Click and pick: Identification of sialoside analogues for siglec-based cell targeting

Article abstract of DOI:10.1002/anie.201205831

Click 'n' chips: Azide and alkyne-bearing sialic acids (purple diamond; see picture) were subjected to high-throughput click chemistry to generate a library of sialic acid analogues. Microarray printing of the library and screening with the siglec family of sialic-acid-binding proteins, led to the identification of high-affinity ligands for siglec-9 and siglec-10. Copyright

Full text of DOI:10.1002/anie.201205831

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DOI: 10.1002/anie.201205831
Cell Targeting
Click and Pick: Identification of Sialoside Analogues for Siglec-Based
Cell Targeting **
Cory D. Rillahan, Erik Schwartz, Ryan McBride, Valery V. Fokin, and James C. Paulson*
The siglec family of sialic-acid binding proteins is comprised
of 15 human and 9 murine members that are primarily
expressed by white blood cells, which mediate innate and
adaptive immune functions.[1] Their restricted expression
pattern and activity as endocytic receptors has made these
proteins attractive molecular targets for directed therapy for
immune-cell-mediated diseases.[2] Although anti-siglec anti-
bodies are already in clinical use, nanoparticles having
sialoside ligands show promise for targeting siglecs in vivo,
thus providing alternatives for delivery of therapeutic cargo.[3]
The difficulty in identifying siglec ligands of suitable
affinity and selectivity has limited the potential of ligand-
bearing nanoparticles.[1a,2,4] Previous reports have demon-
strated that modification of sialic acid (NeuAc) at the C9-
position can produce both increased affinity and selectivity
for sialoadhesin (siglec-1), CD22 (siglec-2), and myelin-
associated glycoprotein (siglec-4).[5] It has also been suggested
that modifications at the C5-position can modulate affinity
and selectivity for several siglecs, however, sialoside ana-
logues modified at this position have not been fully explored
for these properties.[6] Thus, although modifications to sialic
acid at C5 and C9 have potential for yielding promising
sialoside ligands, and these positions are relatively straight-
forward to modify using an enzymatic synthetic approach, the
lack of methods to robustly generate sialoside analogue
libraries and systematically screen them against a library of
siglecs has hampered progress.
synthesized by a convergent chemoenzymatic approach
(Scheme 1, and in the Supporting Information, Scheme S1);
Figure 1. A schematic representation of the click and pick strategy for
identification of high-avidity siglec ligands. This strategy involves the
synthesis of the analogue library by parallel CuI-catalyzed azide–alkyne
cycloaddition (CuAAC), glycan microarray printing, and screening with
fluorescently labeled siglec-Fc chimeras to identify high-affinity ligands.
these compounds have azide or alkyne substituents at the
5-position (A0–D0) or the 9-position (E0–H0) of the sialic
acid moiety, and are attached through an a2-3 or a2-6 linkage
to the penultimate galactose, the two most common linkages
in mammalian glycans. These parent scaffolds were then
subjected to high-throughput CuI-catalyzed azide–alkyne
cycloaddition[7] (CuAAC, click chemistry) with 24–30 cou-
pling partners (Supporting Information, Figures S1 and S2) to
generate a library of 224 sialoside analogues (Supporting
Information, Tables S1–S8), with quantitative conversion for
nearly all couplings. The sialoside products could then be
printed directly onto N-hydroxysuccinimide (NHS) activated
microarray slides without prior purification owing to the
orthogonality of the click reaction with amine acylation
chemistry, thus allowing for far higher screening throughput
and library diversity than in previous efforts.[6b,8]
To address this issue, we devised a facile “click and pick”
strategy involving high-throughput synthesis of a sialoside
analogue library using click chemistry, coupled with micro-
array technology to pick high-affinity “hits” for human and
murine siglecs (Figure 1). To generate the library, eight
sialoside parent compounds with ethyl amine linkers were
[*] C. D. Rillahan, R. McBride, Prof. J. C. Paulson
Department of Chemical Physiology, The Scripps Research Institute
10550 N. Torrey Pines Road, La Jolla, CA 92037 (USA)
E-mail: jpaulson@scripps.edu
Dr. E. Schwartz, Prof. V. V. Fokin
Department of Chemistry, The Scripps Research Institute
10550 N. Torrey Pines Road, La Jolla, CA 92037 (USA)
[**] We would like to thank Prof. K. Barry Sharpless, Prof. Ola Blixt, and
Prof. Jason Hein, for encouragement and help at early stages of this
project as well as Dr. Norihito Kawasaki for advice and assistance in
preparation of human white blood cells. This work was supported by
the NIH (T32AI007606 to C.D.R., R01AI050143 and P01L107151 to
J.C.P., and GM087620 to V.V.F.), a Schering-Plough Research
Institute postdoctoral fellowship (E.S.), and a Rubicon fellowship
from the Netherlands Organization for Scientific Research (E.S.).
To identify high-affinity ligand analogues of individual
siglecs, fluorescently labeled siglec-Fc chimeras were over-
layed on the microarrays (Figure 1). At optimal concentra-
tions of the Fc chimeras there was no binding to native
sialoside controls or the parent scaffolds A0–H0, thus
ensuring that any hits correspond to higher-affinity ligands.[8–9]
Representative microarray data obtained using this approach
is shown for a panel of human and murine siglecs in Figure 2,
Figure 3a, and in the Supporting Information, Figure S3.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201205831.
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Scheme 1. Synthesis of parent azide and alkyne sialosides A0-H0. Reagents and conditions: a) I or II, pyruvate, C. Perfrigens NeuAc aldolase, CTP,
N. Meningitidis CMP-NeuAc synthetase, P. Multocida 2,3-sialyltransferase, 75–90%; b) I or II, pyruvate, C. Perfrigens NeuAc aldolase, CTP,
N. Meningitidis CMP-NeuAc synthetase, P. Damsella 2,6-sialyltransferase, 85–95%; c) IV or V, CTP, N. Meningitidis CMP-NeuAc synthetase,
P. Multocida 2,3 Sialyltransferase, 70–85%; d) IV or V, CTP, N. Meningitidis CMP-NeuAc synthetase, P. Damsella 2,6-sialyltransferase, 85–95%.
CMP=cytidine monophosphate, CTP=cytidine triphosphate.
Remarkably, each siglec exhibits a distinct binding pattern
towards the analogue library, and analogues based on seven of
the eight parent structures A0–H0 yielded high-avidity
ligands for one or more siglec.
In general we found, with few exceptions, that individual
siglecs bound preferably to analogues with substituents at
either C9 or C5, but not both, and the most promising were
those having relatively bulky and hydrophobic substituents.
Furthermore, there appeared to be little preference for the
sialoside linkage (e.g. a2-3 and a2-6) suggesting that the
modified sialic acid, and not the underlying lactose core,
provides most of the binding affinity. We should note that the
sialic acid scaffold, and not the substituent alone, is a key
determinant for binding. Evidence for this is the fact that
analogues with the same substituent linked at either C5 or C9
give drastically different results (for example, siglec-E with an
adamantyl azide at C9, F9 and H9, is a hit but at C5, B9 and
D9, shows no binding).
Figure 2. Siglec screening reveals unique specificity profiles for sialo-
side analogues. Fluorescently labeled murine (siglec-E, siglec-G) and
human (siglec-5, siglec-7, siglec-10) siglecs were applied to the sialo-
side analogue glycan microarray to identify high-affinity analogues.
Exemplary analogue hits are highlighted and denoted with the library
number of the corresponding azide or alkyne substitutent (Tables S1–
S8). The compound nomenclature combines the letter corresponding
to the parent sialoside (Scheme 1, A0-H0) with the number of the
azide or alkyne that it was reacted with (Figure S1 and S2). Controls
include native sialosides and previously identified high-affinity ana-
logues for hCD22, mSn, and rMAG (Figure S7).
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Figure 3. A siglec-9 specific analogue for cell targeting applications was identified through high-throughput screening. a) Comparison of the
binding specificity of fluorescently labeled siglec-9-Fc chimera (top) and siglec-9-expressing CHO cells (bottom). b) An image of fluorescent
siglec-9 CHO cells bound to the analogue microarray. c) The specificity of liposomal nanoparticles incorporating D24-PEG-lipid (siglec-9 targeting;
filled blocks), or no ligand (naked; empty blocks), was assessed, by flow cytometry, for binding to a panel of siglec-expressing cell lines in
triplicate. Inset shows the structure of D24. d) The siglec-9-targeting and naked liposomes were tested for binding to white blood cells isolated
from peripheral human blood and co-stained with an anti-siglec-9 antibody. The two siglec-9-expressing subsets are granulocytes (blue arrow) and
monocytes (red arrow) as shown by forward and side scatter properties.
To explore the potential to use the glycan array to screen
the specificity of siglecs expressed on the surface of intact
cells, we examined the binding of several fluorescently
labeled siglec-expressing cell lines. As shown in Figure 3a,
siglec-9-expressing Chinese hamster ovary (CHO) cells
bound with nearly the same specificity as the siglec-9-Fc
chimera, that is, binding primarily to C5-substituted com-
pounds, and with highest apparent avidity to D24 (see
Figure 3c, inset). Similar results were obtained with human-
CD22-expressing CHO cells and even a human B-cell line
that expresses CD22 (Figure S4). Consistent with the binding
of the hCD22-Fc chimera (Supporting Information, Fig-
ure S3), hCD22-expressing cells bound with highest avidity to
G1, G6, and 9-N-biphenylcarboxamido-NeuAca2-6Galb1-
4Glc (BPCNeuAc), which is a known high-affinity ligand of
CD22.[3e,5] Thus, it is clear that arrays can be probed with
whole cells to identify high-avidity siglec ligands.
As the ultimate goal is to identify ligands that are suitable
for targeting a single human or murine siglec when incorpo-
rated into synthetic multivalent sialoside probes, we tested
exemplary leads for their ability to facilitate selective binding
to their respective siglecs. We selected D24 as a candidate
ligand for siglec-9 owing to the fact that it facilitated binding
to siglec-9 on CHO cells, even in the presence of competing
cis ligands (Figure 3a,b),[3b–e] and it was not recognized by any
other siglec tested in the screen. As a multivalent platform we
chose ligand-bearing liposomal nanoparticles because of their
demonstrated utility for targeting siglecs in vivo.[3b] Accord-
ingly, D24 was covalently attached to a PEGylated lipid
(Supporting Information, Scheme S2), incorporated into lip-
osomal nanoparticles (siglec-9-targeting liposomes), and
these liposomes were assessed for binding to a panel of
siglec-expressing cell lines (Figure 3c and the Supporting
Information, Figure S5). The siglec-9-targeting liposomes
avidly bound to and were rapidly endocytosed by siglec-9-
expressing CHO cells, whereas the control (naked) liposomes
exhibited no detectable binding. In contrast, no binding of the
siglec-9-targeting liposomes was observed to any of the other
seven siglec-expressing cell lines, thus demonstrating their
high selectivity for siglec-9 (Figure 3c).
To assess the ability of the siglec-9-targeting liposomes to
bind to native human leukocytes, additional experiments
were carried out with white blood cells isolated from
peripheral human blood (Figure 3d). While no binding of
the control (naked) liposomes was observed to any cell
population, the siglec-9-targeting liposomes bound avidly to
two siglec-9 positive cell populations in proportion to the
amount of siglec-9-expressed, and exhibited no detectable
binding to cells that were siglec-9 negative. Forward- and side-
scatter analysis showed that the two cell populations were
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granulocytes (blue arrow) and monocytes (red arrow), con-
sistent with previous reports documenting the expression of
siglec-9 on these cell populations.[10]
carried out. Nonetheless, F9 appears to be a promising new
siglec ligand for both mouse and human siglec studies.
As illustrated from these examples, the click and pick
strategy has provided numerous leads for the development of
multivalent ligand-based probes for human and murine
siglecs even in the absence of structural information for the
majority of the siglec family members. Such agents could be
used to explore the functions of siglecs,[3a,13] and for applica-
tions involving targeting of leukocytes in vivo.[3b] Moreover,
the method may be applicable for identifying high-affinity
ligands for other families of glycan-binding proteins of
biological and therapeutic interest such as the C-type
lectins,[14] which are broadly expressed on antigen-presenting
cells (APCs) involved in innate and adaptive immunity.
Recently, chemoenzymatic approaches have been used to
generate azide and alkyne bearing ligands for these recep-
tors,[15] which could serve as starting points for analogue
library generation and subsequent screening efforts to
identify new chemical probes for this protein family and as
vaccine delivery agents to APC subsets.[16]
To further address the general utility of this approach for
identifying high-avidity siglec ligands for cell targeting, we
selected F9, a ligand that bound to human siglec-10, and
prepared the corresponding pegylated lipid (Supporting
Information, Scheme S3) for incorporation into liposomal
nanoparticles. When tested with a panel of human siglec-
expressing cell lines, it was found that these liposomes were
entirely specific for siglec-10 over any other human siglec
(Figure 4a). When these liposomes were incubated with
Received: July 23, 2012
Published online: October 4, 2012
Keywords: carbohydrates · immunology · liposomes ·
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receptors · sialic acids
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