to 0.2 µM (left to right). 9-Acyl substituents of F are
indicated in parentheses (Table S1). (A) Fluorescent image of the array
the R2-6 sialoside F. The six compounds included one with a 9-N-
biphenylcarboxyl (BPC, 15) substituent previously shown to
increase affinity for CD22 by 100-fold.6,10 The glycans were printed
at 10, 2-fold dilutions (10 replicates each). Binding was assessed
with a fluorescently labeled recombinant CD22-Fc chimera, an
antibody-like molecule containing two CD22 ligand binding
domains, that has been demonstrated to bind to glycan microar-
rays.12 Binding of CD22 was sialic acid dependent (Figure 1), with
labeled with a complex of CD22-Fc chimera and secondary FITC-labeled
goat antihuman Fc antibody (see Figure 3). (B) Relative fluorescence
intensity at each printing concentration is compared for the six compounds.
binding observed to the natural Neu5Ac containing compound (c)
and increased binding to each of the three 9-acyl-substituted
compounds (b, d, and f). Scanning the slide to measure relative
fluorescence allowed comparison of binding levels at each printing
† Present address: University of Copenhagen, Denmark.
‡ Present address: Xiamen University, China.
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6680 J. AM. CHEM. SOC. 2008, 130, 6680–6681
10.1021/ja801052g CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
equivalent binding of the biphenyl substituent 15 to both, demon-
strating that the affinity to the acyl substituent is dominant over
the sialic acid to galactose linkage. MAG bound to sialosides with
other substituents including benzoyl- (7) and chlorobenzoyl (8-10)
analogues of either R2-3 (E) or R2-6 (F) sialosides. The results
are consistent with previous studies evaluating the affinity of MAG
toward 9-N-acyl substituents on R-methyl-NeuAc.8
To confirm that the signals for MAG resulted from increased
affinity, a selected series of related sialosides with 9-acyl substit-
uents (7, 8, and 10) of E and F were resynthesized and tested for
binding by MAG and CD22 (Figure S2). While CD22 bound only
the R2-6 sialoside (F) series, MAG bound both, with preference
for the R2-3 (E) series compounds. These compounds were also
assessed for inhibitory potency in an ELISA competition assay.10
Compounds 7E, 8E, and 10E gave IC50 values of 4.9, 9.7, and 2.2
µM, respectively, representing 50-200-fold enhancement of affinity
over the unsubstituted R2-3 sialoside H (IC50 of 490 µM; data
not shown).
Figure 2. Binding of sialoside-specific plant lectins to a sialoside analogue
glycan array. Biotinylated lectins, MAA (top) and SNA (bottom) specific
for Neu5AcR2-3Gal or Neu5AcR2-6Gal linkages, respectively, were
mixed with Alexa-fluor 488 labeled streptavidin, overlaid onto the printed
analogue array, washed, and scanned for fluorescence (see Supporting
Information). Acyl analogues 1-34 of E (yellow) and F (green) correspond
to acyl groups listed in Table S1. Shown is relative fluorescence of lectins
bound to each glycan printed at 5 serial dilutions starting at 100 µM.
“NeuAc” labeled lanes are sialosides with unsubstituted Neu5Ac in R2-3
(H) and R2-6 (I) linkage to N-acetyllactosamine.
In summary, we demonstrate that a sialoside analogue array
allows for the synthesis of sialoside analogues in small quantity
for subsequent screening for high affinity ligands of siglecs and
other sialoside binding GBPs.
Acknowledgment. The authors thank Dr. Paul Crocker for
providing cell lines producing siglec-Fc chimeras of MAG and
sialoadhesin, the Consortium for Functional Glycomics (GM62116)
for the CD22-Fc chimera, Dr. Nicolai Bovin for compound J, and
Ms. Anna Tran-Crie for help in preparing this manuscript. This
research was supported by NIH Grant GM060938.
Supporting Information Available: General procedures for prepa-
rations of the sialoside analogue library, a complete list of printed
analogues, procedures for printing and analysis of GBPs, and analytical
data for MAA and SNA plant lectins. This material is available free of
charge via the Internet at http://pubs.acs.org.
Figure 3. Binding of siglec-Fc chimeras to the sialoside analogue array.
Mouse sialoadhesin-Fc chimera (top) and mouse MAG-Fc chimera (bottom)
complexed with goat antihuman IgG-FITC (2:1; 50 µg/mL total protein)
were overlaid onto the printed analogue array, washed for Figure 2).
Numbered acyl analogues attached to compound E (yellow) or F (green)
are found in Table S1.
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The quality of a glycan array containing the full library was
assessed using the binding of the plant lectins, Maccia amurensis
agglutinin (MAA) and Sambucus nigra agglutinin (SNA), specific
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of neither lectin is blocked by 9-acyl substituents, facilitating their
use to assess the printing efficiency. All 9-acyl-substituted E and
F sialosides were recognized comparably by MAA and SNA,
respectively (Figure 2).
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siglecs, CD22 (Siglec-2), MAG (Siglec-4), and sialoadhesin (Siglec-
1). In keeping with its specificity for R2-6 sialosides, CD22 bound
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Surprisingly, Fc chimeras of sialoadhesin and MAG did not bind
to sialosides with unsubstituted NeuAc but bound R2-3 (E) or
R2-6 sialosides with selective 9-N-acyl substituents in repeated
assays (Figure 3). Sialoadhesin exhibited increased affinities to a
few bisphenyl analogues of E (14, 15), and F (15, 30), with the
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