Protein S-Glyco-Modification through an Elimination-Addition Mechanism

Article abstract of DOI:10.1021/jacs.0c02110

Per-O-acetylated unnatural monosaccharides containing a bioorthogonal group have been widely used for metabolic glycan labeling (MGL) in live cells for two decades, but it is only recently that we discovered the existence of an artificial "S-glycosylation" between protein cysteines and per-O-acetylated sugars. While efforts are being made to avoid this nonspecific reaction in MGL, the reaction mechanism remains unknown. Here, we present a detailed mechanistic investigation, which unveils the "S-glycosylation" being an atypical glycosylation termed S-glyco-modification. In alkaline protein microenvironments, per-O-acetylated monosaccharides undergo base-promoted β-elimination to form thiol-reactive α,β-unsaturated aldehydes, which then react with cysteine residues via Michael addition. This S-glyco-modification produces 3-thiolated sugars in hemiacetal form, rather than typical glycosides. The elimination-addition mechanism guides us to develop 1,6-di-O-propionyl-N-azidoacetylgalactosamine (1,6-Pr₂GalNAz) as an improved unnatural monosaccharide for MGL.

Full text of DOI:10.1021/jacs.0c02110

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Article
Protein S-Glyco-Modification Through an Elimination-Addition Mechanism
Ke Qin, Hao Zhang, Zhenqi Zhao, and Xing Chen
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 27 Apr 2020
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Protein S-Glyco-Modification Through an Elimination-Addition Mechanism
Ke Qin,1,2,# Hao Zhang,1,2,# Zhenqi Zhao, & Xing Chen*
1,2
1,2,3,4,5
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College of Chemistry and Molecular Engineering, Beijing National Laboratory for Molecular
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Sciences, Peking-Tsinghua Center for Life Sciences, Synthetic and Functional Biomolecules
Center, and Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of
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Education, Peking University, Beijing 100871, China.
*
e-mail: xingchen@pku.edu.cn
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ABSTRACT
Per-O-acetylated unnatural monosaccharides containing a bioorthogonal group have been widely
used for metabolic glycan labeling (MGL) in live cells for two decades but it is only until recently
that we discovered the existence of an artificial “S-glycosylation” between protein cysteines and
per-O-acetylated sugars. While efforts being made to avoid this nonspecific reaction in MGL, the
reaction mechanism remains unknown. Here, we present a detailed mechanistic investigation,
which unveils an atypical glycosylation termed protein S-glyco-modification. In alkaline protein
microenvironments, per-O-acetylated monosaccharides undergo base-promoted β-elimination to
form thiol-reactive α,β-unsaturated aldehydes, which then react with cysteine residues via Michael
addition. This S-glyco-modification produces 3-thiolated sugars in the hemiacetal form, rather
than typical glycosides. The elimination-addition mechanism guides us to develop 1,6-di-O-
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propionyl-N-azidoacetylgalactosamine (1,6-Pr GalNAz) as an improved unnatural
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monosaccharide for MGL.
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INTRODUCTION
Exploiting the long-known substrate promiscuity of enzymes involved in sialic acid biosynthesis
and sialylation,1 the Bertozzi group in 2000 developed an unnatural analog of N-
acetylmannosamine (ManNAc), N-azidoacetylmannosamine (ManNAz), which could be
metabolically converted to azido sialic acid (SiaNAz) and incorporated into sialoglycans in live
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cells. Subsequently, the incorporated azide was conjugated with functional probes via Staudinger
ligation, one of the very first bioorthogonal reactions. Since then, various unnatural
monosaccharides containing a bioorthogonal functional group (e.g., the azide and alkyne) have
been developed for labeling sialoglycans as well as other types of glycans.3-5 For example, N-
azidoacetylgalactosamine (GalNAz) can be converted to UDP-GalNAz and UDP-GlcNAz in live
cells, which result in labeling of mucin-type O-linked glycans, N-linked glycans, and O-GlcNAc.
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-alkynyl fucose (FucAl) metabolically labels fucosylated glycans. Furthermore, a series of new
bioorthogonal reactions such as Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC or click
chemistry) and strain-promoted azide-alkyne cycloaddition (SPAAC or copper-free click
chemistry) are now available to choose from.6,7 This strategy, termed metabolic glycan labeling
(MGL), metabolic oligosaccharide engineering (MOE), or metabolic chemical reporter (MCR),
has emerged as a widely used tool for tagging glycans with fluorophores for imaging and with
enrichment probes for glycoproteomic profiling.
Experimentally, the hydrophilic nature of unnatural monosaccharides renders low efficiency
for cellular uptake due to the barrier imposed by the hydrophobic plasma membrane. To improve
cell entry, these unnatural monosaccharides are usually per-O-acetylated to increase
hydrophobicity and membrane permeability. This simple and effective strategy exploits cytosolic
nonspecific esterases to cleave the acetyl groups and produce unprotected free sugars inside the
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cells. For the past two decades, MGL has been predominantly practiced with per-O-acetylated
unnatural monosaccharides. Until very recently, our group unexpectedly discovered that per-O-
acetylated monosaccharides and their unnatural analogs could chemically react with the cysteine
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residues of various proteins in the cells. This so-called artificial cysteine ‘S-glycosylation’ occurs
in parallel with esterase hydrolysis and metabolic incorporation, thus compromising the specificity
of MGL at least to some extent (Figure 1a). One simple solution to this problem is to use
unprotected sugars, but having to suffer from low efficiency. Because the chemical structure of
‘
S-glycosylation’ products and the reaction mechanism remained incompletely understood, we
took a trial and error strategy to develop new unnatural monosaccharides possessing high MGL
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efficiency but no artificial ‘S-glycosylation’. By synthesizing and evaluating a series of partially
protected sugars, we developed 1,3-di-esterified GalNAz including 1,3-di-O-acetyl-GalNAz (1,3-
Ac
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GalNAz) and 1,3-di-O-propionyl-GalNAz (1,3-Pr GalNAz) as satisfying next-generation
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unnatural monosaccharides.
It is remarkable that the ‘S-glycosylation’ has been overlooked in the field of MGL for almost
two decades. To better understand this reaction and further improve MGL, we herein report the
mechanistic investigation of the ‘S-glycosylation’. Interestingly, we found that the reaction
between per-O-acetylated monosaccharides and protein thiols is an atypical glycosylation, which
follows an elimination-addition mechanism and results in 3-thiolated sugars in the reducing
hemiacetal form, rather than the glycosides at the acetal form (Figure 1b). The mechanism of
protein S-glyco-modification guided us to develop 1,6-di-O-propionyl-GalNAz (1,6-Pr GalNAz)
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as a better version of unnatural monosaccharides for MGL.
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Figure 1. Per-O-acetylated monosaccharide-induced protein S-glyco-modification in the cells.
(
a) In traditional MGL, HexNAz are administered in the per-O-acetylated form (e.g., Ac
Ac GalNAz, and Ac ManNAz) to enhance membrane permeability. Once inside the cells, the
cytosolic esterases cleave the acetates and the resulting HexNAz are metabolically incorporated
into cellular glycans. In parallel, Ac HexNAz can directly react with protein cysteine residues and
the protein S-glcyo-modification show an MW of ‘Cys-S-Ac
glyco-modification reaction. Ac HexNAc or Ac HexNAz, abbreviated as Ac
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GlcNAz,
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HexNAz’. (b) Scheme of the S-
HexNAc(z), is first
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hydrolyzed at the anomeric position. The resulted 3,4,6-tri-O-acetylated HexNAc(z) reacts with
the thiol under basic conditions to generate 3-thiolated pyranoses and furanoses bearing two acetyl
groups. The chiral carbons are marked in green.
RESULTS AND DISCUSSION
Mild Base Initiates the Per-O-Acetylated Monosaccharide-Thiol Reaction. Based on the mass
spectrometry (MS)-based proteomic analysis results from the cells metabolically labeled with per-
O-acetylated N-azidoacetylhexosamine (Ac
Ac HexNAz-thiol conjugation had the molecular weight (MW) matching ‘Cys-S-Ac
Figure 1a). The reaction exhibited high selectivity to cysteine among all nucleophilic residues of
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HexNAz),9 the most abundant products of
HexNAz’
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(
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proteins. Sequence analysis around the ‘S-glycosylation’ sites using Two Sample Logo revealed
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high enrichment of lysine residues, suggesting that the reaction required a basic condition (Figure
S1). Furthermore, with the anomeric acetyl group removed, 3,4,6-tri-O-acetyl-ManNAz (3,4,6-
Ac
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ManNAz) and 3,4,6-tri-O-acetyl-GalNAz (3,4,6-Ac GalNAz) exhibited enhanced reactivity
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and resulted in cysteine adducts with the same MW of those by per-O-acetylated ManNAz
GalNAz).10,12 Based on these considerations,
(
Ac
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ManNAz) and per-O-acetylated GalNAz (Ac
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we reasoned that per-O-acetylated N-acetylhexosamine (Ac
per-O-acetylated N-acetylglucosamine (Ac GlcNAc, 1), per-O-acetylated N-acetylgalactosamine
Ac GalNAc), per-O-acetylated ManNAc (Ac ManNAc), per-O-acetylated N-
azidoacetylglucosamine (Ac GlcNAz), Ac GalNAz, and Ac ManNAz are initially converted to
the 3,4,6-tri-O-acetylated form, such as 3,4,6-tri-O-acetyl-GlcNAc (3,4,6-Ac GlcNAc, 2), and
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HexNAc) and Ac
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HexNAz including
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(
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subsequently react with thiol groups to generate adducts bearing two acetyl groups under alkaline
conditions (Figure 1b and S2a).
To test this hypothesis, we established a model reaction using N-acetyl-cysteine (AcCys)
(
Figure S2a). Thin-layer chromatography (TLC) analysis showed that incubation of 2 mM AcCys
with 10 mM 1 and 2 at the pH of 10 for 1 h afforded P1, which showed the MW of AcCys-S-
Ac GlcNAc (Figure S2b, c). Comparing to 1, 2 reacted with AcCys with a much higher yield,
2
indicating that the anomeric deacetylation was a rate-limiting step. Of note, deacetylation is
accelerated by base. At the pH of 7.4, no product was detected for either 1 or 2, confirming that a
basic microenvironment from the protein surfaces and pockets was essential for ‘S-glycosylation’
to occur (Figure S2d). Moreover, the fact that 2 did not react with AcCys at neutral pH indicated
that the alkaline condition played an essential role in steps after the anomeric deacetylation. We
then examined the crude reaction mixtures by liquid chromatography–mass spectrometry (LC-
MS) (Figure S2e). Multiple peaks with the MW of P1 were detected, suggesting that the ‘S-
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glycosylation’ products were isomeric mixtures. At the same time, multiple peaks with MWs
matching partially and completely deacetylated 2 and P1 were observed, which could be attributed
to the well-known deacetylation and migration of the acetyl group on sugars under alkaline
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conditions.13,14 Similar results were observed for 3,4,6-tri-O-acetyl-GalNAc (3,4,6-Ac
) and 3,4,6-tri-O-acetyl-ManNAc (3,4,6-Ac ManNAc, 4) (Figure S3).
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GalNAc,
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Figure 2. Structural determination of the products of reaction of PEM with 2. (a) PEM was
reacted with 2 in 10 mM Na CO buffer for 1 hour, followed by acetylation using acetic anhydride
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to generate P6. The acetylation step was to reduce the complexity of products. (b) Structural and
stereochemical assignments of P6. P6 consist of 4 isomers, three pyranoses and one furanose.
The TOCSY and HMBC correlations used for analysis are marked in red and blue, respectively.
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For P6c, * denotes an undetermined configuration. (c) H, H-TOCSY spectrum of P6. Four
distinct spin systems resulting from the carbohydrate backbone protons are indicated by black lines.
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The full spectrum is shown in Figure S6. (d) H, C-HMBC spectrum of P6. The HMBC
correlations between H of carbohydrate backbones and C of carbonyl groups are indicated. δ
proton NMR chemical shift (in ppm); δ , carbon NMR chemical shift (in ppm).
H
,
C
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Structural Determination of the Reaction Products. We then sought to purify and characterize
the structure of reaction products. To facilitate the purification and NMR analysis, we used another
thiol compound, phenylethyl mercaptan (PEM), to react with two equivalents of 2 (Figure 2a, b
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and S4a). Similar to AcCys, PEM-S-Ac
obtained as mixtures (Figure S4b). To reduce the product complexity, we per-O-acetylated the
mixtures and isolated PEM-S-Ac GlcNAc (P6) with an overall yield of 61% (Figure S4a). HPLC
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GlcNAc (P4) and its deacetylated product P5 were
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analysis indicated that P6 probably contained four isomers (P6a, P6b, P6c and P6d) in a ratio of
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:6:2:1 (Figure S4c). P6 was then subjected to analysis by NMR spectroscopy. The H spectrum
of P6 exhibited the aromatic protons with chemical shifts at around 7.2 ppm, acetyl protons at
around 2.0 ppm, and aliphatic protons between 2.5 and 6.5 ppm, most of which were from the
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carbohydrate backbone (Figure S5a). In H, H-total correlation spectroscopy (TOCSY), the
carbohydrate backbone protons showed four distinct spin systems, which confirmed the presence
of the four isomers for P6 (Figure 2c and S6).
By analyzing the heteronuclear multiple bond correlation (HMBC), homonuclear correlation
spectroscopy (COSY), and heteronuclear single quantum coherence (HSQC) spectra (Figure 2d,
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S7 and S8a), the H and C chemical shifts of the sugar moiety of P6 were mostly assigned (Figure
S5b and S8b) and the chemical structures of four P6 isomers were determined (Figure 2b, see
Supporting Information for details). For P6b, the major isomer, the HMBC spectrum showed that
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H-1, H-2, H-4 and H -6 displayed clear cross-signals with the carbonyl C-signals, demonstrating
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that C-1, -4 and -6 connected with an acetoxy group while the C-2 connected with an acetamido
group (Figure 2d). H-5 had no 3JCH-coupling with any carbonyl carbon and therefore the downfield
chemical shift of C-5 at 70.87 ppm should be attributed to connection to the electron-withdrawing
oxygen within the six-membered ring. Therefore, the remaining C-3 was thiolated. Of note, in
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the initial report of the discovery of ‘S-glycosylation’, we intuitively proposed a thioglycosidic
linkage based on various known nucleophilic substitution reactions at the anomeric center. The
NMR results herein amended the structure to have a C-S linkage at C-3. Furthermore, compared
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with the H and C chemical shifts of other protons and carbons in the carbohydrate backbone, the
higher field shift of H-3 (2.84 ppm) and C-3 (46.89 ppm) was owing to relatively poor deshielding
effect of the sulfur atom, which further confirmed the C-S bond at C-3 (Figure S5b and S8b). In
addition, the 3JH-1,H-2 coupling constant of 3.4 Hz and 3JH-2,H-3 coupling constant of 12.1 Hz
confirmed the α-anomeric configuration and the equatorial orientation of C-3 thiol of P6b,
respectively. Taken together, the chemical structure of P6b was unambiguously determined as a
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-thiolated α-pyranose (Figure 2b). By analyzing the NMR spectra for P6a, P6c, and P6d in the
similar way, their chemical structures were determined (Figure 2b). As two pyranose isomers,
P6a had the β-anomeric configuration and P6d contained an axial C-3 thiol substitution.
Interestingly, P6c was a furanose. Unlike the six-membered ring, the five-membered ring is more
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flexible, making definitive depiction of configuration based on J coupling values difficult. Thus,
an energetically favorable structure was assigned for P6c.
Capture and Identification of Thiol-Reactive Intermediates. Base-catalyzed isomerization has
long been explored to convert biomass-derived monosaccharides to rare monosaccharides.16 An
undesirable feature of this conversion is that monosaccharides are prone to degrade to over 50
different byproducts in strong alkaline conditions.17 Considering that the S-glyco-modification
demanded base, we hypothesized that per-O-acetylated monosaccharides after anomeric
deacetylation might be degraded or converted to thiol-reactive intermediates in alkaline conditions.
To capture the possible intermediates in LC-MS, we incubated 2 with diluted NaHCO
3
/Na
2
CO
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buffer (pH = 10, 25 mM). Four peaks corresponding to 5 (including isomers 5a and 5b) and 6
(
including isomers 6a and 6b) with the same molecular weight were observed (Figure S9a).
Adding AcCys gradually converted 5 to P1, while 6 were not significantly decreased (Figure S9b).
The slight decrease of 6 was probably due to interconversion with 5. These results indicate that 5
are thiol-reactive intermediates in the S-glyco-modification reaction.
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We then sought to purify and identify 5. 10 mM 2 was incubated with NaOH at increasing
concentrations (Figure 3a). With 1 µM NaOH, a small portion of 2 was converted to 5, with no 6
observed. Increasing NaOH resulted in more 5. Generation of 6 started with 0.1 mM NaOH. Of
note, deacetylation occurred simultaneously with more NaOH leading to more deacetylated
products. When the NaOH concentration was increased to 10 mM, 5 was completely deacetylated.
1
00 mM NaOH resulted direct deacetylation of 2 as the major reaction, leading to no 5 or 6
produced. Similar results were observed by using Na CO at varied concentrations (Figure S10).
Based on these results, we isolated 5 and 6 by using 5 and 10 mM Na CO , respectively (Figure
S11). The chemical structures of 5 and 6 were determined by H, H-COSY, H, C-HSQC and
2
3
2
3
1
1
1
13
1
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H, C-HMBC (Figure S12). The acyclic 5a and 5b contained an α,β-unsaturated aldehyde, which
can react with thiols via Michael addition in water.18 By contrast, the 2,3-unsaturated glycals 6a
and 6b in the pyranose and furanose forms, respectively, should not be thiol-reactive. The
formation of 6 were probably through ring closure of 5 (Figure 3b and S13).
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Figure 3. Thiol-reactive intermediates and proposed mechanism for S-glyco-modification. (a)
UV-Vis chromatograms showing 10 mM 2 incubated with NaOH at increasing concentrations for
1
h, followed by analysis by LC-MS equipped with a UV-Vis detector. Note that the peaks of a 2
isomer and a 6 isomer overlapped with each other. The deacetylated mixtures contained
deacetylated intermediates and substrates. Absorbance was recorded at l = 220 nm. a.u., arbitrary
units. The MS spectra were used to confirm peak assignments as necessary. (b) Conversion of 2
to the thiol-reactive intermediates 5. Note that 5 can be interconverted with 6 in the ring form, and
deacetylation occurs simultaneously under basic conditions. Deacetylated intermediates are shown
in Figure S13. For 6, * denotes an undetermined configuration. (c) Proposed two-step elimination-
addition mechanism for S-glyco-modification with 2. In the β-elimination step, ring opening
occurs to produce acyclic monosaccharides, which can then undergo based-promoted E1cb. In the
Michael addition step, acyl migration between 4-OH and 5-OH results in the pyranose and
furanose isomers. Note that base is not required for Michael addition.
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Mechanism of the Per-O-Acetylated Monosaccharide-Thiol Reaction. With the thiol-reactive
intermediates 5 identified, we proposed an elimination-addition mechanism (Figure 3c). Starting
from the per-O-acetylated monosaccharides, the anomeric acetate is first hydrolyzed in aqueous
solution. The resulting hemiacetal 2 then establish an equilibrium with the open-chain aldehyde
form via the ring-chain tautomerism in water. The 3-acetoxy group of the aldehyde 8 undergoes
base-promoted β-elimination to form the α,β-unsaturated aldehydes 5a. Acyl migration between
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- and 5-OH results in an equilibrium of interconversion between two α,β-unsaturated aldehydes
5
a and 5b. Michael addition with thiols, followed by ring closure, gives the 3-thiolated pyranoses
and furanoses, respectively.
Since an electron-withdrawing group (EWG) such as the aldehyde is a prerequisite for β-
elimination, the monosaccharide must first undergo ring opening to become the open-chain
aldehyde form, which is then eliminated to produce the thiol-reactive Michael acceptor. This also
confirms that 6 are produced from 5, rather than from direct β-elimination of 2. With a strong
EWG at α-carbon and a poor nucleofuge at β-carbon, β-elimination adopts the E1cB mechanism,
in which formation of the carbanion and dissociation of the leaving group are not concerted.19
Because the acetoxy is generally considered to be a poor nucleofuge, we reasoned that β-
elimination of 8 follows E1cb.
Mechanism of Protein S-Glyco-Modification. Having demonstrated the elimination-addition
mechanism for model reactions, we next evaluated protein S-glyco-modification. We synthesized
2
3
-O-acetyl-4,6-di-O-[ H
3
]acetyl-GlcNAc (9), in which the C-4 and C-6 acetates were
differentiated from the C-3 acetate by deuterium labeling (Figure S14a). According to the
mechanism, β-elimination would selectively occur for C-3 acetoxy, and both of the deuterated C-
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and C-6 acetates should remain, which can be detected by MS. LC-MS analysis on the reaction
of 9 with AcCys showed that the thiol-reactive intermediates and the products possessed a MW
increase of 6 Da (Figure S14b, c). These results confirmed that 9 could serve as a probe for
investigating the protein S-glyco-modification mechanism.
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Figure 4. Protein S-glyco-modification follows the elimination-addition mechanism. (a) Scheme
of proteins reacted with 9 via the elimination-addition mechanism, resulting in modification of
cysteines with an increase of MW by 293.138 Da. (b) Representative MS2 spectrum of the peptide
containing modified Cys-139 in COLF1 reacted with 9 in PBS buffer for 1 h. The modification
showed an MW shift of 293.138 Da.
We then incubated purified cofilin 1 (COLF1) protein with 9 in PBS buffer (pH = 7.4) for 1 h
(
Figure 4a). Modifications of C139, C80, C39 with a MW increase of 293.1 Da, matching the
elimination-addition mechanism, were detected (Figure 4b and S15). All three Cys were located
adjacent to Lys residues, confirming the need of basic microenvironment on proteins for the β-
elimination reaction to occur. In principle, the Lys residues enriched around the modified Cys
using per-O-acetylated monosaccharides might come from both the anomeric deacetylation and β-
elimination steps (Figure S1). We therefore employed 3,4,6-tri-O-acetyl-GlcNAz (3,4,6-
Ac GlcNAz, 11) as a warhead to reacted with MCF7 cell lysates (Figure S16a). The sequences
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around the modification sites were enriched with Lys, demonstrating that the β-elimination step
demanded base (Figure S16b). Consistent with this conclusion, acidic conditions with pH of 5 and
6, which neutralized the lysine reside, significantly diminished reaction of 11 with MCF-7 cell
lysates via S-glyco-modification (Figure S17).
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Figure 5. Mechanism-based design and development of 1,6-Pr
azidosugar for MGL. (a) Structure of 14. (b) In-gel fluorescence scanning showing HeLa cell
lysates treated with 14 or Ac GalNAz at varied concentrations for 1 h, followed by reaction with
alkyne-Cy3. (c) Cell counting assay by CCK-8 kit showing viability of HeLa cells incubated with
4 at varied concentrations for 48 h. Error bars represent mean ± s.d. Results are from four
2
GalNAz (14) as an improved
4
1
independent experiments. n.s., not significant (one-way ANOVA). (d) In-gel fluorescence
scanning showing HeLa cells incubated with 14 and GalNAz at varied concentrations for 48 h,
followed by reaction with alkyne-Cy3. Coomassie Brilliant Blue (CBB)-stained gels in (b) and (d)
demonstrate equal loading.
Mechanism-Based Design of 1,6-Pr
2
GalNAz for MGL. The S-glyco-modification serves as a
side reaction in MGL, which might cause certain false-positives and should be avoided.
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Previously, we found that 1,3-Ac
2
GalNAz and 1,3-Pr GalNAz did not induce S-glyco-
2
modification and could be used as a satisfactory unnatural monosaccharide for metabolic labeling
of O-GlcNAc in various cell lines.10 According to the elimination-addition mechanism, if 1,3-
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Ac
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GalNAz and 1,3-Pr GalNAz were selectively deacylated at the anomeric position by cytosolic
2
esterases, the resulting 3-O-acetyl-GalNAz (3-AcGalNAz) and 3-O-propionyl-GalNAz (3-
PrGalNAz) bearing C-3 acetoxy and propionyloxy, respectively, could in principle be eliminated
to generate thiol-reactive intermediates. To test this hypothesis, 10 mM 1,3-di-O-acetyl-GalNAc
(
1,3-Ac
mM AcCys at pH of 10 for 1 h. TLC and MS analysis showed detectable products with MW of
AcCys-S-GalNAc, indicating that slight S-glyco-modification of AcCys occurred for both 12 and
3 (Figure S18). The fact that 1,3-Ac GalNAz and 1,3-Pr GalNAz do not induce detectable S-
2
GalNAc, 12) and 1,3-di-O-propionyl-GalNAc (1,3-Pr GalNAc, 13) were incubated with
2
5
1
2
2
glyco-modification in cell lysates and living cells can be attributed to: (i) lower concentrations are
used in cell lysates and needed for MGL in living cells, (ii) cytosolic esterases have an
approximately 50% probability to first cleave the C-3 ester, and (iii) 3-AcGalNAz and 3-
PrGalNAz with a half chance to be generated are quickly further deacylated to free sugars with
ignorable elimination products. Nevertheless, a perfect unnatural monosaccharide should avoid
protection at 3-OH, based on the mechanism. We therefore designed and synthesized 1,6-
Pr
2
GalNAz (14; Figure 5a) with an overall yield of 8% (Scheme S1).
HeLa cell lysates were incubated with 14, followed by reaction with alkyne-Cy3. In-gel
fluorescence scanning showed that no artificial S-glyco-modification was detected for 14 up to 2
mM (Figure 5b). No significant cytotoxicity was observed in HeLa cells incubated with 14 at
concentrations up to 500 µM for 48 h (Figure 5c). We then tested the metabolic labeling efficiency
by treating HeLa cells with 14 or GalNAz. The metabolic labeling efficiency of 14 at 100 µM was
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higher than 2 mM GalNAz in HeLa cells (Figure 5d). These results demonstrate that 14 designed
based on the mechanism is a highly specific and efficient unnatural monosaccharide for metabolic
labeling.
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CONCLUDDING DISCUSSION
In developments of chemical probes (i.e., small-molecule inhibitors and activators), off targets
and non-specific activities have been widely appreciated, and called for systematic
characterizations.20 Conversely, MGL has seldomly been bothered by off targets because
unnatural monosaccharides are by design chemically inert. Our discovery of “S-glycosylation”,9
now amended to be “S-glyco-modification”, was unexpected and has raised concerns on possible
false-negatives in studies using per-O-acetylated unnatural monosaccharides. We have also
pointed out that the level of interference caused by S-glyco-modification depends on particular
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MGL experiments, and in many cases might not be significant. The reaction mechanism revealed
in this work supports this hypothesis. The ring-chain tautomerism of monosaccharides in aqueous
2
1
solution only produces a very little amount of open-chain aldehydes (e.g., < 0.01% for glucose ).
It is likely that acylation could slowly shift the equilibrium towards the open-chain form by
imposing ring strain, although the cyclic forms were still dominant (Figure S19). Furthermore,
base is a prerequisite for β-elimination, which limits S-glyco-modification within the perimeter of
local alkaline microenvironments generated by basic amino acids of proteins. Taken together, S-
glyco-modification is a slow reaction under physiological condition. Therefore, within the cellular
environments, the stoichiometry of protein S-glyco-modification induced by per-O-acetylated
unnatural monosaccharides in MGL experiments is probably low relative to that of metabolic
labeling, particularly for unnatural sugars with high labeling efficiency. On the other hand, it is
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important to be always aware of the possible false-negatives, particularly for large-scale
glycoproteomic profiling without identifying the modification sites.
It is worthwhile to discuss whether the per-O-acetylated unnatural monosaccharides can
continue to be used for MGL. Although the protein S-glyco-modification is relatively slow at
physiological pH, it is not straightforward to quantify the extent of nonspecific reaction and
interference with the metabolic labeling results. Therefore, unnatural monosaccharides with no
artificial S-glyco-modification are always the best choice. For GalNAz, one should use 1,6-
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Pr
2
GalNAz developed in this work or 1,3-Pr
GalNAz reported previously.10 With the mechanism
2
clearly revealed, this partially protection strategy should be readily applied to other azido or
alkynyl sugars to make all kinds of unnatural sugars with high efficiency and specificity available
to the community. The second-best choice would be to use unprotected unnatural sugars, with the
trade-off of having to use high concentrations of unnatural sugars.
From the perspective of carbohydrate chemistry, the elimination-addition mechanism
represents a unique way to make 3-thio-sugars in aqueous solution. Interestingly, 3-thio-sugars
have previously been prepared through thiol addition with α,β-unsaturated aldehydes and α,β-
unsaturated lactones, which were derived from glycals via allylic rearrangement and oxidation,
receptivity.22,23 Fundamentally, S-glyco-modification is a one-pot synthesis of 3-thio-sugars in
aqueous condition from readily prepared per-O-acetylated monosaccharides or their derivatives.
The S-glyco-modification is of great value for protein chemistry. We have exploited it for
cysteine profiling and large-scale identification of cysteine modifications by the anti-inflammatory
metabolite itaconate.12 We found that S-glyco-modification matched better with cysteine
modification by itaconate, indicating that per-O-acetylated monosaccharides were distinct from
other thiol probes, such as iodoacetamide and α,β-unsaturated ketones. From the elimination-
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addition mechanism, we now know that the distinct cysteine reactivity is probably because S-
glyco-modification is driven by the alkaline microenvironments. It will be of great interest to
investigate whether cysteine profiling by per-O-acetylated monosaccharides target cysteine sites
with distinct chemical and biological properties resulted from the alkaline microenvironments.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at
DOI:xxxxxxx.
Materials, Methods, Scheme S1, Figures S1 to S43, and Supplementary References (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Email: xingchen@pku.edu.cn.
Author Contributions
#
These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
Part of the MS experiments were performed at the mass spectrometry facility at the National Center
for Protein Sciences at Peking University. This work is supported by the National Key Research
and Development Projects (No.2018YFA0507600) and the National Natural Science Foundation
of China (NO.91753206, NO.21521003).
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