Chemical remodeling of cell-surface sialic acids through a palladium-triggered bioorthogonal elimination reaction

Article abstract of DOI:10.1002/anie.201409145

We herein report a chemical decaging strategy for the in situ generation of neuramic acid (Neu), a unique type of sialic acid, on live cells by the use of a palladium-mediated bioorthogonal elimination reaction. Palladium nanoparticles (Pd NPs) were found to be a highly efficient and biocompatible depropargylation catalyst for the direct conversion of metabolically incorporated N-(propargyloxycarbonyl)neuramic acid (Neu5Proc) into Neu on cell-surface glycans. This conversion chemically mimics the enzymatic de-N-acetylation of N-acetylneuramic acid (Neu5Ac), a proposed mechanism for the natural occurrence of Neu on cell-surface glycans. The bioorthogonal elimination was also exploited for the manipulation of cell-surface charge by unmasking the free amine at C5 to neutralize the negatively charged carboxyl group at C1 of sialic acids.

Full text of DOI:10.1002/anie.201409145

.
Angewandte
Communications
International Edition: DOI: 10.1002/anie.201409145
German Edition: DOI: 10.1002/ange.201409145
Chemical Decaging
Chemical Remodeling of Cell-Surface Sialic Acids through
a Palladium-Triggered Bioorthogonal Elimination Reaction**
Jie Wang, Bo Cheng, Jie Li, Zhaoyue Zhang, Weiyao Hong, Xing Chen,* and Peng R. Chen*
Dedicated to Professor Qing Huang
Abstract: We herein report a chemical decaging strategy for the
in situ generation of neuramic acid (Neu), a unique type of
sialic acid, on live cells by the use of a palladium-mediated
bioorthogonal elimination reaction. Palladium nanoparticles
glycoproteins containing a high density of negatively charged
Sias. The resulting repulsion between these late-stage cancer
[5]
cells facilitates their entry into the blood stream. Neu5Ac
can be modified at the C5 position or at the C4, C7, C8, and
C9 hydroxy groups, thus contributing to the diversity of the
(
Pd NPs) were found to be a highly efficient and biocompat-
[1]
ible depropargylation catalyst for the direct conversion of
metabolically incorporated N-(propargyloxycarbonyl)neur-
amic acid (Neu5Proc) into Neu on cell-surface glycans. This
conversion chemically mimics the enzymatic de-N-acetylation
of N-acetylneuramic acid (Neu5Ac), a proposed mechanism
for the natural occurrence of Neu on cell-surface glycans. The
bioorthogonal elimination was also exploited for the manip-
ulation of cell-surface charge by unmasking the free amine at
C5 to neutralize the negatively charged carboxyl group at C1 of
sialic acids.
Sia family. Besides these modifications, Neu5Ac could
potentially be de-N-acetylated to give the core Neu. Recent
studies supported the presence of this monosaccharide in
[6]
certain cell types, particularly nerve cells and cancer cells.
Nevertheless, Neu is rarely encountered in nature, and
whether it exists as a constituent of cell-surface oligosacchar-
ides remains debatable. Neu is chemically unstable and thus
unlikely to be enzymatically installed into glycans by the
[
7]
biosynthetic machinery like Neu5Ac. Alternatively, Neu
might be directly generated from Neu5Ac on cell-surface
glycans through de-N-acetylation, presumably by certain de-
[
7]
S
ialic acids (Sias) are a family of nine-carbon-atom mono-
N-acetylases. Although this mechanism seems plausible, the
existence of such enzymes remains elusive. A means to
generate Neu on cell surfaces would facilitate the elucidation
of the biological functions and biosynthetic mechanism of
Neu. Therefore, we sought to develop such a strategy by
exploiting palladium-mediated elimination reactions and
methodology for metabolic glycan labeling (Figure 1).
saccharides that often reside at the outmost end of cell-
[
1]
surface glycans. Sias have more than 50 naturally occurring
forms, among which Neu5Ac is the most abundant in
[1,2]
humans.
Found at all cell surfaces of vertebrates as well
as certain bacteria, Sias play critical roles in diverse cellular
processes, including cell differentiation, host–pathogen inter-
[3]
actions, and signaling transduction. Furthermore, the highly
prevalent Sias create negative charges on the cell mem-
Palladium is a powerful transition metal in catalyzing
diverse chemical transformations ranging from carbon–
[4]
[8]
brane. For example, metastatic cancer cells often express
carbon bond formation to versatile cleavage reactions.
Recently, some palladium-mediated elimination reactions
have been exploited for unmasking an array of chemically
[
+]
[+]
[+]
[
*] J. Wang, B. Cheng, J. Li, Z.-Y. Zhang, W.-Y. Hong,
[9]
[10]
Prof. Dr. X. Chen, Prof. Dr. P. R. Chen
caged molecules, including fluorophores, nucleotides, and
Synthetic and Functional Biomolecules Center
Beijing National Laboratory for Molecular Sciences
College of Chemistry and Molecular Engineering, Peking University
Beijing 100871 (China)
[11]
amino acid side chains of intact proteins, within live cells.
Such methods provided a powerful bioorthogonal activation
strategy for: I) turning on the fluorescence of chemically
masked fluorophores (e.g. propargyl-derived fluorescein) in
E-mail: xingchen@pku.edu.cn
[
9]
pengchen@pku.edu.cn
living cells, II) spatially controlled activation of nucleotide
prodrugs (e.g. 5-fluoro-1-propargyluracil) with enhanced
[+]
J. Wang, Prof. Dr. X. Chen, Prof. Dr. P. R. Chen
Peking-Tsinghua Center for Life Sciences
Beijing (China)
[
10]
local therapeutic effects,
and III) intracellular activation
of enzymes to probe the downstream signaling pathways (e.g.
[
+]
[11]
J. Wang
chemically caged phosphothreonine lyase). Together, these
Academy for Advanced Interdisciplinary Studies
Peking University (China)
palladium-catalyzed biocompatible cleavage reactions have
expanded the scope and applications of bioorthogonal
+
[
] These authors contributed equally to this work.
[12]
reactions, which have been mainly focused on biocompat-
[
**] We thank Prof. Michael Pawlita for sharing K20 cells. This research
was supported by the National Key Basic Research Foundation of
China (2012CB917301 to P.R.C.; 2012CB917303 to X.C.) and the
National Natural Science Foundation of China (21225206,
ible conjugation reactions in the past decade. We envisioned
that such a decaging strategy could be exploited to chemically
generate Neu from a metabolically incorporated caged-Neu
precursor on cell-surface glycans.
9
1313301, and 21432002 to P.R.C.; 21172013 and 91313301 to
X.C.).
The metabolic glycan-labeling method exploits the under-
lying biosynthetic machinery to metabolically incorporate
chemically modified unnatural sugars into cellular glycans for
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201409145.
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introduce Neu5Proc onto cell-sur-
face glycans throughout the rest of
our study.
We then searched for an effi-
cient palladium-based depropargy-
lation catalyst by using the N-Proc-
caged fluorogenic coumarin dye 3 as
a fluorogenic reporter. Compound 3
is virtually nonfluorescent, but can
be converted into the highly fluo-
rescent compound 4 by the cleavage
of its Proc group (Figure 2a). The
increased fluorescence signal from
decaged coumarin 4 was recorded to
obtain a kinetic curve and the
decaging efficiency. Because this
Proc group can be eliminated by
palladium species with all possible
Figure 1. A chemical decaging strategy based on palladium-mediated elimination and metabolic
glycan labeling for generating Neu on live cells. a) Neu5Proc serves as an analogue of Neu5Ac that
can be converted into the core neuramic acid by a palladium-mediated depropargylation reaction.
b) Neu5Proc can be metabolically incorporated into cell-surface sialylated glycans. Upon the addition
of a Pd catalyst, the Proc group is cleaved to form Neu in situ.
0
II
oxidation states (Pd , Pd , and
IV
Pd ), we surveyed the depropargy-
lation activity of a series of Pd
catalysts in all three oxidation
[13]
[9b,17]
diverse labeling purposes.
For example, the Neu5Ac
states.
A total of eight Pd reagents were surveyed: C1
0
biosynthetic pathway is permissive to Neu5Ac analogues
containing functional groups such as azides and alkynes at the
stands for Pd nanoparticles (Pd NPs), which were prepared
according to the method based on reduction with sodium
borohydride and characterized by transmission electron
[
14]
N-acyl position. In many cases, the same labeling handles
can be installed by use of the corresponding analogues of N-
acetylmannosamine (ManNAc), the biosynthetic precursor of
[
15]
sialic acid.
The incorporated unnatural sugars can be
subsequently conjugated with fluorescent probes for glycan
imaging by bioorthogonal chemistry (e.g. click chemistry).
Instead of bioconjugation, we sought to eliminate the N-acyl
substituent at C5 of Neu5Ac analogues to generate Neu. In
the search for an appropriate Neu5Ac analogue, we turned
our attention to a ManNAc analogue, N-(propargyloxycar-
bonyl)mannosamine (ManNProc), which was recently dem-
onstrated to be metabolically converted into the correspond-
[16]
ing sialic acid (Neu5Proc) with high efficiency. Realizing
that Neu5Proc also contains a Proc group that is sensitive to
the Pd catalyst, we envisioned that the cell-surface Neu5Proc
could be chemically unmasked to generate Neu in situ, in
a process which may resemble the aforementioned de-N-
acetylation process (Figure 1b).
We
first
prepared
peracetylated
ManNProc
[16]
(
Ac ManNProc) by organic synthesis
and Neu5Proc by
4
enzymatic conversion with recombinant Escherichia coli K12
aldolase. The metabolic incorporation of the resulting com-
pounds was examined in CHO cells: Cells treated with
Figure 2. Catalyst screening for the palladium-triggered depropargyla-
tion reaction. a) Coumarin-based fluorogenic reporter for the depropar-
gylation reaction. The N-Proc-caged coumarin derivative 3 exhibits
quenched fluorescence that can be reactivated upon the removal of
Neu5Proc for 24 h or Ac ManNProc for 72 h at 378C were
4
labeled with biotin azide by copper-catalyzed azide–alkyne
cycloaddition (CuAAC) and stained with Alexa Fluor 488–
streptavidin. A robust fluorescence signal was detected by
flow cytometry in a dose-dependent manner. The incorpo-
ration of Neu5Proc increased sharply as its concentration was
increased from 0.025 to 5 mm; the incorporation efficiency
reached saturation at 3 mm (see Figure S1 in the Supporting
the Proc group to yield the coumarin dye 4 (l =380 nm,
ex
lem =450 nm). Palladium species examined in this study are shown
below the scheme. For water-insoluble palladium species, stock
solutions (10 mm) in dimethyl sulfoxide were prepared and were
subsequently diluted into an aqueous buffer before usage. tapAd:
1,3,5-triaza-7-phosphaadamantane, tppts: tris(3-sulfophenyl)phosphine
trisodium salt, [Pd allyl Cl ]: allylpalladium(II) chloride dimer, dba:
2
2
2
dibenzylideneacetone. b) Depropargylation activity of palladium cata-
lysts (100 mm) as determined by the increase in fluorescence (corre-
sponding to the conversion of 3 (100 mm) into compound 4). FTR:
fluorescence turn-on ratio.
Information). When Ac ManNProc was used, highly efficient
4
incorporation was observed at 50 mm (see Figure S2). We
therefore used Neu5Proc (3 mm) or Ac ManNProc (50 mm) to
4
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0
0
microscopy (see Figure S3); C2 and C3 are two active Pd
catalysts with water-soluble phosphine ligands; C4 is a Pd
0
precursor, whereas C7 is an air-stable Pd catalyst; C5 and C6
II
are two additional Pd catalysts without or with a phosphine
IV
ligand; C8 is a widely used and commercially available Pd
catalyst. C1–C3 exhibited higher reactivity than the other
catalysts (Figure 2b). In particular, Pd NPs (C1) demonstrated
the highest reactivity, with a fluorescence turn-on ratio (FTR)
as high as 563-fold that of the control, and reached saturation
within 4 min. The high cleavage efficiency may be due to
active Pd atoms or Pd clusters that have been leached from
this heterogeneous catalyst. The depropargylation activity of
these Pd species was next examined directly on Neu5Proc by
LC–MS analysis (see Figure S4). As expected, Pd NPs exhib-
ited the best cleavage efficiency (> 95% yield within 4 min).
Finally, a toxicity study indicated that CHO cells and Jurkat
cells had high viability even in the presence of Pd NPs at
a concentration of 500 mm (10 times of our working concen-
tration; see Figures S5 and S6). Taken together, these results
show that Pd NPs are highly efficient and biocompatible
catalysts for the depropargylation of Neu5Proc.
We next applied Pd NPs for the direct conversion of
Neu5Proc into Neu on cell-surface glycans. We developed
a fluorescence assay to characterize the depropargylation
reaction on cell surfaces by monitoring the diminishment of
the terminal alkyne (Figure 3a). The cells incorporating
Neu5Proc were treated with biotin azide and stained with
Alexa Fluor 488–streptavidin. Analysis by flow cytometry
showed that the fluorescence intensity of palladium-treated
cells was significantly lower than that of those without Pd
treatment. In particular, Pd NPs (C1) exhibited the highest
cleavage efficiency of the Pd catalysts tested (Figure 3b; see
also Figure S7), in agreement with the in vitro assay results
described above. Furthermore, the depropargylation on cell
surfaces was imaged by confocal fluorescence microscopy.
Significantly lowered fluorescence was observed when the
Figure 3. Palladium-mediated Neu5Proc depropargylation on live cells.
a) Scheme describing the fluorescence assay for Neu5Proc depropargy-
lation. The cells with incorporated Neu5Proc were treated with a Pd
catalyst or with a vehicle. The presence of alkynes on the surface of
cells was then assessed by CuAAC-mediated fluorescence labeling,
followed by flow cytometry and confocal fluorescence imaging. b) CHO
cells were incubated with Neu5Proc (3 mm) for 24 h, followed by
treatment with a Pd catalyst or a vehicle for 5 min. The cells were then
treated with biotin azide, stained with Alexa Fluor 488–streptavidin,
and analyzed by flow cytometry. c) CHO cells were treated with
Ac ManNProc (50 mm) or Ac ManNAl (50 mm) for 72 h, followed by
4
4
treatment with Pd NPs or a vehicle for 5 min. After CuACC-mediated
fluorescence labeling, the cells were imaged by confocal fluorescence
microscopy. The nuclei were visualized by staining with Hoechst 33342
(blue signal). Scale bars: 20 mm.
glycoconjugates (see Figure S10). Moreover, DMABA-deriv-
atized Neu showed a distinct MS peak due to the presence of
the dimethylamino group. CHO cells displaying Neu5Proc
were depropargylated by Pd NPs and treated with DMABA-
NHS. Cells were then lysed, and Sias were released and
collected for LC–MS analysis in the selected-ion recording
(SIR) mode. The single quadrupole detector was set for
recording only the anion of interest (m/z 348: [Neu5Proc-
CHO cells incubated with Ac ManNProc were treated with
4
Pd NPs, in agreement with the results of flow cytometry
(
Figure 3c). When CHO cells treated with peracetylated N-(4-
À
À
pentynoyl)mannosamine (Ac ManNAl), a ManNAc analogue
H] ), and the ion intensity of [Neu5Proc-H] decreased 2.4-
fold after depropargylation triggered by Pd NPs on Neu5-
Proc-displaying CHO cells (Figure 4c). The same sample was
4
containing a noncleavable terminal alkyne moiety, was used as
a negative control, no significant fluorescence change was
observed upon treatment with Pd NPs (Figure 3c; see also
Figure S8). Similar results were observed in HeLa cells (see
Figure S9). These results collectively confirmed that the
fluorescence decrease was caused by palladium-mediated
depropargylation rather than damage of the alkynyl group
by Pd species.
To further confirm the reaction products of palladium-
triggered Neu5Proc depropargylation on cell surfaces and
quantify the reaction efficiency, we developed a LC–MS
analysis method (Figure 4a). The cell-surface Sia could be
released from sialylated glycoproteins and quantified by LC–
MS. For Neu, we designed a MS tag, 4-(dimethylamino)ben-
zoic acid succinimidyl ester (DMABA-NHS), to facilitate
detection (Figure 4b). DMABA-NHS forms an amide bond
with Neu to block the free amino groups, therefore avoiding
side reactions of amines with ketones and aldehydes in the
acidic hydrolysis medium used for releasing Neu from
then subjected to selected-cation LC–MS analysis for [(Neu ꢀ
+
MS tag)+H]
(m/z 415;
Figure 4d).
The
[(Neu ꢀ
+
MS tag)+H] peak from CHO cells treated with Pd NPs
increased by a factor of 2.6 after depropargylation as
compared to the control samples. The decrease in the
À
[Neu5Proc-H] anion peak and the increase in the [(Neu ꢀ
+
MS tag)+H] cation peak both confirmed that the depro-
pargylation reaction occurred through the desired process.
The cleavage efficiency from two independent experiments
was averaged to 71% according to the decline in the
À
[Neu5Proc-H] peak of cell-surface glycans.
Finally, we sought to demonstrate that the palladium-
mediated conversion of Neu5Proc into Neu could be utilized
to manipulate the cell-surface charge. The negatively charged
carboxyl groups on Sias create negative charges on the plasma
[
4]
membrane; these charges are particularly important for
producing strong cell–cell repulsion among Sia-overexpress-
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a free amine in the negatively charged
Sias, thus offering a chemical strategy
for the manipulation of cell-surface
charge and cell clustering.
In summary, bioorthogonal decag-
ing strategies offer a facile approach for
the in situ activation or liberation of
biomolecules under physiological con-
ditions. Although the photodecaging
strategy is another efficient and non-
invasive method for the rapid uncaging
[19]
of biomolecules, such as proteins, the
bulky photocaging groups are usually
not compatible with metabolic engi-
neering, which relies on the limited
tolerance of the cellular biosynthetic
machinery to modifications of natural
[
2]
substrates. In contrast, the Proc group
utilized in this study is small in size and
tolerated by the sialic acid biosynthetic
pathway. Proc is chemically inert and
becomes sensitive only in the presence
of a palladium catalyst. To our knowl-
edge, the reported in situ conversion of
Neu5Proc into Neu is the first example
of the creation of the core nine-carbon-
atom sugar Neu on the surface of live
Figure 4. Quantification of Neu5Proc-depropargylation efficiency on the surface of live cells by
LC–MS. a) Work flow for quantifying the efficiency of palladium-mediated Neu5Proc depropargy-
lation by LC–MS analysis. Compound 6, which can block the liberated free amine after the
depropargylation of Neu5Proc, was used as the MS tag. A selected-ion recording mode was then
used to remove the MS signals due to unwanted molecules. b) Structure of the DMABA MS tag.
À
+
c,d) Chromatographic signal of selected ions ([Neu5Proc-H] and [NeuNHꢀMS-tag+H] ). CHO cells. This transformation may facilitate
cells were incubated with Ac ManNProc (50 mm) for 72 h, and the surface Neu5Proc and Neu
the study of the functional roles of Neu
under various physiological and/or
4
(
with MS tag) were detected by LC–MS after the depropargylation process.
pathological
conditions.
Notably,
[
5]
ing cancer cells. We envisioned that the depropargylation of
Neu5Ac is a key residue on cell-surface glycans that mediates
the interaction between influenza virions and host cells.
[20]
Neu5Proc (pK = 2.74; see Figure S11) would produce Neu
a
containing a free amine at the C5 position with strong proton
Whether Neu on host cells can be directly recognized by the
Neu5Ac-binding proteins of the virion coat remains to be
investigated. Finally, metabolic engineering of cell-surface
sialic acids has been previously exploited for modulating cell–
affinity (pK = 2.44, pK = 9.33; see Figure S11), thus parti-
a1
a2
ally neutralizing the carboxyl-derived negative charge on cell
surfaces (Figure 5a). We tested this hypothesis on K20 cells,
a subclone of the human B lymphoma cell line BJA-B that is
[
21]
[22]
cell interactions
and controlling stem-cell fate.
Our
[18]
deficient in the de novo biosynthesis of Sias.
K20 cells
strategy provides a simple and straightforward approach to
trigger cell clustering by manipulating the cell-surface charge,
with temporal control enabled by chemical decaging. Future
applications of our method include cell and tissue engineering
therefore contain no Sias on the cell surface and are able to
uptake Neu5Ac or its analogues from the culture medium and
incorporate them into cell-surface glycans. K20 cells incor-
porating Neu5Proc were treated with Pd NPs, and the
z potential was measured before and after the Pd-NP-
triggered depropargylation. The cell-surface z potential was
shifted to a less negative value, thus indicating the generation
of free amine groups (Figure 5b, top). As a control, K20 cells
with no Neu5Proc supplementation exhibited a less negative
z potential, which was not affected by treatment with Pd NPs
[
23]
for regenerative medicine.
Keywords: bioorthogonal elimination · cell surfaces ·
metabolic oligosaccharide engineering · palladium · sialic acids
How to cite: Angew. Chem. Int. Ed. 2015, 54, 5364–5368
Angew. Chem. 2015, 127, 5454–5458
(
Figure 5b, bottom). The modulation of cell-surface charge
was also possible in Jurkat cells (see Figures S12 and S13).
Interestingly, neutralization of the negative charge may
render the cells prone to clustering (Figure 5a). As expected,
Jurkat cells incorporating Neu5Proc formed significantly
larger clusters after treatment with Pd NPs (Figure 5c; see
also Figure S14), whereas for cells without Neu5Proc incor-
poration, the treatment with Pd NPs did not alter clustering.
Taken together, these results show that the palladium-
mediated depropargylation on Neu5Proc could liberate
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Figure 5. Palladium-triggered Neu5Proc decaging for the manipulation of cell-surface charge and cell
clustering. a) The palladium-mediated in situ conversion of Neu5Proc into Neu could generate a free
amine at C5 and thus alter cell-surface charge and cause increased propensity for cell clustering.
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