Contents
1. Introduction Page No.
2. Method Development towards Efficient Assembly of Glycoproteins Page
No.
2.1. Development and Application of Synthetic Methods Page No.
2.2 Development and Application of Enzymatic Methods Page No.
2.3 Efforts towards Effort-less Purification
3. Comprehensive Study of Protein Glycosylation Using Synthetic
Glycoforms
3.1 Synthetic Glycoforms with Single Glycosylation Site Page No.
3.2 Synthetic Glycoforms with Multiple Glycosylation Sites Page No.
4. Conclusion and Perspectives Page No.
1. Introduction
Protein glycosylation is a most complicated type of post-translational
modifications.[1-3] The variety of glycan
structure, glycosidic sites and glycosidic linkage, added up to the
complexity of protein glycosylation. Protein glycosylation, unlike pure
protein and oligonucleotide synthesis, is not template mediated; hence,
the glycosylation outcome depends on the activity and concentration of
glycan substrates, the structural and conformational properties of the
catalytic domain and the different reactivities of each enzyme involved
in the glycosylation process.[3] In this way, the
resulting mixture of glycoproteins, termed glycoforms, with likely
varied glycan structure as well as site occupancy, are regarded as
valuable materials towards better understanding of protein glycosylation
in live system. Despite the challenges in accessing well-defined
homogeneous glycoproteins, it has been one of the most frequently
studied area in the past decades, thus enabling the ongoing structural
and functional studies of protein glycosylation. Researchers from
synthetic chemistry and chemical biology devoted numerous efforts in
this regard, and significant achievement was
realized.[4] Apparently, we are getting closer to
the code of life: novel synthetic methods were developed, and various
decent-sized glycoproteins were synthesized, which had never been
achieved before. Of particular significance is the breakthrough of
synthetic glycoforms: we can now probe the effect of protein
glycosylation systematically through a library of glycoproteins, each in
homogeneous form, with distinct glycan chains attached to the protein
respectively, and we believe that with additional efforts we will be
able to connect such information with the actual outcome in cells and
even in individual people. Hereby it is our great honor to give a brief
summary of the progress made in this field. Rather than lengthy
comprehensive prose, we would like to highlight the breaking news in
recent three years.
Novel synthetic methods is fundamental for the successful assembly of
glycoproteins.[5-10] Synthesis of each component,
being glycans and proteins, as well as the glycosidic linkage in
between, remains a demanding task. In order to develop viable and
efficient strategies to chemically construct homogeneous complex
glycopeptides and glycoproteins, extensive efforts have been made in the
field. To date, protein chemical synthesis and semisynthesis, enabled by
solid phase peptide synthesis and various chemical ligation
strategies,[10-14] have made it possible to access
native proteins of decent size. Furthermore, considering the presence of
various functionalities within the glycoprotein molecule, synthetic
tactics are generally required regarding the efficiency and beauty in
the successful assembly of glycoproteins.
Of particular significance is the breakthrough of synthetic glycoforms:
we can now probe the effect of protein glycosylation systematically
through a library of glycoproteins, each in homogeneous form, with
distinct glycan chains attached to the protein respectively. Apparently,
we are getting closer to the code of life: novel synthetic methods were
developed, and various decent-sized glycoproteins were synthesized,
which had never been achieved before. and we believe that with
additional efforts we will be able to connect such information with the
actual outcome in cells and even in individual people. Hereby a brief
summary of the progress made in this field is presented. Rather than
lengthy comprehensive prose, we would like to highlight the breaking
news in recent three years.
2. Methods Development towards Efficient Assembly of Glycoproteins
2.1. Development and Application of Synthetic Methods
Better understanding of the essence of protein glycosylation calls for
more methodological developments and synthetic efforts. Based on the
glycosidic linkage, glycoproteins can be categorized different types,
commonly known as N-linked, O-linked, S-linked and C-linked
glycoproteins, as well as phosphoglycans and glypiated
proteins.[1] To date, chemical synthesis of
glycoproteins mostly focused on the N- and O-linked glycoproteins, which
represents a majority of glycoproteins in nature. While chemical
ligation methods contribute to join peptide fragments toward respective
proteins, glycans are mostly introduced at the stage of peptide
synthesis (Figure 1). The linear approach is quite straightforward,
where the glycosyl amino acids are pre-formed as individual building
blocks, and are incorporated for the assembly of glycoproteins (Figure
1, Approach A).[5] Alternatively, a convergent
approach denotes that the glycan and the protein are prepared
separately, and then conjugated together to give the glycoprotein
(Figure 1, Approach B). Generally, N-linked glycoproteins, where the
glycosyl amine is conjugated to the protein at asparagine site with the
formation of an amide bond, can be performed in a convergent
manner.[15-16] In this case, an orthogonally
protected Asp residue is introduced initially at the glycosylation site,
which is selectively unmasked and subjected to aminolysis by glycosyl
amine to give the N-glycosylated Asn. Otherwise, N-linked glycoproteins
can be obtained in linear fashion as well, incorporating the
structurally defined N- glycosyl-Asn building blocks into solid
phase peptide synthesis to generate glycopeptides. These building
blocks, obtained either through the chemical coupling of a glycosyl
amine precursor to an Asp derivative, or through isolation from natural
sources followed by suitable protection,[17-18]have made a number of N-linked glycopeptides and glycoproteins with
various glycan structures accessible. For example, the asparagine with a
homogeneous complex-type biantennary sialyloligosaccharide can be
isolated from egg yolk or egg powder,[17, 19]whereas ones with high mannose type N-glycan can be isolated from
soybean flour,[20-21] generally on
milligram-to-gram scale. However, O-linked glycoproteins, where the
O-glycans are attached to the protein via serine, threonine or tyrosine
residues, are more frequently subjected to stereoselectivity issue
during the formation of O-glycosidic linkage, and are thus preferably
prepared in a linear way (namely, Approach A). The O-linked glycosyl
amino acids, generally termed “cassettes”, are prepared as building
blocks and then incorporated into the peptide
fragments.[22] Based on their respective chemical
property, novel methods towards the preparation of S-linked and C-linked
glycopeptides are also developed, adding to the toolbox of glycopeptide
and glycoprotein synthesis.
Figure 1 Approaches to N- and O-linked glycopeptides.
To improve the synthetic efficiency of convergent aspartylation, a
modified aminolysis protocol was introduced using aspartyl active
esters.[23-24] Compared with the thio- or oxoester
counterparts, aspartyl selenoesters demonstrated enhanced reactivity,
among which the seleno-phenyl ester afforded an optimal balance between
high reactivity and sufficient stability. In particular, the
selenoester-mediated glycosylation takes place without additional
coupling reagents or catalysts, and is highly compatible with free
C-terminal carboxylic acid, as well as multiple functional groups within
the complex-type sialylundecasaccharide. Facilitated by these method, a
couple of N-linked glycopeptide fragments bearing mono-, di-, and
oligosaccharide were synthesized in high yields, including ones derived
from human interleukin-5 and erythropoietin (EPO).
Another interesting discovery for the convergent synthesis of N-linked
glycopeptides is the thioamide-directed activation of aspartate strategy
(Figure 1, Approach C).[25] Basically, the
incorporation of a thioamide N-terminal to an Asp residue facilitates
Ag(I)-promoted conversion into respective N- glycosylated Asn
residue in the presence of a glycosyl amine. An optimal result was
obtained when using 1.2 equiv of Ag2CO3,
and a moderate excess (4 equiv.) of glycosyl amine, with (oxo)amide
observed as a minor byproduct. This thioamide-triggered approach is
highly chemoselective, and leads to topographically-defined
N-glycosylated asparagine derivatives. Expectedly, further
investigations are welcome such as incorporation of more complex
glycans, and potential application to on-resin glycosylations.
The frequent occurrence of aspartimide formation represents a formidable
challenge in N-linked glycopeptide synthesis, especially during the
process of convergent aspartylation (Figure 2A). Continuous efforts were
engaged in this regard, with modified protocols developed over years.
For example, pseudoproline-assisted aspartylation was devised as a
general approach.[26-27] By converting the
consensus sequence for N-glycosylation NXS/T residues into a
pseudoproline moiety, a twist in the protein backbone can be
generated,[28] thus preventing the formation of
aspartimides, both in peptide elongation and convergent aspartylation
(Figure 2B). At the end of glycopeptide synthesis, global acid treatment
regenerated the native sequence concomitantly. This finding is crucial
to the success of convergent aspartylation, that it has become the
method of choice for N-linked glycoprotein synthesis, with the
construction of some most challenging synthetic targets such as N-linked
RNase,[26] erythropoietin
(EPO)[26-27] and
E-cadherin[29] glycopeptides. Considering that the
protecting group of aspartic acid had an impact on the reaction outcome,
a cyanosulfurylide moiety was developed as a novel carboxylic acid
protecting group for Asp, to prevent aspartimide formation during
peptide synthesis (Figure 2C); yet, the applicability is to be
demonstrated in a broader context of glycopeptide
synthesis.[30] To avoid the exposure of aspartic
acid residues during peptide synthesis, a late-stage oxidation strategy
was introduced, employing homoserine as a precursor of aspartic acid
(Figure 2D).[31] Upon the full assembly of peptide
sequence, oxidation using (2,2,6,6-tetramethyl-piperidin-1-yl)oxyl
(TEMPO) and bis(acetoxy)iodobenzene (BAIB) led to the conversion into
aspartic acid at respective sites. The method extended to the synthesis
of a glycopeptide containing aspartic acids and an O-sulfated glycan.
Figure 2 (A) Aspartimide formation and (B‒D) preventative
measures
A proteoglycan is a subtype of O-linked glycoprotein found in cell
membranes within mucus and connective tissue; it is composed of core
proteins covalently-bonded to glycosaminoglycans
(GAGs).[32] Owing to the acid lability of
sulfates, proteoglycans are recognized as a most difficult synthetic
target integrating the sulfated glycosaminoglycans with the peptide
backbone.[33-36] Syndecan-1 is a prototypical
transmembrane proteoglycan commonly found on cell surface, which carries
heparan sulfates (HS) and chondroitin sulfate (CS) glycan chains at its
ectodomain.[32] While the sulfation patterns of HS
and CS dictate many activities, the core protein can potentially impact
HS functions. Hence, the preparation of a collection of
site-specifically modified syndecan glycoforms and sulfo-forms is highly
desirable. However, the presence of sulfates poses severe challenge to
its synthesis: as sulfates are highly acid labile, common solid phase
peptide synthesis is not applicable. To this end, suitable protection on
sulfates led to successful synthesis of syndecan
glycopeptides.[37] Towards better understanding of
the interplay between the glycosaminoglycan chain and core protein
backbone, construction of larger syndecan molecules up to glycoprotein
sized level, was highly desirable. In this regard, a novel
“glycopeptide cassette” approach was
introduced,[38] where a short-sequenced,
orthogonally functionalized glycopeptide was designed as a cassette to
allow for site-specific sulfation and subsequent chemical ligation;
starting there, microwave-assisted Ag(I) mediated ligation, along with
native chemical ligation, Ser/Thr ligation and desulfurization to give
ectodomain (23‒120) of syndecan-1, representing the longest O-sulfated
syndecan-1 molecule synthesized ever (Figure 3).
Figure 3 “Glycopeptide cassette” approach towards O-sulfated
glycoprotein synthesis
Well-established chemical ligation strategy is fundamental to the
successful preparation of glycoproteins with site-specific
glycosylation. Distinct from the conventional linear and convergent
approaches, Kajihara et al. developed a novel bifunctional
thioacid-mediated strategy, for the (semi)synthesis of N-linked
glycoproteins (Figure 4).[39] The key point rests
on a well-designed bifunctional glycosyl asparagine thioacid, which can
couple two peptide fragments at its N- and C-termini, respectively, thus
leading to the target glycoproteins within short steps. The strategy
takes place via a two-step transformation: 1) a diacyl disulfide
coupling (DDC) between a glycosyl asparagine thioacid and a peptide
α-thioacid, which affords a native amide bond via a disulfide bond
intermediate without any polymerization reactions; and 2) the resultant
glycopeptide has a thioacid form at its C-terminus, which will undergo
thioacid capture ligation (TCL) with another peptide bearing a disulfide
functional group at its N-terminus to afford the full-length
glycoprotein backbone.
Figure 4 (Semi)synthesis of N-glycoproteins via a bifunctional
glycosyl asparagine thioacid
Likely due to the steric hindrance of glycans, which could slow the
competitive polymerization of the thioacid moiety, the DCC reaction
proceeds chemoselectively with the glycosyl asparagine thioacid;
subsequently, an intramolecular S→N acyl transfer readily occurs on the
diacyl disulfide intermediate, yielding the corresponding amide product
predominantly. Yet, all internal peptide cysteines should be protected
by the acetamidomethyl (Acm) or phenylacyl (Pac) protecting group
because their thiol groups can prevent essential disulfide bond
formation in DDC and TCL. The stereochemistry of this chemical
transformation was carefully characterized, via extensive NMR and
reverse-phase HPLC analysis, which confirmed that each individual DDC
and TCL reaction did not give rise to epimerization. The established
method was extended to the synthesis of the cytokine glycoproteins CCL1
and interleukin-3 (IL-3), consisting of 73 and 133 amino acid residues,
respectively. Together, this thioacid-mediated strategy represents an
additional strategy along with existing protein chemical synthesis
strategies, such as native chemical ligation (NCL) and expressed protein
ligation (EPL).
Considering the high nucleophilicity, cysteine residues have become a
popular handle in protein modification. Thus, to directly install a
glycosyl unit onto the thiol group of cysteine residues, preferably in
aqueous media, is a conceptually straightforward and attractive task. A
radical-based glycosylation approach was developed, which
chemoselectively afforded axial S-glycosidic bonds under photocatalytic
conditions (Figure 5A).[40] The approach utilized
bench-stable allyl glycosyl sulfones as the glycosylating agents, to
react with an S pyridinyl masked disulfide as a cysteine surrogate.
O2 should be avoided in the reaction system, as it
interferes with the radical process. Moreover, a second-generation was
developed with the reactive disulfide generated in situ . The
utility of this approach successfully was exemplified with the
glycosylation of various peptides, including a cell penetrating
peptide-R8, Tyr-amyloid P component (27−38) and Amyloid β/A4 protein
precursor 770 (135−155), and a series of MUC1 peptide 20-mer bearing
S-linked Tn-antigen mimetics. Alternatively, glycosyl Bunte salts were
utilized for the selective “tagging” of cysteine residues, which
created glycomimetics with a disulfide bond at the conjugation site
(Figure 5B).[41] The reaction takes place in water
under basic condition, under an argon atmosphere. Moreover, a modified
two-step protocol was developed, for donors of relatively low
reactivities. Via the newly formed disulfide linkage, it has been shown
that the GlcNAc-attached analogue, exhibited better blood Glc-lowering
activity and higher enzymatic stability in vivo compared with the
native GLP-1, whereas the conjugation of other sugars, such as Glc and
SiaLac, did not always lead to the enhanced pharmacological activity.
This information should be useful for the future design of glycosylated
GLP-1 analogues. Worth mentioning, the native disulfides within the
peptide substrate are well preserved during these two types of cysteine
selective transformation.
Figure 5 Strategies for S-modified glycoproteins.
C-Mannosylation is a rare type of protein glycosylation and occurs in
the endoplasmic reticulum in a protein co-translational
manner.[42] An α-mannose is attached to C2 atom of
the indole ring of Trp7 via a carbon-carbon bond. C -Mannosyl
tryptophan was firstly discovered in human RNase
2,[43] and later in various glycoproteins such as
interleukin-12, properdin, thrombospondin, mucins (MUC5AC and MUC5B),
and erythropoietin receptor.[44] Owing to the
knowledge gap in the biological profile of protein C-mannosylation, it
has been a synthetic target of pursuit. Recently, metal-catalyzed C-H
glycosylation strategy was extended to the preparation of C-mannosyl
tryptophan and C-mannosyl glycopeptides. Using a new isoquinoline acid
auxiliary installed on the N terminus of tryptophan, a Pd-catalyzed
auxiliary-directed remote C–H glycosylation of tryptophan was
developed, employing mannosyl chloride as a glycosyl
donor.[45] Thus, a C2-mannosyl tryptophan was
obtained with excellent α-stereoselectivity, giving rise to the first
total synthesis of insect C-glycopeptide hormone Cam-HrTH-I (Figure 6A).
Alternatively, late-stage C−H glycosylations of structurally complex
amino acids and peptides was accomplished. A Ni-catalyzed reductive
hydroglycosylation reaction was developed, where glycosyl bromides could
be coupled with a variety of acetylenic amino acids and peptides with
high regioselectivity and stereoselectivity (Figure
6B).[46] No epimerization of the amino acids was
observed in the reaction, as determined by careful HPLC analysis for the
present C-glycosylation. In particular, a highly favorable
1,2-trans diastereoselectivity was obtained for the C2-axially
substituted mannopyranosides, rendering the present protocol a promising
method for the preparation of C-glycosyl peptides of biological and
therapeutical significance.
Figure 6 (A) Pd-catalyzed C-H glycosylation for the preparation
of C-mannosyl Trp and glycopeptides. (B) Ni-catalyzed reductive
hydroglycosylation for the preparation of C-linked glycopeptides.
2.2. Development and Application of Enzymatic Methods
Despite the prosperity in the chemical synthesis of glycoproteins, it
still encounters challenges. First of all, it requires an excess use of
glycosyl building blocks, which are usually obtained through
sophisticated preparation steps, thus leading to low overall efficiency.
In addition, the added steric hindrance of glycans may account for
decreased reactivity for the coupling of glycosyl building blocks, and
even chemical ligation as well.[47] In addition to
the chemical reactions discussed above, enzymatic and chemoenzymatic
synthesis is emerging as an attractive approach that complements and
expands the chemical methods for constructing homogeneous
glycoproteins.[9] Clearly, to meet the intrinsic
diversity of carbohydrates and glycoproteins in nature, the pursuit of
novel enzymatic approaches has been an ongoing trend. In particular,
since the currently available in vi vo techniques
generally provide heterogeneous mixtures of different glycoforms,
enzymatic in vitro methodologies have been pursued.
In addition to their hydrolytic activity, endoglycosidases can also
effectively transfer oligosaccharides onto corresponding
hydroxyl-containing substrates by
transglycosylation.[48] This dual capability makes
endoglycosidases, especially the ENGases, valuable tools for the
convergent synthesis of oligosaccharides and glycoconjugates: once the
initial GlcNAc moiety is installed, either from de novo chemical
synthesis or from enzymatic degradation of the naturally occuring
heterogeneous structure, the following enzymatic transformation readily
takes place to afford various glycan structures (Figure 7). As N-linked
glycoproteins adopt a pentasaccharide “core region” common to all
N-glycans, ENGases-catalyzed en bloc transfer of respectively
activated glycosyl donor appears an ideal choice for glycan remodeling,
leading to N-linked glycoforms with increased diversity. Approaches to
effect improvements in ENGase catalyzed glycosylation processes include
the use of N-glycan oxazolines as activated sugar
donors,[49-50] in combination with mutant variants
of ENGases (known as “glycosynthase’) with altered activity towards
product hydrolysis.[51-52]
Figure 7 ENGase catalyzed preparation of N-linked glycoproteins
In addition to endoglycosidase-catalyzed en bloctransglycosylation, glycosyltransferases mediated glycan chain
elaboration appears as another approach.[53] Such
glycosyltransferases can be used to add individual glycan units to give
various synthetic glycoforms. A prominent example is the successful
preparation of a core m1 structured O-mannosyl glycopeptide, reported by
Flitsch et al., employing human protein O -linked mannose
β1,2-N -acetylglucosaminyltransferase 1 (POMGnT1) and bovine
β1,4-galactosyltransferase (β1,4-GalT)
respectively.[54] Similar strategy was reported by
Nishimura et al., on the preparation of a synthetic library of core m1
O-mannosyl glycopeptides, which revealed specific binding pattern and
conformational change at O -mannosylated
position.[55] Recently, Li et al. reported a
divergent synthesis of core m1, core m2 and core m3 O-mannosyl
glycopeptides via a chemoenzymatic approach.[56]Taking advantage of an orthogonally protected mannosyl module, three
core structured O-mannosyl amino acid building blocks can be accessible
within minimal steps. Subsequent enzymatic galactosylation and
sialylation, mediated by bacterial β1,4-galactosyltransferase fromNeisseria meningitidis (NmLgtB) and a α2,3-sialyltransferase 1
M144D mutant from Pasteurella multocida (PmST1 M144D), further
expanded the diversity of synthetic O-mannosyl glycopeptides.
Moreover, development of enzymatic tools towards broader application
scenario has been pursued. For example, to facilitate the proteoglycan
synthesis, the utility of human xylosyltransferase I (XT-I), the enzyme
responsible for installation of initial xyloside on protein backbone,
was investigated.[57] Furthermore, the utility of
human β-1,4-galactosyltransferase 7 (β4GalT7) was extended to the
synthesis of Gal-Xyl bearing glycopeptides.[58]Together, XT-I can be coupled with β4GalT7 for one pot synthesis of
glycopeptides bearing galactose-xylose disaccharide, thus opening the
door for expeditious chemoenzymatic synthesis of proteoglycan type
glycopeptides and glycoproteins. Recently, yeast spores were developed
as novel natural carriers of
glycosyltransferases.[59] Encapsulation of
β1,4-galactosyltransferase from Homo sapiens (HsGalT),
α2,3-sialyltransferase 1 from Pasteurella multocida (Pm2,3ST1)
and α2,6-sialyltransferase from Photobacterium damselae(Pd2,6ST), on the surface of yeast spores, enabled facile assembly of
diverse naturally occurring sialyl-galactosylated glycans via tandem
reaction cascade. Furthermore, O-GlcNAcase (OGA) was adopted to aid the
chemical synthesis and folding of disulfide-rich proteins, based on the
establishment of a removable glycosylation modification (RGM)
strategy.[60] In line with the knowledge that
protein glycosylation contributes to the maintenance of its correct
geometry in vivo , the attachment of simple O-GlcNAc moiety at the
Ser/Thr sites was found to effectively facilitate protein folding
process by stabilization of their folding intermediates. Upon successful
folding, the RGM groups can be cleanly removed by O-GlcNAcase (OGA) to
afford the correctly folded proteins with multiple disulfide bonds.
Using this strategy, the first total synthesis of native interleukin-5
(IL-5) was achieved, which was otherwise unsuccessful due to the
formation of misfolded IL-5 isoform (Figure 8).
Figure 8 Preparation of disulfide-rich proteins via removable
glycosylation modification (RGM) strategy
2.3. Efforts towards Effort-less Purification
Material loss during the purification of peptide fragments, especially
the multiple rounds of HPLC purification, accounts for the accumulative
low yield of glycoprotein synthesis. To circumvent the possible
purification of ligation intermediate, a one-pot four-segment ligation
strategy was employed for the preparation of O-glycosylated histone H2A
carrying an O -GlcNAc at Ser40 (Figure
9).[61] The synthesis involves a peptide thioester
and two orthogonally activatable thioester equivalents, cysteinyl prolyl
ester (CPE) and N-alkylcysteine (NAC). These equivalents were
demonstrated to have different optimal pH conditions for
thioesterification (CPE: pH > 7.8; NAC: pH 4–6), thus
allowing for the sequential ligation to take place in one-pot manner.
For the attachment of O-GlcNAc, methylbenzyl (MBn) was utilized due to
its lability in TFA cocktail during the global deprotection stage. Even
though slight hydrolysis of glycosidic linkage was noted during TFA
deprotection, the glycosylated segment was obtained in reasonably high
yield (21%), which led to the successful assembly of the whole
polypeptide sequence via one-pot ligation, with an overall yield of
54%; the following metal-free desulfurization afforded the
O-glycosylated histone H2A.
Figure 9 Preparation of O-GlcNAc modified histone H2A via a
one-pot four-segment ligation strategy
To save on the labors and the severe handling loss due to multiple
rounds of HPLC purifications, an N-terminal bifunctional
protection/purification handle (PPH) was developed (Figure
10A).[62] The handle was designed to serve dual
roles: a photo-labile linker basically works as a temporary protection
of N-terminal Cys, and a His6 tag allows for quick and easy purification
of the peptide fragments via immobilized metal affinity chromatography
(for example, Ni-NTA beads). A pH switch was installed, which enabled
facile loading and elution of the peptide by adjusting the pH under mild
conditions (pH 7.0 to 3.0). This protection/purification handle strategy
is practically successful for the construction of glycoproteins with
tandem repeats, via a couple of sequential cycles of chemical ligation
and affinity purification. A quick illustrative example is the
preparation of an 80-mer mucin 1 (MUC1) peptide fragment from 20-amino
acid tandem repeats. Furthermore, an antifreeze glycoprotein (AFGP)
bearing α-linked O-GalNAc at its Thr residue within each ATA tripeptide
repeating unit,[63] was assembled up to 240-mer,
which represents the largest chemically synthesized protein decorated
with glycans reported to date. Driven by similarly practical
considerations, a tag-assisted strategy was introduced to facilitate the
step-by-step purification during the assembly of glycopeptides (Figure
10B).[64] A hydrophobic tag was initially
introduced at the C-terminus, followed by peptide chain assembly. Thus,
after each round of (glycosyl) amino acid coupling and Fmoc removal, the
tag was there to enable quick purification via centrifugation; upon the
completion of peptide chain assembly, the tag could be selectively
removed using a cocktail of TFA/TFE/DCM. Thus, the resulting
glycopeptides can be subjected to fragment coupling to elongate the
peptide backbone, or undergo enzymatic glycan modification to obtain
more sophisticated glycan structures. Following this protocol, a
collection of O-glycopeptides containing various glycan chains were
successfully obtained, including the SARS-CoV-2 glycopeptides, the
20-mer MUC1 glycopeptides, and a 31-mer glycosylated glucagon-like
peptide-1 (GLP-1).
Figure 10 To ease the purification, (A) protection/purification
handle (PPH) and (B) hydrophobic tag were introduced to glycopeptide
synthesis.
3. Comprehensive Study of Protein Glycosylation Using Synthetic
Glycoforms
Among the variety of post-translational modifications on protein,
glycosylation is one of the most complicated modifications. Since the
biosynthesis of glycans is regulated by the substrate specificity of
glycosyltransferases and glycosidases, resulting in considerably diverse
glycan structures (glycoforms), to understand the biological processes
arising from each glycoform is never a simple routine work. Despite that
the biological activity of a glycoprotein is typically modified by the
glycan part, homogenous glycoproteins (glycoforms) are rarely accessible
from natural sources and need to be prepared by synthesis.
Apart from chemical synthesis, semisynthesis approach exhibits
advantages to obtain synthetic glycoforms with expanded structural
diversity.[10, 13] Protein expression, generally
carried out in E. coli , is able to offers unglycosylated
fragments in large scale. Additionally, expression of certain peptide
fragment may overcome the solubility problem encountered in chemical
synthesis. Thus, with the encouraging development in synthetic chemistry
and biology, allowing scientists to precisely control the subtle
differences in glycan structure and amino acid sequence, it is now more
possible than ever before to quantitatively interrogate the
structure−property relationships of protein glycosylation, and in
particular, to uncover the molecular basis of such relationships. The
improved understanding promises future medical translation.
3.1 Synthetic Glycoforms with Single Glycosylation Site
The improved understanding promises future medical tr Interleukin 6
(IL-6) is a cytokine that exerts both immunostimulating and regenerating
effects through binding to the IL-6 receptor with different
localization.[65] Although many of the biological
effects of human IL-6 have been studied in detail, little is known about
the influence of its glycans. A systematic study was carried out with a
representative set of IL-6 glycoforms, including complex-type and
oligomannosidic N-glycans.[66] The chemoenzymatic
semisynthesis of the library of IL-6 glycoforms involved three
segments,[67] where the short glycopeptide segment
was synthesized via by pseudoproline-assisted convergent aspartylation;
the N-terminal and C-terminal segments were obtained recombinantly from
the corresponding fusion protein (Figure 11). Moreover, the sialylated
glycopeptides were obtained by enzymatic sialylation using the bacterial
sialyltransferases PdST6 or PmST3 and purified by RP-HPLC. In all cases
the sialylations required optimization of the reaction conditions. Of
note, as the recombinant IL-6 peptide (49–183) is not compatible due to
low recovery from preparative RP-HPLC, it was prepared by means of
corresponding SUMO fusion protein followed by proteolysis. The obtained
synthetic IL-6 glycoforms were then compared with the nonglycosylated
reference IL-6 E. coli.
It was found that the native helical fold of the set of IL-6 glycoforms
was independent of the glycan, where only the overall intensity of the
circular dichroism spectra varied to a small extent. In addition, the
N-glycan of IL-6 appears no significant effect on the binding to the
cellular IL-6 receptor, judged by a proliferation assay using an
IL-6-dependent cell line. Strikingly, all the IL-6 glycoforms showed
different in vivo plasma clearance rates in rats. The unexpected
differences in the biological properties of the various IL-6 glycoforms
could be further revealed by providing sufficient amounts of a
systematically varied library of synthetic IL-6 glycoforms.
Figure 11 Chemoenzymatic semisynthesis of IL-6 glycoforms with
N-glycans at Asn44
Interleukin-2 (IL-2) is a cytokine that exerts a wide spectrum of
effects on the immune system, and it plays crucial roles in regulating
both immune activation and homeostasis.[68]
Moreover, it is clinically applied for immunotherapy against various
diseases. Proleukin ®, the recombinant human IL-2, is indicated for the
treatment of adults with metastatic melanoma and metastatic renal cell
carcinoma.[69] Hence, IL-2 remains one of the most
extensively studied cytokines to date. Native human IL-2 is a 133 amino
acid cytokine that is glycosylated at Thr 3. To expand the understanding
of the structure-activity relationship, particularly with regard to
glycosylation of IL-2, a concise synthetic route was
devised.[70] Briefly, a synthetic glycopeptide
fragment (1‒5) salicylaldehyde ester was coupled with a recombinant
protein fragment (6‒133) via Ser/Thr ligation (Figure 12). Using this
semisynthetic protocol, the poor solubility issue, recognized as a
significant hurdle in previous reports, was carefully circumvented.
Thus, apart from the non-glycosylated IL-2, three different IL-2
glycoforms were prepared on multi-milligram scale, bearing O-linked
monosaccharide GalNAcα→, disaccharide Galβ1→3GalNAcα→, or trisaccharide
Neu5Acα2→3Galβ1→3GalNAcα→ at Thr3. Biological evaluation of the
homogenous glycoprotein library reveals that the activity of IL-2 in
activating individual T cell subset is glycan dependent, thus
highlighting the possibility of further improvement in clinical
application.
Figure 12 Semisynthesis of IL-2 glycoforms with O-glycans at
Thr3
Interestingly, the same set of O-glycans were found to have an effect on
the progression of Alzheimer’s disease. The study on O-glycosylation of
amyloid-β (Aβ) peptides was carried out using three synthetic
glycoforms, bearing O-linked monosaccharide GalNAcα→, disaccharide
Galβ1→3GalNAcα→, or trisaccharide Neu5Acα2→3Galβ1→3GalNAcα→, at
Tyr10.[71] Each glycosyl amino acid building block
was chemically synthesized with defined structure. A modified solid
phase peptide synthesis protocol was devised, with a removable
poly-lysine tag to aid the purification and improve the overall yield.
Amongst these synthetic glycoforms, a concentration-dependent inhibitory
effect was noted, induced by Tyr10 O-glycosylation against the fibril
formation of Aβ42. Besides, fewer β-sheets were observed with
glycosylated Aβ42 than unmodified Aβ42; especially for trisaccharide
modified Aβ42, a random coil secondary structure was clearly indicated.
Moreover, increased susceptibility of disaccharide modified Aβ42 fibril
toward degradation by extracellular degradation enzymes indicates a
possible protective role of O-glycosylation in Aβ pathological
aggregation.
Interleukin-17A (IL-17A) is a proinflammatory cytokine that plays
essential roles in regulating both innate immunity and host defense in
human body.[72] To address the functions of
N-glycosylation at Asn45 of IL-17A, synthetic IL-17A glycoforms were
prepared, each bearing O-GlcNAc, GlcNAcβ1→4GlcNAc or complex type
sialylundecasaccharide, respectively.[73] A
four-fragment chemical ligation strategy was adopted, where O-GlcNAc or
GlcNAcβ1→4GlcNAc building blocks were introduced at respective site,
followed by Endo-M-catalyzed
transglycosylation[74] to elaborate the glycan
chain to give the corresponding complex type sialylundecasaccharide
(Figure 13). Of note, modified transglycosylation protocol was necessary
to afford an improved efficiency. Compared with the glycosylated IL-17A,
the nonglycosylated isoform exhibited poor stability and a much higher
tendency to aggregate during the preparation, indicating protective
effect induced by glycosylation against aggregation; in addition,
relatively high thermal stabilities were observed for the glycosylated
IL-17A. Moreover, N-glycosylation was found to interfere with the
interactions between IL-17A and its receptor IL-17RA, where the binding
affinity decreased as the N-glycan gets larger. Although speculations
remain to be uncovered due to the absence of detailed structural
information regarding N-glycans in natural IL-17A, the current research
set an example to follow for interrogating the functions of
glycosylation.
Figure 13 Synthesis of IL-17A glycoforms with N-glycans at
Asn45
3.2 Synthetic Glycoforms with Multiple Glycosylation Sites
Adiponectin is a circulating glycoprotein mainly produced from
adipocytes. It is a key regulator of glucose and lipid metabolism, thus
increasing the systemic insulin sensitivity and energy
homeostasis.[75] The mammalian adiponectin
collagenous domain contains four 5-(2S,5R)-hydroxyl lysine residues,
which are glycosylated with a glucosyl-galactose disaccharide. To fill
in the knowledge gap regarding its glycosylation, and towards the
therapeutic application, the effect of glycosylation on adiponectin
collagenous domain was explored.[76] An exhaustive
collection of homogeneously glycosylated adiponectin collagenous domains
with all of the possible 15 glycoforms have been chemically and
individually synthesized (Table 1), using stereoselective glycan
synthesis and chemical peptide ligation.
Table 1 Synthetic glycoforms of adiponection collagenous domain
Briefly, a facile, large-scale synthesis of the (2S,5R)-hydroxylysine
was developed; starting there, stereoselective glycosylation afforded
Glcα1→2Galβ1→(2S,5R)-hydroxylysine building block for the glycopeptide
fragment preparation. Ser/Thr ligation joined the two peptide fragments
(39‒60) and (61‒107) together. Subsequently, the evaluation of
biological activities and pharmacological properties indicates that the
glycan plays an important role of the collagenous domain of adiponectin,
in the inhibition of cancer cell growth as well as the regulation of
systemic energy metabolism. The current work showcases the power of
chemical synthesis in systematically addressing the role of
glycosylation on activity and specificity, and opens the door to explore
the opportunity of using the synthetic glycopeptide as a potential
supplementary towards drug development.
To tackle the challenge in biomass transformation, Tan et al.
systematically probed the effects of O-mannosylation of a family 1
carbohydrate binding module (CBM) of Trichoderma reeseicellobiohydrolase I (Tr Cel7A), using a panel of 23 individual
synthetic glycoforms (Table 2).[77] To elucidate
the potential site-specific, size-specific and synergistic effects of
CBM glycans, regarding its binding affinity for a variety of
lignocellulose-derived cellulose and lignin substrates, the synthetic
glycoforms were divided into six sets, including the unglycosylated CBM
1, monoglycosylated CBMs (2–4, 5–7, 8–10), multi-glycosylated CBMs
(11–20), and CBM glycoforms with varied amino acid sequence or glycan
structure. It appears that glycoforms that contain the same mannose
structures distributed across all available sites (13, 19, and 20), have
the most beneficial glycosylation pattern for improving binding
selectivity to cellulose over lignin. Although a single dimannosyl
residue at the Ser3 site could also be helpful for increasing the
preference for cellulose binding, the binding specificity of CBM 6 is
not as high as those of the multi-mannosylated variants. These data led
to further investigation using molecular dynamic
simulation.[78] Thus, via synthesizing,
characterizing, and systematically analyzing a collection of synthetic
CBM variants, the beneficial effects of glycosylation on CBM binding
preferences were clearly indicated across multiple biomass-derived
cellulose and lignin substrates.
Table 2 Synthetic glycoforms of carbohydrate binding module ofTr Cel7A
The outbreak of global pandemic COVID-19 spurred the investigation of
the glycosylation on spike protein of coronavirus SARS-CoV-2,
particularly regarding its receptor binding domain
(RBD).[79-82] Driven by this idea, a collection of
RBDs containing homogeneous N- and O-glycans were prepared via a
semisynthesis approach.[83] Namely, the
glycosylated fragments (319‒335) and (336‒360) were chemically
synthesized and the rest part was prepared recombinantly (Figure 15).
The N-linked glycans at Asn331 and Asn343 was introduced as GlcNAc,
Man3GlcNAc2 pentasaccharide or the
complex-type nonasaccharide; O-glycan at Thr323 was an O-linked GalNAc.
The binding of these synthetic glycoforms of RBD to its host receptor,
angiotensin converting enzyme 2 (ACE2), as well as to anti-SARS-CoV-2
RBD monoclonal antibodies (mAbs) was studied. The established platform
represents a versatile strategy for accessing synthetic glycosylated
RBDs, which facilitates the understanding the specific role of
glycosylation on these viral RBDs and paves the way for the future
development of effective therapeutics and vaccines.
Figure 14 Synthetic RBD glycoforms with various N- and
O-glycans
ADP-ribosylation of nuclear proteins is a critical feature of various
DNA damage repair pathways.[84-85] Muir et al.
reported the development of an efficient and modular semisynthetic route
to full-length ADP-ribosylated histones H2B and H3, installed at Ser6
and Ser10, respectively (Figure 15).[86] The
synthetic route involved chemically synthesized ADP ribosylated histone
tail peptide thioester (H2B(1-16)S6ADPr and
H3(1-14)S10ADPr), and recombinant truncated histone
(H2B(17-125)A17C and H3(15-135)A15C)
expressed in E. coli . Native chemical ligation of the peptide
thioesters with the corresponding recombinant fragments, followed by
free radical-mediated desulfurization smoothly afforded the native
full-length ADP-ribosylated histones H2BS6ADPr and H3S10ADPr.
Furthermore, these histones were used to generate various
ADP-ribosylated chromatin substrates. To this end, three different
ADP-ribosylated histone octamer complexes were refolded, which contained
either ADP-ribosylated H2B, ADP-ribosylated H3, or both, in addition to
a control octamer complex in which all histones are unmodified.
Figure 15 Semisynthesis of ADP ribosylated histones
Analysis by native polyacrylamide gel electrophoresis (PAGE) indicated
that the bulky, anionic ADP-ribose moiety did not impede efficient
mononucleosome formation; similar thermal stability was observed among
the ADP-ribosylated and unmodified mononucleosomes, further indicating
that proper nucleosome assembly was maintained. However,
ADP-ribosylation exhibited inhibitory effect on chromatin fiber
compaction, which implied its role in chromatin relaxation in the
context of DNA repair. Furthermore, the crosstalk between
ADP-ribosylation and lysine methylation was studied, where
ADP-ribosylation of H3S10 inhibits G9a-mediated H3K9 methylation,
indicating the modulation of the H3-tail dynamics by ADP-ribosylation.
Collectively, native full-length ADP-ribosylated H2B and H3 were
prepared through protein semisynthesis, and were utilized to generate
chemically defined ADP-ribosylated mononucleosomes and nucleosome
arrays. This work sheds light on a role of histone mono-ADP-ribosylation
in DNA repair and also highlights the value of designer ADP-ribosylated
chromatin substrates in further investigation.
In the era of antibiotic resistance, the discovery of new antibiotics
with new skeletons and mode of action is of high pursuit. As a group of
nonribosomal cyclic glycopeptide natural products, mannopeptimycins
demonstrate promising activities against clinically important resistant
Gram-positive pathogens like methicillin-resistant Staphylococcus
aureus (MRSA) and vancomycin-resistant Enterococci(VRE).[87] However, the high structural complexity
of mannopeptimycins poses a formidable obstacle to their total
synthesis.[88] Mannopeptimycins comprise six amino
acids with alternating D- and L-configurations, four of which are
nonproteinogenic: D- and L-β-hydroxyenduracididines
(βhEnd),[89] L-β-methyl phenylalanine (βMePhe),
and D-tyrosine (D-Tyr), where D-βhEnd and D-Tyr were glycosy: Novel
Antibacterial Glycopeptides lated (Figure 16). Chen et al. finished the
first total synthesis of mannopeptimycins α and β, a convergent (3+3)
assembly of the cyclic peptide backbone.[90] The
N-linked mannose moiety was installed on cyclic guanidine substrates
(D-βhEnd) via the gold catalyzed glycosylation of the mannosyl
ortho-alkynylbenzoate donor, and Boc-βMePhe-OH was prepared via
Pd-catalyzed C−H arylation chemistry. Li et al. reported the total
synthesis based on a streamlined peptide assembly strategy, and
proceeded macrocyclization via β-hydroxyenduracididine
ligation.[91] These impressive achievements shed
light on the synthesis of nonribosomal cyclic glycopepitdes with high
structural complexity, which will enable exploration of previously
inaccessible mannopeptimycin derivatives, and promote the development of
new analogues with enhanced antibacterial activity.
Figure 16 by Chemical synthesis of mannopeptimycins: novel
antibacterial glycopeptides
4. Conclusions and Perspectives
Owing to the importance of protein glycosylation, rigorous evaluation of
the relationship between the precise structure and biological function
of glycoproteins has to be performed, which heavily relies on the access
to structure-defined glycoproteins with single glycoform. Since these
single glycoforms are hard to obtain from natural sources, innovative
synthetic methods and technologies are highly valued in this regard.
The size, physical properties, high structural diversity and complexity
of glycoproteins poses significant challenge in the preparation of
glycoproteins. Recent efforts in this area summarized herein include 1)
efficient chemical synthesis, chemoenzymatic synthesis and semisynthesis
methods, 2) simplified purification protocols, and 3) glyco-conjugation
and glyco-engineering strategies. On this basis, a great number of
native glycoproteins are now chemically accessible, as illustrated with
the massive successful cases discussed above. The obtained synthetic
glycoforms furnish new insights into the role of glycosylation.
Furthermore, the repertoires of synthetic tools facilitate the ongoing
research with a broader health-promoting application scenario. For
example, significant progress is witnessed on the
glyco-remodeling/glycoengineering of immunotherapeutics, including
therapeutic antibodies,[92-94] cell-based
immunotherapies (e.g. chimeric antigen receptor (CAR)-T cell
therapy[95]), and synthetic antitumor vaccines
(e.g. MUC1 glycopeptide based vaccines[96]), which
exhibits generally improved immunological profile, and holds promise for
clinical translation. Besides, Glycosylation of peptides and proteins
has emerged as a promising strategy to improve the pharmacokinetic
profile of peptide- and protein-based therapeutics. Hence, it is
expected that with the facile access to structurally defined synthetic
glycoforms, future endeavors will help unravel the associations between
glycosylation and human diseases, and will open up novel therapeutic
avenues.
Apparently, preparation of glycoproteins to mimic nature complexity is a
fascinating target, and also an attractive, arduous and problem-driven
journey, calling for continuous efforts and wisdom to come. While the
challenge in protein synthesis can be overcome via introduction of
glycosylation, it can be anticipated that the ongoing development of
protein chemical synthesis and semisynthesis tools, exemplified with
various chemical ligation, conjugation and purification technologies,[97-104] will contribute to the progress of
glycoprotein synthesis. Meanwhile, a primary concern under development
is the construction of diverse forms of glycosidic linkage, between each
glycan unit and also between the glycan chain and protein backbone,
where stereoselectivity and regioselectivity issues are frequently
interrogated. It is foreseeable that the prosperity in carbohydrate
chemistry will lead to new progress in glycoprotein synthesis, to an
unprecedent future.
Acknowledgement
This work is financially supported by
Shenzhen Fundamental Research Program (RCBS20200714114957255), National
Natural Science Foundation of China (Grant No. 22177061), the National
Key Research and Development Program of China (2018YFA0902000), the Open
Projects Fund of Shandong Key Laboratory of Carbohydrate Chemistry and
Glycobiology (2021CCG01).
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