Probing the frontiers of glycoprotein synthesis: The fully elaborated β-subunit of the human follicle-stimulating hormone

Article abstract of DOI:10.1002/anie.201107482

Ambitious undertaking: The β-subunit of the human follicle-stimulating hormone (hFSH) displaying a N-linked consensus sequence oligosaccharide at each of the two wild-type sites was synthesized. The glycoprotein has been designed with acetamidomethyl prot

Full text of DOI:10.1002/anie.201107482

Angewandte
Chemie
DOI: 10.1002/anie.201107482
Chemical Synthesis of Glycoproteins
Probing the Frontiers of Glycoprotein Synthesis: The Fully Elaborated
b-Subunit of the Human Follicle-Stimulating Hormone**
Pavel Nagorny, Neeraj Sane, Bernhard Fasching, Baptiste Aussedat, and Samuel J. Danishefsky*
The human follicle-stimulating hormone (hFSH) belongs to a
family of hormones responsible for the maintenance of
essential reproductive processes (gonadotropins).[1] FSH is
produced in the anterior pituitary, and the binding of FSH to
its receptor stimulates the maturation of follicles and the
production of estrogen in females, and maintains spermato-
genesis in males.[2] Consequently, FSH is clinically used in the
treatment of anovulatory disorders associated with infertil-
ity.[3] Administration is usually in the form of subcutaneous
injections, often once a day, over prolonged periods of time.
Side effects of this treatment range from allergic reactions
and nausea, to mood swings and fatigue.[4] Presently, FSH is
mainly derived from recombinant technologies, specifically,
from Chinese Hamster Ovary (CHO) cells.[5] The material so
obtained is a complex mixture of hormone glycoforms, that is,
highly heterogeneous with respect to the carbohydrates on
the peptide backbone.[6] In normal adult humans, the FSH
receptor (FSH-R) is expressed only on the ovarian granulosa
cells of females and the testicular Sertoli cells of males.
However, in a recent discovery it was found that the FSH-R is
ubiquitously expressed on the endothelial cells of the
peripheries of the tumors of the breast, prostrate, colon,
pancreas, kidney, stomach, testis, and ovary.[7] Earlier studies
in mice have indicated that the effect of FSH on the growth of
tumors is at least as potent as that of epidermal growth factor
(EGF).[8] Although there was a concentration dependency of
this effect, what might be an interesting study of the relative
roles of the various glycoforms is presently stymied by the
unavailability of homogeneous forms of FSH.
Structurally, FSH is a heterodimeric glycoprotein com-
posed of two non-covalently associated subunits (a and b) (1,
Figure 1).[9] Each of the subunits contains two N-linked
oligosaccharides—the a-subunit at Asn52 and Asn78, and the
Figure 1. a) Structure of hFSH heterodimer (1) with glycans (see inset
legend). b) b-Subunit (2) displaying the N-linked consensus sequence
oligosaccharide at the wild-type sites. c) Structure of the consensus
sequence oligosaccharide. Acm=acetamidomethyl, Ac=acetyl.
b-subunit at Asn7 and Asn24, which are incorporated in the
rough endoplasmic reticulum (RER) through co-translational
modifications of the peptide backbone. The structures of the
oligosaccharides play a crucial role in the proper folding,
subunit assembly, secretion, and activation of the target
receptor and, ultimately, the metabolic fate of the molecule.[10]
Clearly, a method for gaining access to homogeneous glyco-
forms of FSH would be highly desirable for establishing a
structure–activity relationship (SAR). Due to a lack of viable
techniques for separating such complex mixtures of glyco-
forms,[11] chemical and chemoenzymatic methods[12] have
emerged as a viable option for the preparation of homoge-
neous glycoproteins.
[*] Dr. P. Nagorny,[+] Dr. N. Sane,[+] Dr. B. Fasching, Dr. B. Aussedat,
Prof. Dr. S. J. Danishefsky
Laboratory for Bioorganic Chemistry
Sloan-Kettering Institute for Cancer Research
1275 York Avenue, New York, NY 10065 (USA)
and
Department of Chemistry, Columbia University
Havemeyer Hall, 3000 Broadway, New York, NY 10027(USA)
E-mail: s-danishefsky@ski.mskcc.org
The seminal work by Kent and co-workers in developing
the native chemical ligation (NCL) reaction[13] has led to the
synthesis of very challenging protein targets such as a
ubiquitin diastereomer,[14] RNase,[15] integral membrane
kinase,[16] and tetraubiquitin protein,[17] among others, that
are inaccessible by conventional peptide synthesis. The scope
of the NCL reaction has been vastly expanded now, to enable
ligations at Met, Ala, Phe, Ser, Val, Thr, Lys, Leu and Pro.[18]
[+] These authors contributed equally to this work.
[**] This work was supported by NIH grant CA103823 (to S.J.D.) and
NIH grant CA125934-02 (to P.N.). B.F. is grateful to the Austrian
Fonds zur Fçrderung der wissenschaftlichen Forschung for a
fellowship. We thank Rebecca Wilson for assistance with the
preparation of the manuscript.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201107482.
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Yet, the ability to gain access to complex glycoproteins
through de novo chemical synthesis remains a rather daunting
task.[12a] The difficulties can be attributed in part to the
challenges of obtaining complex carbohydrates, particularly
those containing sialic acid, fucose and high mannose
residues, by chemical synthesis, in sufficient quantities. With
respect to a possible synthesis of FSH, we had recently
described an approach to the synthesis of the b-subunit using
the disaccharide chitobiose as a model building block for our
glycoprotein assembly.[19] Herein, we present the results of a
rather ambitious undertaking, that is, the synthesis of the b-
subunit of FSH (2) containing a consensus sequence oligo-
saccharide at each of the two N-linkage sites, endowed with
high mannose, fucose and sialic acid presentation, using
natural motifs in all of the glycosidic linkages. The system has
been synthesized with protected cysteines anticipating folding
and association with the a-subunit.[20] The particular bianten-
nary consensus dodecasaccharide chosen for this purpose was
found to be abundant in batches of recombinant FSH
displaying high bioactivity. This oligosaccharide is also
found to exist on other glycoprotein hormones such as,
Chorionic gonadotropin (hCG), Luteinizing hormone (hLH),
and Thyroid-stimulating hormone (hTSH) as well as a-
fetoprotein (associated with human hepatocellular carci-
noma).[21] Consequently, the protocol presented here should
also be extendable to the synthesis of these and other
glycoproteins.
assembly, the construction was performed from the C to the
N terminus of the subunit. In order to engage in NCL, the
C terminus of each individual peptide fragment was function-
alized as a thioester by single amino acid coupling. The
peptide fragments were obtained by Fmoc-based solid phase
peptide synthesis (SPPS) and the protecting groups on the
amino acid side chains during SPPS were chosen in a way that
the aspartic acid residue that will bear the dodecasaccharide
was protected orthogonally to those residues that were likely
to interfere during the glycan attachment. Of the twelve
cysteines on the protein, the nine that were not required for
NCL were protected with acetamidomethyl (Acm) protecting
groups. Additionally, these cysteine side chain protections
prevent undesirable cross-linkages, by oxidation, during the
course of the synthesis. Cleavage from the resin and selective
deprotection of the aspartic acid side chain provided a free
carboxylic acid, which was then coupled with the glycosyl-
amine 7 by HATU-mediated Lansbury aspartylation.[22]
The dodecasaccharide 7 was obtained by chemical syn-
thesis, following the program outlined in Scheme 2.[19,23] The
strategy exploited the symmetry in the dodecasaccharide, in
that a bis-glycosylation reaction on the hexasaccharide
acceptor 13 with the trisaccharide donor 12 was executed in
the late stages of the synthesis. The hexasaccharide core 13
was obtained through a highly convergent series of glyco-
sylations using the known building blocks 8–11. In each case,
the protecting groups were carefully selected to maximize the
diastereoselectivity during the glycosylations and to minimize
the number of deprotection steps that would need to be
performed towards the end of the synthesis. Global depro-
tection of all the protecting groups present on the sugar
moieties provided the dodecasaccharide 14. The anomeric
hydroxy group was converted into a primary amine under
Kochetkov amination conditions[19,24] by treatment with
saturated ammonium bicarbonate solution. Excess ammoni-
The b-subunit of FSH consists of 111 amino acids and the
N-linked sugars are present at Asn7 and Asn24.[9a] The
abundance of cysteine residues in the peptide backbone, at
fairly regular intervals, speaks to the possibility of assembling
the glycoprotein using NCL. The key disconnections are
depicted in Scheme 1. The two key ligation sites chosen were
Phe19–Cys20 and Trp27–Cys28. To enable the incorporation of
the more precious glycopeptides in the final stages of
Scheme 1. Retrosynthetic strategy for the construction of the fully elaborated b-subunit of hFSH (2). HATU=O-(7-azabenzotriazol-1-yl)-
tetramethyluronium hexafluorophosphate.
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free. Cocktail B[28] treatment removed all the acid-labile
protecting groups on the amino acid side chains. This three-
step protocol afforded [bFSH1–19] 17 in 21% yield after HPLC
purification. The material so obtained was treated with the
glycosyl amine 7 under HATU-mediated coupling conditions.
The aspartylation proceeded in 73% conversion as indicated
by LC-MS, along with a trace amount of undesired asparti-
mide formation. Treatment with [Pd(PPh3)4] and phenylsilane
removed all the allyl and alloc protecting groups on the amino
acid side chains, in a one-flask procedure. The mixture was
purified by HPLC to provide the [bFSH1–19] glycopeptide 5 in
17% yield (averaged over nine trials).
Similarly, [bFSH20–26] 18 was obtained by Fmoc-based
SPPS on Fmoc-Thr(tBu)-TGT resin and cleavage from the
resin. Treatment of peptide 18 with tryptophan phenyl-
thioester 19, and subsequent deprotection with Cocktail B,
provided [bFSH20–27] 6 in 66% yield (3 steps) (Scheme 4).
Scheme 2. Schematic representation of the synthesis of the dodeca-
saccharide 7.
um bicarbonate was removed by repeated lyophilization of
the material with minimal exposure to moisture.[25]
The protected [bFSH1–18] 15 was obtained through Fmoc-
based SPPS starting from the commerically available Fmoc-
Arg(Pbf)-TGT resin. Pseudoproline dipeptides (denoted by
y) were incorporated into the peptide synthesis sequence to
improve the yield of this aggregation-prone fragment.[26] The
C-terminal carboxylic acid of the peptide was coupled to
phenylalanine phenylthioester 16 under Sakakibara condi-
tions (Scheme 3),[27] which are known to be epimerization-
Scheme 4. Synthesis of [bFSH20–27] 4. a) 19, HOOBt, EDC, TFE/CHCl3
1:3; b) Cocktail B; c) 7, HATU, DIEA, DMSO.
Lansbury aspartylation of this peptide with the glycosyl amine
7 proceeded in 50% conversion with some aspartimide
formation (ca. 8%). The [bFSH20–27] glycopeptide 4 was
isolated by HPLC purification in 27% yield (averaged over
five trials).
The last segment required for the assembly was
[bFSH28–111] (3, Scheme 5). Fragment 3 is devoid of glyco-
sylation sites and could have been obtained entirely by SPPS.
However, we were unable to obtain useful yields of this
peptide on solid support and we therefore opted to accom-
plish its synthesis through native chemical ligation of two
smaller peptide fragments of roughly equal length. Gly65–
Cys66 was chosen as the ligation site, for two reasons. First,
ligations at glycines are particularly effective, likely due to the
lack of steric bulk at the a-position. Additionally, conversion
of terminal glycine carboxylic acids to their thioesters is not
subject to epimerization. Thus, two shorter peptide fragments
were synthesized. [bFSH28–65] was obtained by SPPS on Fmoc-
Gly-TGTresin. The terminal glycine residue was converted to
glycine phenylthioester and treated with Cocktail B to
remove all the acid-labile protecting groups, providing frag-
ment 20 in 48% yield (over three steps). The fully depro-
Scheme 3. Synthesis of [bFSH1–19] 5. a) 16, HOOBt, EDC, TFE/CHCl3
1:3; b) Cocktail B; c) 7, HATU, DIEA, DMSO; d) [Pd(PPh3)4], PhSiH3,
NMP. HOOBt=3-hydroxy-1,2,3-benzotriazin-4(3H)-one, EDC=N’-(3-
dimethylaminopropyl)-N-ethylcarbodiimide, TFE=2,2,2-trifluoroetha-
nol, DIEA=ethyldiisopropylamine, DMSO=dimethyl sulfoxide,
NMP=N-methyl-2-pyrrolidone.
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Scheme 5. Final ligation of the glycopeptide fragments. a) 20, 21, PhSH, Gnd·HCl, Na2HPO4, TCEP·HCl, H2O (pH 7.4); b) NH2OMe·HCl,
Gnd·HCl, TCEP·HCl (pH 4.8); c) 4, PhSH, Gnd·HCl, Na2HPO4, TCEP·HCl, H2O (pH 7.4); d) NH2OMe·HCl, Gnd·HCl, TCEP·HCl (pH 4.8); e) 5,
PhSH, Gnd·HCl, Na2HPO4, TCEP·HCl, H2O (pH 7.3). Mass spectrum of compound 2 (inset). Gnd·HCl=guanidine hydrochloride, TCEP·HCl=
tris(2-carboxyethyl)phosphine hydrochloride.
tected peptide [bFSH66–111] 21 was obtained on solid support
using Fmoc-Glu(OtBu)-TGT resin, cleavage and treatment
with Cocktail B. The two fragments, 20 and 21, were coupled
under native chemical ligation conditions, using thiophenol as
an additive. Upon completion of the reaction as monitored by
LC-MS, the terminal Thz protecting group was removed using
methoxylamine hydrochloride, in a one-flask procedure, to
provide [bFSH28–111] 3 in 38% yield after HPLC purification.
The final assembly of the individual peptide fragments
commenced with the coupling of fragment [bFSH20–27] 4 with
the fragment [bFSH28–111] 3 under NCL conditions in a pH 7.3
buffer (Scheme 5). Subsequently, the N-terminal Thz group
was cleaved using methoxylamine hydrochloride at pH 4.8, to
free the N-terminal cysteine required for the final ligation, in
a one-flask procedure. Purification by HPLC provided the
desired glycopeptide [bFSH20–111] 22 bearing the dodecasac-
charide, in 26% yield (averaged over three trials).
compound 2 represents the largest realistically glycosylated
glycoprotein with all natural-type linkages to have been
synthesized in a homogeneous state, using strictly chemical
methods.
Much encouraged by this demonstration of feasibility, we
continue our studies through which we hope to synthesize
hFSH itself. There remains deprotection of the Acm protect-
ing groups[29] on the cysteine side chains such as to allow for
folding and subsequent association with the a-subunit. The
total synthesis of the latter is already well advanced. The
results of these ongoing studies will be disclosed shortly.
Received: October 24, 2011
Published online: December 9, 2011
Keywords: follicle-stimulating hormone · glycoproteins ·
.
native chemical ligation · solid-phase peptide synthesis
Finally, the glycopeptide [bFSH1–19] 5 was coupled to the
glycopeptide [bFSH20–111] 22 under NCL conditions. Gratify-
ingly, we obtained the full chain b-subunit of FSH (2)
containing the two dodecasaccharides in 27% yield (averaged
over three trials) after HPLC purification. Interestingly, the
ligation was facile despite the fact that the two bulky
dodecasaccharides are merely 16 residues apart during the
ligation event. Also noteworthy is the fact that the final
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