Chemical synthesis of the β-subunit of human luteinizing (hLH) and chorionic gonadotropin (hCG) glycoprotein hormones

Article abstract of DOI:10.1021/ja503545r

Human luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) are human glycoprotein hormones each consisting of two subunits, an identical α-subunit and a unique β-subunit, that form noncovalent heterodimers. Structurally, β-hCG shares a high degree of sequence similarity with β-hLH, including a common N-glycosylation site at the N-terminus but differs mainly in the presence of an extended C-terminal portion incorporating four closely spaced O-linked glycans. These glycoproteins play important roles in reproduction and are used clinically in the treatment of infertility. In addition, the role of hCG as a tumor marker in a variety of cancers has also attracted significant interest for the development of cancer vaccines. In clinical applications, these hormones are administered as mixtures of glycoforms due to limitations of biological methods in producing homogeneous samples of these glycoproteins. Using the powerful tools of chemical synthesis, the work presented herein focuses on the highly convergent syntheses of homogeneous β-hLH and β-hCG bearing model glycans at all native glycosylation sites. Key steps in these syntheses include a successful double Lansbury glycosylation en route to the N-terminal fragment of β-hCG and the sequential installation of four O-linked glycosyl-amino acid cassettes into closely spaced O-glycosylation sites in a single, high-yielding solid-supported synthesis to access the C-terminal portion of the molecule. The final assembly of the individual glycopeptide fragments involved a stepwise native chemical ligation strategy to provide the longest and most complex human glycoprotein hormone (β-hCG) as well as its closely related homologue (β-hLH) as discrete glycoforms.

Full text of DOI:10.1021/ja503545r

Article
pubs.acs.org/JACS
Chemical Synthesis of the β‑Subunit of Human Luteinizing (hLH) and
Chorionic Gonadotropin (hCG) Glycoprotein Hormones
Alberto Fernandez-Tejada,†,∥ Paul A. Vadola,†,∥,§ and Samuel J. Danishefsky*,†,‡
́
†Laboratory for Bioorganic Chemistry, Molecular Pharmacology and Chemistry Program, Sloan Kettering Institute for Cancer
Research, 1275 York Avenue, New York, New York 10065, United States
‡Department of Chemistry, Columbia University, 3000 Broadway, New York, New York 10027, United States
S
* Supporting Information
ABSTRACT: Human luteinizing hormone (hLH) and human
chorionic gonadotropin (hCG) are human glycoprotein hor-
mones each consisting of two subunits, an identical α-subunit and
a unique β-subunit, that form noncovalent heterodimers.
Structurally, β-hCG shares a high degree of sequence similarity
with β-hLH, including a common N-glycosylation site at the N-
terminus but differs mainly in the presence of an extended C-
terminal portion incorporating four closely spaced O-linked
glycans. These glycoproteins play important roles in reproduction
and are used clinically in the treatment of infertility. In addition,
the role of hCG as a tumor marker in a variety of cancers has also
attracted significant interest for the development of cancer vaccines. In clinical applications, these hormones are administered as
mixtures of glycoforms due to limitations of biological methods in producing homogeneous samples of these glycoproteins.
Using the powerful tools of chemical synthesis, the work presented herein focuses on the highly convergent syntheses of
homogeneous β-hLH and β-hCG bearing model glycans at all native glycosylation sites. Key steps in these syntheses include a
successful double Lansbury glycosylation en route to the N-terminal fragment of β-hCG and the sequential installation of four O-
linked glycosyl-amino acid cassettes into closely spaced O-glycosylation sites in a single, high-yielding solid-supported synthesis
to access the C-terminal portion of the molecule. The final assembly of the individual glycopeptide fragments involved a stepwise
native chemical ligation strategy to provide the longest and most complex human glycoprotein hormone (β-hCG) as well as its
closely related homologue (β-hLH) as discrete glycoforms.
The gonadotropic hormones hLH and hCG are required for
normal development and secretory activity of the gonads and
stimulate the endocrine and gametogenic functions.7 hLH is
produced by gonadotroph cells in the pituitary gland and plays a
key role in the reproductive process, including regulating the
menstrual cycle, triggering ovulation and development of the
corpus luteum, and stimulating the production of testosterone.8
hCG is produced primarily in the human placenta and has similar
physiological functions to hLH (i.e., upregulating progesterone
and testosterone production and inducing ovulation and
spermatogenesis).9 It also plays a role in the protection of the
fertilized embryo and development of the fetus during
pregnancy.10 In clinical settings, marketed forms of these
hormones (often co-administered) are used in human
reproductive medicine for the treatment of infertility and in in
vitro fertilization.11
INTRODUCTION
■
Protein glycosylation is one of the most frequent and relevant
post-translational modifications.1 It is present in more than half
of all proteins in nature2 and plays a key role in the modulation of
protein properties and function.3 One of the fastest growing
areas in the pharmaceutical industry is the field of therapeutic
glycoproteins.4 They are currently being marketed as natural
mixtures of glycoforms (i.e., conserved peptide backbone but
highly variable as to the site and pattern of glycosylation). Our
laboratory has a long-standing interest in the synthesis of
homogeneous glycoproteins, as exhibited in our recently
reported chemical synthesis of an erythropoietin glycoform
with the wild-type polypeptide backbone and well-defined N-
and O-glycosides.5 We have also been interested in the human
glycoprotein hormones (hGPH), which are composed of two
noncovalently associated subunits, a common α-subunit (α-
hGPH) and a hormone-specific β-subunit, each containing
diverse oligosaccharides at defined glycosylation sites. Recently,
we described the synthesis of the α- and β-subunits of human
follicle-stimulating hormone (hFSH or follitropin).6 Herein, we
have focused our synthetic efforts on the gonadotropins, human
luteinizing hormone (hLH or lutropin) and human chorionic
gonadotropin (hCG).
Interestingly, the β-subunit of hCG (β-hCG) is also
overexpressed as particular glycoforms in certain types of tumors
(epithelial, pancreatic, colorectal) and has been found to inhibit
apoptosis in cancer tissues and promote metastasis.12 Clinical
Received: April 9, 2014
Published: May 7, 2014
© 2014 American Chemical Society
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Figure 1. (A) Fully elaborated β-subunit of hLH with chitobiose at the N-glycosylation site (Asn30) and proposed disconnection positions. (B)
Homogeneous full-length sequence of β-hCG bearing two chitobiose (Asn13 and Asn30) and four GalNAc moieties (Ser121, Ser127, Ser132, and
Ser138) at the N- and O-glycosylation sites, respectively. Key ligation sites and envisioned peptide fragments for final assembly are also indicated. 3D
structure of α/β-hCG heterodimer (inset). (C) Structure of N-linked carbohydrates at defined glycosylation sites on hLH and hCG polypeptide
backbone; elaborated dodecasaccharide (left) and chitobiose as model glycan (right). (D) Structure of representative O-linked glycans present in hCG
peptide sequence: glycophorin tetrasaccharide (left) and simpler GalNAc.
trials using anti-β-hCG cancer vaccines have been carried out in
patients with epithelial, pancreatic, and colorectal cancer.13 Thus,
β-hCG constitutes a promising target for cancer immunotherapy
and antitumor vaccine development.14 Furthermore, antibodies
against β-hCG and one type of hCG glycoform (hyper-
glycosylated hCG) have been shown to inhibit tumor cell
growth and metastasis.15 There have also been studies, though
conflicting, on the antiproliferative properties of hCG against the
Kaposi’s Sarcoma (KS) in HIV patients as well as its biological
activity against HIV-1.16 These conflicting studies may be due to
the heterogeneity of different clinical-grade preparations of hCG,
which contain mixtures of complex glycoforms.17 Since certain
glycoforms are more active than others, access to specific,
discrete glycoforms by chemical synthesis can confer significant
therapeutic advantages. Moreover, the synthesis of chemically
pure glycoproteins bearing defined glycans can help determine
the specific roles of glycosylation in biosynthesis and function.
Structurally, the β-hLH subunit consists of 121 residues with
one N-linked glycan at Asn30 (Figure 1A).7 The more complex
β-subunit of hCG shares 85% sequence homology with β-
hLH.7,18 However, it has a unique serine-rich, 30 amino acid
carboxy terminal extension with 4 additional sites of O-
glycosylation at these residues. Thus, β-hCG is made up of a
145-amino acid protein backbone bearing two N-linked glycans
at Asn13 and Asn30 and four O-linked glycans at Ser121, Ser127,
Ser132, and Ser138 (Figure 1B).19 The carbohydrate content
represents about one-third of the molecular weight of hCG, and
glycosylation is of structural and functional importance, affecting
both its half-life in circulation and signal transduction induced by
this hormone.20 Numerous investigations, mostly on hCG,
suggest that the sugar chains are not required for hormone-
receptor binding but play a critical role in receptor activation.21
The major structural differences in the Asn-linked carbohydrates
of hLH and hCG are in the terminal sugar moieties of the
sialylated, biantennary, complex-type N-glycans (Figure 1C).
The observed diversity of terminal glycosylation sequences
suggests that the peripheral sugar residues are not essential for
the biological action of these hormones.22 This observation,
together with the fact that structures lacking N-glycosylation
exhibit almost complete loss of hormone function,23 indicates
that the underlying core structures may play a more relevant role
in the biological activity of both hormones.24 However, the entire
β-hCG carboxy-terminus, which contains four O-glycosylation
sites with mucin-type O-glycans (Figure 1D), has been found to
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be a common epitope for hCG-based monoclonal antibodies,
which can inhibit tumor cell growth and metastasis.25 Detection
of hCG isoforms by these antibodies has been used clinically to
screen for Down Syndrome, to identify pregnancy disorders, and
to monitor trophoblastic disease.26
Scheme 1. Synthesis of N-linked Glycopeptide Fragment 1: β-
hLH(chitobiose)[S1−Y37] (A) and Peptide Fragment 2: β-
a
hLH[Z38−G71] (B)
In this work, we report the first total synthesis of the two
longest β-subunits of the human glycoprotein hormones (β-hLH
and β-hCG), which were accessed as pure glycoproteins bearing
defined carbohydrate structures. The successful approach is
exemplified herein with the use of the disaccharide chitobiose at
the two N-glycosylation sites and the monosaccharide N-acetyl-
galactosamine (GalNAc) at the four O-glycosylation sites as
model glycans for our glycoprotein assembly. The system has
been synthesized with protected cysteines anticipating folding
and its association with the previously synthesized, common α-
subunit of the human glycoprotein hormones.6a Based on our
extensive experience with similar glycoprotein targets, final
cysteine deprotection and subsequent folding of the correspond-
ing β-subunits are expected to be successfully accomplished in
forthcoming studies to provide fully synthetic materials for future
biological evaluation.
RESULTS AND DISCUSSION
■
The complexity of the hCG β-subunit stems not only from its
size (the longest of all human glycoprotein hormones) and the
presence of the two N-glycosylation sites but also from the
position of its unique, closely spaced four O-glycosylation sites at
the carboxy-terminus of the molecule. Fortunately, the relatively
high content of cysteine residues in the peptide backbone at fairly
regular intervals enables the assembly of both full-length
glycoproteins from appropriately selected peptide fragments
using the native chemical ligation (NCL) reaction.27 Our plan for
the construction of β-hLH and β-hCG was based on maximum
convergence, and thus, we identified the key ligation sites
depicted in Figure 1. β-hLH was envisioned to arise from three
fragments of between 30 and 50 amino acids long, whereas our
synthetic strategy for hCG relied on the assembly of five peptide
segments, each bearing the corresponding N-/O-linked glycans
in a highly modular fashion.
Given the high sequence homology between both targets and
the relatively simpler structure of β-hLH, in comparison with β-
hCG, we chose to start with the synthesis of the former, en route
to β-hCG. In addition to providing access to hLH as one of the
important glycoproteins within the family, this synthetic
sequence could also serve as a proof of concept for the feasibility
of the approach in terms of the following strategic steps:
glycosylation via Lansbury aspartylation,28 global deprotection of
the glycopeptide segments, and the final union of these
fragments by NCL.
Chemical Synthesis of the β-Subunit of hLH as a
Validation of the Synthetic Strategy. For the preparation of
β-hLH, we initially envisioned a retrosynthetic disconnection of
the molecule in three fragments: β-hLH(chitobiose)[S1−Y37],
β-hLH[C38−G71], and β-hLH[C72−L121] (Figure 1A). The
corresponding peptide fragments were obtained by Fmoc-based
solid-phase peptide synthesis (SPPS) with acid-labile protecting
groups on the amino acid side chains, with the exception of the
non-NCL-participating cysteine residues, in which case the
acetamidomethyl (Acm) group was chosen. For the glycopeptide
segment (β-hLH(chitobiose)[S1−Y37]), a pseudoproline
dipeptide (Ile27-Thr28) was introduced in the sequence to
improve the synthetic efficiency of the subsequent glycosylation
at Asn30 under Lansbury conditions.29 In addition, the aspartic
a
Amino acid residues bearing acid-labile protecting groups shown in
deep red, pseudoproline dipeptides shown in orange (underlined), and
Acm-protected Cys residues shown in purple. Chitobiose glycan
shown in blue, and thioester functionalities shown in green. Cocktail
B: 88% TFA, 5% H2O, 5% phenol (PhOH), 2% triisopropylsilane
(TIPSH).
acid that will bear the chitobiose moiety was protected with a
quasi-orthogonal O-2-phenylisopropyl ester (O-2-PhiPr or OPp)
group that can be removed selectively in the presence of tBu-
based protecting groups by treatment with 1−2% trifluoroacetic
acid (TFA) in dichloromethane.30 Thus, after cleavage from the
resin, the C-terminal carboxylic acid of the protected peptide was
first coupled to tyrosine phenylthioester (HCl·H-Tyr-SPh)
under Sakakibara conditions,31 which are known to be
epimerization free, and subsequent selective deprotection of
Asp30 provided the free carboxylic acid side chain (pF1:β-
hLH[S1−Y37]). Next, HATU-mediated Lansbury aspartylation
with chitobiose glycosyl amine followed by treatment with
Cocktail B (88% TFA, 5% H2O, 5% phenol, 2% TIPSH) for
removal of all acid-labile protecting groups on the amino acid
side chains afforded the corresponding glycopeptide fragment β-
hLH(chitobiose)[S1−Y37] in 22% yield (over four steps) after
only one HPLC purification (Scheme 1A). For the synthesis of
the second fragment β-hLH[Z38−G71], the N-terminal
cysteine was protected as a thiazolidine (Thz, Z), and the C-
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a
Scheme 2. Synthesis of β-hLH[C72−L121] by NCL Between β-hLH[Z72−D99] and β-hLH[C100−L121] Fragments
a
Amino acid residues bearing acid-labile protecting groups shown in deep red, pseudoproline dipeptides shown in orange (underlined), Acm-
protected Cys residues shown in purple, and thioester functionality shown in green. NCL buffer: guanidine·HCl (6 M), Na2HPO4 (0.2 M), tris(2-
carboxyethyl)phosphine hydrochloride (TCEP·HCl) (0.02 M), 4-mercaptophenylacetic acid (MPAA) (0.2 M), aqueous TCEP solution (0.5 M), pH
7.2−7.4. Thz-deprotection buffer: MeONH2·HCl (0.3 M), guanidine·HCl (6 M), pH 4.0−4.5. Cocktail B: 88% TFA, 5% H2O, 5% phenol (PhOH),
2% triisopropylsilane (TIPSH).
a
Scheme 3. Final Assembly by NCL of Full-Length β-Subunit of hLH Bearing Chitobiose at the N-Glycosylation Site
a
NCL buffer: guanidine·HCl (6 M), Na2HPO4 (0.2 M), tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl) (0.02 M), 4-mercaptophenyl-
acetic acid (MPAA) (0.2 M), aqueous TCEP solution (0.5 M) pH 7.2−7.4.
terminal glycine residue was converted to glycine phenyl-
thioester using PyBOP as a coupling agent. Finally, global
deprotection under acidic conditions with Cocktail B provided β-
hLH[Z38−G71] in 72% yield (over two steps) (Scheme 1B).
The last segment required for the assembly was β-hLH[C72−
L121] (Scheme 2). This fragment is devoid of glycosylation sites,
and our initial approach was to synthesize it entirely by SPPS.
However, despite the incorporation of three pseudoproline
dipeptides29 and the use of the Hmb (2-hydroxy-4-methox-
ybenzyl) protecting group,32 aspartimide formation was
predominant, and we were unable to obtain useful yields of
this peptide on solid support. We therefore approached this
synthesis through NCL of two smaller peptide fragments of
roughly similar size. Thus, protected peptides β-hLH[Z72−S98]
and β-hLH[Z100−L121] were accessed using SPPS following
cleavage from the resin. In the first case, the terminal serine
residue (Ser98) was coupled to allyl-protected aspartic acid
ethylthioester [HCl·H-Asp(OAllyl)-SEt]33 under Sakakibara
conditions, and subsequent treatment with Cocktail B gave β-
hLH[Z72−D99] in 58% yield (over two steps). The fully
deprotected peptide β-hLH[C100−L121] was obtained in two
steps (64%), involving global acid deprotection (Cocktail B) and
conversion of the N-terminal thiazolidine (Thz100) to cysteine
using methoxyamine hydrochloride (MeONH2·HCl) at pH
4.0−4.5.34 The two small fragments were then coupled under
NCL conditions (phosphate buffer solution containing
guanidine·HCl and TCEP·HCl at pH 7.2−7.4) with 4-
mercaptophenylacetic acid (MPAA) as an additive, and the
allyl protecting group on the Asp99 side chain was subsequently
removed [PdCl2(dppf), phenylsilane, DMSO]. Finally, the N-
terminal Thz group was cleaved using a buffer solution of
MeONH2·HCl and guanidine·HCl in water at pH 4.0−4.5 to
provide β-hLH[C72−L121] in 55% yield (over three steps)
(Scheme 2).
The final assembly of the individual peptide fragments
commenced with the coupling of segment β-hLH[C72−L121]
with β-hLH[Z38−G71] through NCL, followed by removal of
the Thz group to free the N-terminal cysteine required for the
final ligation, in a one-flask procedure. Purification by HPLC
provided the desired peptide β-hLH[C38−L121] in 43% yield
(two steps). Finally, β-hLH[C38−L121] was coupled to the
chitobiose-containing glycopeptide β-hLH(chitobiose)[S1−
Y37] under NCL conditions to afford the full-length β-subunit
of hLH β-hLH(chitobiose)[S1−L121] in 35% yield following
HPLC purification (Scheme 3).
Chemical Synthesis of the β-Subunit of hCG Bearing
Four O-Linked Glycans. Having identified a successful route
that featured ligation steps proceeding with complete conversion
in reasonable reaction time frames, we set out to synthesize the
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larger and more complex β-subunit of hCG incorporating two N-
linked glycans and four closely spaced O-glycosylation sites.
Our initial approach to β-hCG relied on the key
disconnections shown in Figure 1B, whereby five simpler
fragments, each bearing the corresponding N-/O-glycans could
be merged together via NCL. This highly modular strategy would
allow us to investigate the effect of introducing various defined
carbohydrates at different sites on the protein scaffold. As for the
glycosylation points, each of the N-linked glycans was coupled to
the complementary peptide fragment via Lansbury aspartylation,
as shown earlier for β-hLH. Interestingly, a more challenging
approach was also considered, consisting of the double
incorporation, simultaneously, of the two N-linked carbohy-
drates on a longer peptide backbone comprising both N-
glycosylation sites. With respect to the more complicated O-
glycopeptide fragment, we envisioned the installation of all four
O-linked glycans on solid support as glycosyl-serine “cassettes”
during the synthesis of the carboxy-terminal segment.
Scheme 4. Synthesis of N-Glycopeptide Fragments 1: β-
hCG(chitobiose)[S1−G22] (A) and 2: β-
hCG(chitobiose)[Z23−Y37] (B) and Convergent Approach
to Bis-Glycosylated N-Glycopeptide Fragment 1.2: β-
hCG(chitobiose)2[S1−Y37] via a Double Lansbury
a
Aspartylation (C)
Our initial target glycoform presents the readily available
disaccharide chitobiose as a model N-linked glycan for the native
complex bianntenary dodecasaccharide (Figure 1B and C),
similar to that previously described for hLH. We began our
approach with the synthesis of protected pF1:β-hCG[S1−G22]
and pF2:β-hCG[Z23−Y37] using standard Fmoc-based SPPS
followed by cleavage from resin, C-terminus derivatization, and
selective aspartic acid deprotection (Scheme 4A and B). In the
event, the glycine residue (Gly22) in Fragment 1 was reacted
with PhSH under PyBOP-mediated coupling conditions to give
the corresponding C-terminal thioester, whereas Fragment 2 was
subjected to single amino acid attachment with HCl·H-Tyr-SPh
under epimerization-free Sakakibara conditions (EDC,
HOOBt). In both cases, selective removal of the OPp protecting
group (at Asp 13 and Asp30, respectively) revealed the free
aspartic acid to be coupled with chitobiose amine. Thus, pF1:β-
hCG[S1−G22] and pF2:β-hCG[Z23−Y37] were then sub-
jected to the two-step Lansbury aspartylation/Cocktail B
deprotection protocol described earlier29a to afford glycopeptide
fragments β-hCG(chitobiose)[S1−G22] and β-hCG[Z23−
Y37] in 28% and 25% yield, respectively, (over four steps)
after HPLC purification. Since the same N-glycan (chitobiose)
was to be installed at both fragments, a double Lansbury
aspartylation strategy was also explored for the synthesis of the
entire bis-glycosylated segment β-hCG(chitobiose)2[S1−Y37]
(Scheme 4C). First, SPPS of the protected fragment, followed by
cleavage from the resin, C-terminal thioesterification (PhSH,
PyBOP), and subsequent selective Asp(OPp) deprotection (2%
TFA/DCM) provided peptide segment pF1.2:β-hCG[S1−
Y37] having the two aspartic acid (Asp13 and Asp30) side
chains free for further coupling with two chitobiose units. In the
event, 6 equiv of chitobiose amine, with respect to the peptide,
and 6 equiv of HATU as the coupling agent were employed to
give an encouraging 18% yield (over four steps) after global
deprotection with Cocktail B. A 9% yield of monoglycosylated
product was also isolated under these reaction conditions.
Importantly, in the case of incorporating the same glycan unit,
this approach represents a significant improvement over the
synthesis of the two smaller individual fragments in terms of
convergency and overall synthetic efficiency. Thus, this strategy
was preferentially applied to access the corresponding β-
hCG(chitobiose)2[S1−Y37] fragment (Scheme 4C).
a
Amino acid residues bearing acid-labile protecting groups shown in
deep red, pseudoproline dipeptides shown in orange (underlined), and
Acm-protected Cys residues shown in purple. Chitobiose glycan
shown in blue and thioester functionalities shown in green. Cocktail B:
88% TFA, 5% H2O, 5% phenol (PhOH), 2% triisopropylsilane
(TIPSH).
tection with Cocktail B, β-hCG[Z38−G71] was isolated in 75%
yield over two steps (52% overall from resin) (Scheme 5A). The
synthesis of Fragment 4, β-hCG[Z72−T109], was initially
hampered by significant aspartimide formation. Fortunately, the
use of a piperidine/oxyma pure cocktail for the Fmoc-
deprotection during the SPPS entirely suppressed this problem,
and after single amino acid attachment (HCl·H-Thr-SPh)6a at
the C-terminus, followed by subsequent treatment with Cocktail
B, the desired peptide fragment β-hCG[Z72−T109] was
obtained in 45% yield (over two steps) with no aspartimide
formation (Scheme 5B).
Finally, access to the last segment β-hCG(Ac3-Gal-
NAc)4[C110−Q145] required a dramatically different approach
than that described above. The presence of four O-linked glycans
prohibits the late-stage attachment of these carbohydrates to the
fully elaborated peptide. Alternatively, a more linear strategy,
termed the “cassette-based” approach was followed,35 wherein
the O-glycan, linked to a properly protected serine residue, is
directly introduced in the peptide sequence as a conveniently
protected glycosyl amino acid building block (“cassette”), during
solid-supported synthesis (Scheme 6A). This process not only
allowed for assembly of this complex glycopeptide fragment but
Fragment 3 was prepared in a very straightforward manner,
beginning with SPPS, followed by cleavage from the resin and
thioesterification of the C-terminus with PhSH. After depro-
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We then targeted the synthesis of this fragment bearing the α-
N-acetylgalactosamine moiety (GalNAc) on all four serine
residues, which corresponds to the “Tn antigen” structure
(Scheme 6B). This system is the minimal common constituent of
larger O-linked glycans and has been shown to be relatively
abundant at Ser121, 127, 132, and 138 in wild-type β-hCG.24
Following known procedures, we synthesized the conveniently
protected, glycosylated Fmoc-Ser building block bearing O-
acetyl groups in the GalNAc residue and having a free carboxylic
acid on the serine.35a With this cassette in hand, we performed
the synthesis of the required fragment via automated SPPS, with
manual coupling of the corresponding glycosyl-serine residue
(1.5 equiv) under PyAOP/HOAt coupling conditions. Gratify-
ingly, after treatment with Cocktail B, we were able to obtain the
entire fragment β-hCG(Ac3-GalNAc)4[C110−Q145] with O-
acetyl protected sugars in a single SPPS with great purity in 22%
overall yield after HPLC purification (Scheme 6B). Importantly,
this successful synthesis was amenable to scale-up, which
afforded up to more than 50 mg of the most complex fragment
of this synthetic version of β-hCG bearing four closely spaced Tn
antigen structures.
Scheme 5. Synthesis of Peptide Fragments 3: β-hCG[Z38−
a
G71] (A) and 4: β-hCG[Z72−T109] (B)
This strategy is likely to be applicable to the installation of
alternative O-linked glycan cassettes at the four O-glycosylation
sites for the assembly of different glycoforms. In this regard, a
number of glycosyl amino acids incorporating more elaborated
glycans, such as tumor-associated carbohydrate antigens (TF,
STn, STF) or mucin-related core structures 1−4, have been
synthesized through the cassette approach. Importantly, using
these O-linked glycosylated building blocks, the cassette
methodology has been successfully applied for the solution-
and solid-phase synthesis of a series of mucin-derived O-
glycopeptides bearing larger oligosaccharides.36 These examples
constitute an important precedent for the applicability of this
strategy toward more complex glycosylated versions of β-
hCG[C110-Q145]. Practical synthetic access to the carboxy-
terminus fragment of β-hCG is an important step in itself as it has
been found to be a common epitope for hCG-based monoclonal
antibodies.25 Screening of the specificity of different glycoforms
and their role in binding to these antibodies or the development
of new glycoforms could be used as a powerful tool in important
clinical applications.
a
Amino acid residues bearing acid-labile protecting groups shown in
deep red, pseudoproline dipeptides shown in orange (underlined),
Acm-protected Cys residues shown in purple, and thioester
functionalities shown in green. Cocktail B: 88% TFA, 5% H2O, 5%
phenol (PhOH), 2% triisopropylsilane (TIPSH).
Scheme 6. Cassette Approach Employed for Incorporation of
the O-Linked Glycans on Solid Support (A) and Synthesis of
Glycopeptide Fragment 5: β-hCG(Ac3-GalNAc)4[C110−
Q145] Bearing N-acetylgalactosamine (GalNAc) at the Four
O-Glycosylation Sites (B)
With all the prerequisite fragments in hand, assembly of the β-
subunit of hCG relied on the coupling of the individual peptide
segments by NCL, starting with fragments β-hCG[Z72−T109]
and β-hCG(Ac3-GalNAc)4[C110−Q145]. Thus, these two
fragments were joined together under standard NCL conditions,
using MPAA as an additive. Upon completion of the reaction as
monitored by ultraperformance liquid chromatography (UPLC),
the terminal Thz (Z) protecting group was removed using
MeONH2·HCl in a one-flask procedure. Subsequently, size-
exclusion centrifugal filtration of the previous crude mixture
followed by treatment with 5% aqueous hydrazine led to clean
and complete removal of the acetate groups to provide β-
hCG(GalNAc)4[C72−Q145] in 50% yield over three steps
(Scheme 7).
With the in situ ligation and Thz opening followed by acetate
deprotection successfully performed in the synthesis of half of
the full-length glycoprotein, we next used another NCL to bring
β-hCG(GalNAc)4[C72−Q145] and β-hCG[Z38−G71] to-
gether. The coupling reaction was completed within 3 h with
no starting material remaining, as assessed by LC-MS. Then,
MeONH2·HCl was added, and the pH was adjusted to 4.0−4.5
to induce conversion of the N-terminal Thz to cysteine. Happily,
also, in principle, enables the installation of different O-linked
glycans by simply applying the desired cassette during peptide
backbone elongation on SPPS.
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Scheme 7. Synthesis of Glycopeptide Fragment 4.5: β-hCG(GalNAc)4[C72−Q145] by NCL of β-hCG[Z72−T109] and β-
hCG(Ac3-GalNAc)4[C110−Q145] Followed by Thz Opening and Subsequent De-O-Acetylation
Scheme 8. Final Ligation of the Glycopeptide Fragments to Access Full-Length, Homogeneously Glycosylated β-Subunit of hCG
after size-exclusion centrifugal filtration, this procedure enabled
good recovery of β-hCG(GalNAc)4[C38−Q145] for the final
ligation reaction.37 In the critical ligation event, O-glycosylated
fragment β-hCG(GalNAc)4[C38−Q145] was combined with
N-glycopeptide β-hCG(chitobiose)2[S1−Y37] under standard
NCL conditions to give the target glycoprotein β-hCG(chito-
biose)2(GalNAc)4[S1−Q145] after 4 h. Upon HPLC purifica-
tion, the primary sequence of the 145-residue β-subunit of hCG
containing homogeneous N- and O-linked glycans was obtained
in a gratifying 33% yield (Scheme 8)38 [see Figure 2 for mass
spectrum and LC trace (UV) of β-hCG containing chitobiose at
Asn13 and Asn30, and N-acetylgalactosamine at Ser121, Ser127,
Ser132, and Ser138].
Thus, compound β-hCG(chitobiose)2(GalNAc)4[S1−
Q145], bearing not only two N-linked carbohydrates but also,
and even more challenging, four O-linked sugars, represents the
largest human glycoprotein hormone to have been synthesized in
homogeneous form using strictly chemical means. While the
current work has involved chitobiose and GalNAc as
representative N-/O-linked model glycans, this demonstration
of feasibility through the modular glycoform assembly presented
herein opens the door to the application of this technology to the
synthesis of a library of homogeneous glycoproteins via chemical
synthesis by installing alternative glycans on the corresponding
peptide fragments
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Figure 2. Mass spectrum and UV trace of β-hCG(chitobiose)2(GalNAc)4[S1−Q145] glycoprotein. Calcd for C759H1239N213O252S13, 17797.08 Da
(average isotopes) [M + 9H]9+ m/z 1978.45, found 1979.24; [M + 10H]10+ m/z 1780.71, found 1781.27; [M + 11H]11+ m/z 1618.92, found 1619.29;
[M + 12H]12+ m/z 1484.09, found 1484.67; [M + 13H]13+ m/z 1370.01, found 1370.42; [M + 14H]14+ m/z 1272.22, found 1272.55; [M + 15H]15+ m/z
1187.47, found 1187.79; [M + 16H]16+ m/z 1113.32, found 1113.54; [M + 17H]17+ m/z 1047.89, found 1048.16; [M + 18H]18+ m/z 989.73, found
990.02; [M + 19H]19+ m/z 937.69, found 938.02; [M + 20H]20+ m/z 890.85, found 891.14.
biological studies of these well-defined glycoforms to better
understand the specific roles of certain glycans in the function
and bioactivity of hCG in clinical settings. This knowledge may,
in turn, lead to the development of more efficacious and
improved therapeutics.
CONCLUSION
■
In summary, we have synthesized the glycosylated primary
sequence of two complex glycoprotein hormones (β-hLH and β-
hCG) in homogeneous form using the current innovations of
peptide and glycopeptide chemistry. The first approach
described for the preparation of β-hLH served to validate the
synthetic strategy en route to the more complex hCG β-subunit.
The synthesis of the latter featured two challenging aspects that
were successfully executed. First, a double Lansbury glyco-
sylation was accomplished to provide the N-terminal fragment of
the molecule in a highly modular fashion. Second, practical access
to the carboxy-terminus was gained by sequential installation of
four O-linked glycosyl-amino acid cassettes into closely spaced
O-glycosylation sites in a single and high-yielding solid-
supported synthesis. This O-linked glycopeptide fragment was
then successfully advanced by further coupling, deprotection,
and subsequent ligations with the remaining peptide segments to
provide the full-length β-subunit of hCG. Interestingly, the
highly modular assembly exemplified herein sets the stage for
accessing more complex, chemically pure β-hCG glycoforms by
synthesizing collections of each individual fragment containing a
number of different, elaborated glycans and bringing them
together following the successful ligation strategy outlined in this
work. While the late-stage processes involving final Acm removal
and oxidation/folding remain to be validated for β-hLH and β-
hCG, building on our previous successes with erythropoietin and
other glycoprotein targets, we have confidence in the ability to
generate correctly folded β-subunits of these glycoprotein
hormones. This important prospect should allow future
ASSOCIATED CONTENT
* Supporting Information
■
S
A detailed description of general experimental procedures,
including spectroscopic and analytical data for new compounds is
provided. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
s-danishefsky@ski.mskcc.org
■
Present Address
§Department of Chemistry, DePaul University, 1110 West
Belden Avenue, Chicago, IL 60614.
Author Contributions
∥These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the National Institutes of Health
(CA103823 to S.J.D.). A.F.-T. thanks the European Commission
(Marie Curie International Outgoing Fellowship) for funding.
Postdoctoral support is acknowledged by P.A.V. (NIH,
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