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An Orthogonal Double-Linker Resin Facilitates the Efficient Solid-Phase Synthesis of Complex-Type N-Glycopeptide Thioesters Suitable for Native Chemical Ligation.

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Glycopeptide Synthesis
An Orthogonal Double-Linker Resin Facilitates
the Efficient Solid-Phase Synthesis of ComplexType N-Glycopeptide Thioesters Suitable for
Native Chemical Ligation**
Stefano Mezzato, Manuela Schaffrath, and
Carlo Unverzagt*
In memory of Murray Goodman
Recombinant therapeutic glycoproteins frequently bear complex oligosaccharides (N-glycans) connected to asparagine.[1]
The variability of N-glycosylation leads to glycoprotein
microheterogeneity rendering the isolation of pure glycoforms extremely difficult. It has been demonstrated that the
biological activity of glycoproteins depends on the nature of
the attached glycans.[2] For medicinal and pharmaceutical
purposes the generation of uniform glycoproteins with a
tailored glycosylation pattern is of high interest. In parallel to
the creation of special expression systems,[3] synthetic methodologies[4] have been developed to generate homogenous
natural glycoproteins by remodeling of glycans, transglycosylation, reverse proteolysis, or native chemical ligation
(NCL),[5] of which the latter promises the highest potential
and flexibility. To obtain building blocks suitable for the
assembly of complex-type N-glycoproteins by native chemical
ligation, we have developed a high-yielding solid-phase
synthesis of glycopeptide thioesters that bear complex-type
The native chemical ligation is based on the chemoselective reaction between a peptide a-thioester and a peptide
with an N-terminal cysteine residue. Although several
examples have demonstrated the use of this chemistry for
synthesis of glycopeptides and glycosylated proteins,[6] the
efficient generation of glycopeptide thioesters that contain
complex-type N-glycans on the solid phase has not been
[*] S. Mezzato, Dr. M. Schaffrath, Prof. C. Unverzagt
Bioorganische Chemie, Gebude NW1
Universitt Bayreuth
95440 Bayreuth (Germany)
Fax: (+ 49) 921-555-365
[**] We thank the Deutsche Forschungsgemeinschaft and the Fonds der
Deutschen Chemischen Industrie for funding.
Supporting information for this article is available on the WWW
under or from the author.
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
reported.[7] However, a solution to this problem was required
for the envisioned synthesis of bovine RNase B as a model
glycoprotein that consists of 124 amino acids with an Nglycosylation site at Asn 34 and the nearest ligation site at
Cys 40. The glycosylated fragment 30–68 of RNase B (C) was
selected to establish the required chemistry and was strategically split into the thioester segment A and the cysteine
peptide B (Scheme 1). For the challenging solid-phase synthesis of the thioester fragment RNase 30–39 (A), which bears
a heptasaccharide N-glycan, the sulfonamide-based safetycatch approach[8] appeared most attractive among the methods developed for the synthesis of thioesters.[9]
The safety-catch linker is particularly stable to nucleophiles, basic and acidic conditions,[10] whereas, after completion of the synthesis, selective sulfonamide activation renders
the linker susceptible to nucleophilic attack and allows the
release of the final product. However, the inertia of the
sulfonamide linker requires time-consuming two-step microcleavages to monitor the reaction efficiency by LC–MS. As a
consequence, only the final products are usually checked after
final release from the resin.[11] To overcome this problem and
to allow easy step-by-step control, especially for the critical
glycopeptide formation, we took into consideration the
attachment of an additional linker.[12] The second handle
should enable easy detachment of the peptide chain as well as
be completely orthogonally stable to the safety-catch linker.
These requirements are fulfilled by the Rink-amide linker,[13]
as it can be cleaved easily with trifluoroacetic acid (TFA)
(Scheme 2). The peptides thus released are free of acid-labile
protecting groups which therefore increases the solubility and
simplifies HPLC and LC–MS analysis. A hydrophobic spacer
facilitates reversed-phase HPLC detection of the products
and the double-linker fragment.
As a solid support we chose an amino PEGA[14] resin to
carry the double-linker array. First, the Fmoc protected Rinkamide linker 1 was attached to the resin by using HCTU and
DIPEA (Scheme 3). The Fmoc protecing group was then
cleaved with piperidine in NMP to furnish compound 3, which
was elongated with Fmoc phenylalanine as a spacer. After
cleavage of the Fmoc group, the safety-catch linker 3carboxypropanesulfonamide[8] (4) was coupled with DIC
and HOBt to give the fully functionalized resin 5. Reaction
efficiencies were monitored by the Kaiser test[15] and the
coupling reactions went to completion.
The loading of the safety-catch linker has often been a
matter of concern,[16] as poor coupling yields and racemization
of the first amino acid impair further synthesis. The first
amino acid loaded onto the resin was arginine by using DIC
and 1-MeIm[17] in 80 % yield as determined by UV detection
of the dibenzofulvene–piperidine adduct at 290 nm.[18]
Double coupling led to nearly quantitative acylation of the
sulfonamide linker. The following peptide chain elongation
by four Fmoc amino acids was carried out manually under
commonly used conditions,[19] and pentapeptide 7 was
obtained in high yield and purity.
From previous work it was known[7b, 20] that the coupling of
Fmoc asparagine attached to complex-type N-glycans is
difficult, especially on the solid phase, and tends to give low
yields, presumably owing to a number of factors including the
DOI: 10.1002/anie.200461125
Angew. Chem. Int. Ed. 2005, 44, 1650 –1654
Scheme 1. Retrosynthesis of complex-type glycosylated RNase B fragment 30–68.
Scheme 2. The combination with the orthogonal Rink-amide linker facilitates the LC–MS monitoring of reactions based on the safety-catch linker.
resin matrix and the activation. The coupling efficiencies of
the synthetic glycosyl amino acid 8[20b, 21] carrying an unprotected biantennary complex-type heptasaccharide N-glycan
were monitored under equivalent reaction conditions for
different resins. The PEGA resin probed superior to other
polystyrene-based matrices which may be related to the
better swelling properties of PEGA. In parallel, the initially
low coupling yields of 8 were optimized by comparing various
activation reagents. It was found that PyBOP in the presence
of DIPEA gave the best yields when using only 0.8 equivAngew. Chem. Int. Ed. 2005, 44, 1650 –1654
alents of the precious glycosyl amino acid 8 (with respect to
the loading of the resin). The glycosyl amino acid 8 was first
dissolved in DMSO/NMP (1:1), added to the resin, and
activated in situ with solid PyBOP and DIPEA to afford the
glycopeptide 9 in excellent yield (95 %, UV detection of
cleaved Fmoc).
Protection of the hydroxy groups of the carbohydrate was
necessary to prevent their acylation[20d, 22] by activated amino
acids during the subsequent chain elongation of 9. Furthermore, unprotected oligosaccharides can be unstable during
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Scheme 3. Synthesis of glycopeptide thioester A: a) 1, HCTU, DIPEA, DMF; b) 20 % piperidine in NMP; c) 1. Fmoc-Phe-OH, HCTU, DIPEA; 2. b);
3. 4, DIC, HOBt, DMF; d) Fmoc-Arg(Pbf)-OH, DIC, 1-MeIm, CH2Cl2, DMF; e) manual solid-phase peptide elongation by conventional Fmoc
chemistry with Fmoc amino acid (4 equiv), HOBt (4 equiv), TBTU (4 equiv), DIPEA (9 equiv); f) 8 (0.8 equiv), PyBOP, DIPEA, DMSO, NMP;
g) Ac2O, pyridine, HOAc; h) TMSCHN2, n-hexane, CH2Cl2 ; i) ethyl 3-sulfanylpropionate, sodium thiophenolate, DMF; j) TFA, H2O, TES, ethyl 3-sulfanylpropionate, room temperature, 2 h (46 % from 8); k) A (1 equiv), B (1.3 equiv), 6 m guanidinium chloride, thiophenol (2 %), phosphate buffer
(pH 7.6); l) hydrazine hydrate (10 %), DTT (5 %). HCTU = N-[(1H-6-chlorobenzotriazol-1-yl) (dimethylamino)methylene]-N-methylmethanaminium
hexafluorophosphate N-oxide; DIPEA = N,N-diisopropylethylamine; DMF = N,N-dimethylformamide; NMP = N-methylpyrrolidone; Fmoc = 9-fluorenylmethyloxycarbonyl; DIC = N,N’-diisopropylcarbodiimide); HOBt = 1-hydroxybenzotriazole; Pbf = 2,2,4,6,7-pentamethyldihydrobenzofuran-5sulfonyl; MeIm = N-methylimidazole; TBTU = N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium tetrafluoroborate
N-oxide; PyBOP = benzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate; DMSO = dimethyl sulfoxide; TMS = trimethylsilyl;
TFA = trifluoroacetic acid; TES = triethylsilane; DTT = 1,4-dithiothreitol.
final deprotection of the peptide backbone with TFA,[23a]
whereas acetylation provides complete TFA stability.[23b]
Subsequently, acetylation using acetic anhydride and pyridine
(1:1) protected the OH groups of the heptasaccharide and
simultaneously capped the remaining N-terminal amino
groups. However, after the acetylation step, the loading of
the resin appeared to be significantly lower (2/3 of the peptide
was cleaved within 16 h of reaction time); TFA cleavage and
LC–MS showed that a major side product was identified as
the N-acetylated sulfonamide linker completely lacking the
peptide chain. Most likely, an additional N-acetylation of the
safety-catch handle leads to this outcome (Scheme 4).
In solution the N-acylation of N-acylsulfonamides was
found to occur to a small extent.[24] Under basic conditions the
N-acylsulfonamide (pKa = 2.5[25]) is easily deprotonated and
the imide anion is susceptible to further acetylation. This
activates the handle, which is subsequently attacked by
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
nucleophiles (e.g. pyridine), thus regenerating the starting
material (Scheme 4, path a) or irreversibly disconnecting the
peptide chain from the linker (Scheme 4, path b) with
complete loss of the product. We assumed that the acylation
of the acylsulfonamide should be pH-dependent, and, by
avoiding the deprotonation of the N-acylsulfonamide, an
overacylation should be precluded. Optimization studies
revealed that the initial neutralization of the pyridine catalyst
with acetic acid (Ac2O/py/AcOH 5:3:2 v/v/v) afforded the
fully acetylated glycopeptide without any side product being
observed. The protected glycohexapeptide 10 was conveniently elongated by Fmoc solid-phase peptide synthesis to
give the glycodecapeptide 11 with Boc methionine as the final
The controlled release of the glycopeptide as a thioester
requires the prior activation of the safety-catch linker by
alkylation with iodo acetonitrile or trimethylsilyldiazome-
Angew. Chem. Int. Ed. 2005, 44, 1650 –1654
To assure that the thioester A was
reactive in a native chemical ligation,[5]
elongation with the synthetic RNase 40–
68 peptide B[28] was examined on an
analytical scale. Compounds A and B
were dissolved in 6 m guanidinium hydrochloride (GnHCl) buffered at pH 7.6
and the ligation was started by addition
of thiophenol (final concentration of
2 %).[5b] The reaction progress was followed by LC–MS and showed that the
ligation had proceeded to 50 % after 1 h
and was complete within 8 h. Compound
A was completely consumed and no
hydrolyzed thioester was detectable in
the crude reaction mixture, thus indicating efficient ligation. The analogous
reaction in aqueous 1,4-dioxane[11a] gave
rise to considerable amounts of hydrolyzed thioester. The acetate groups were
then removed in a one-pot reaction with
hydrazine hydrate (10 %) containing
DTT (5 %) to prevent disulfide formation. Ester hydrolysis was finished after
Scheme 4. Side-reactions of the safety-catch linker during acetylation under basic conditions.
1 h and yielded the desired unprotected
glycopeptide C[27] as the target molecule
(identified by LC–MS). The glycopeptide C obtained by native chemical ligation represents the
thane (TMSCHN2).[8] TMSCHN2 was selected as the more
homogeneously glycosylated fragment 30–68 of RNase B
powerful and selective reagent;[26] this commercially available
carrying a complex-type N-glycan.
and shelf-stable 2 m solution in n-hexane was diluted with
Herein we have established a general and efficient solidTHF (1:1) before use. In the first experiments the safety-catch
phase synthesis of complex glycopeptide a-thioesters as
linker of the PEGA resin was methylated to about 50 %,
fragments of N-glycoproteins. The orthogonal combination
whereas similar activation performed on a polystyrene matrix
of a safety-catch linker with a Rink amide gave a novel
went to completion. We assumed that the PEGA resin did not
analytical construct that facilitates rapid LC–MS analysis and
swell enough in the nonpolar THF/n-hexane mixture and that
optimization of several key reactions. It was found that
peptide aggregation would further impair the activation of the
activation of glycosyl-asparagine in situ gives the highest
linker. The use of more-polar solvent mixtures (tBuOH/ncoupling yields and that the OH groups can be acetylated
hexane 2:1 or CH2Cl2/n-hexane 2:1) proved very effective and
(capping) without activating the safety-catch linker. With the
furnished the methylated product 12 in quantitative yield
availability of complex-type glycopeptide thioesters the
after 2 h without side-chain modification of methionine.
chemical synthesis of fully N-glycosylated glycoproteins
Activation of the safety-catch linker was conveniently monimoves closer into reach. Work is currently in progress to
tored by LC–MS after selective TFA cleavage of the
provide the fragments required for the synthesis of full-length
glycopeptide bound to the methylated linker. After complete
RNase B.
activation the glycopeptide thioester was released with ethyl
3-sulfanylpropionate and catalytic sodium thiophenolate.[11a]
Received: June 30, 2004
However, complete detachment of the peptide required
Revised: September 13, 2004
3 equivalents of sodium thiophenolate and 100 equivalents
Published online: February 3, 2005
of ethyl 3-sulfanylpropionate. The displacement efficiency
was indirectly monitored (LC–MS) by TFA-mediated release
Keywords: glycopeptides · glycoproteins · native chemical
of the linker after treatment with thiol, which led to only the
ligation · oligosaccharides · solid-phase synthesis
deacylated N-methylsulfonamide scaffold.
Subsequently, the acid-labile protecting groups were
completely removed from the crude thioester at room
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[27] ESI-TOF-MS (CH3CN/H2O): A(C141H219N23O70S2): Mcalcd
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the deprotected peptide.
Angew. Chem. Int. Ed. 2005, 44, 1650 –1654
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