Synthesis of an S-linked glycopeptide analog derived from human Tamm-Horsfall glycoprotein

Article abstract of DOI:10.1039/b311583f

Direct base catalyzed S-glycosylation of a cysteine and a homocysteine containing peptide with O-acetyl protected bromides in DMF-water solution furnished two glycopeptide fragments. The two glycopeptide fragments were linked to the target glycopeptide with two S-glycosyl residues mimicking a part of Tamm-Horsfall glycoprotein.

Full text of DOI:10.1039/b311583f

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Synthesis of an S-linked glycopeptide analog derived from human
Tamm–Horsfall glycoprotein†
Xiangming Zhu, Tobias Haag and Richard R. Schmidt*
Fachbereich Chemie, Universität Konstanz, Fach M 725, D–78457 Konstanz, Germany.
E-mail: Richard.Schmidt@uni-konstanz.de; Fax: (ϩ49) 7531 883135
Received 19th September 2003, Accepted 7th November 2003
First published as an Advance Article on the web 18th November 2003
Direct base catalyzed S-glycosylation of a cysteine and a
homocysteine containing peptide with O-acetyl protected
bromides in DMF–water solution furnished two glyco-
peptide fragments. The two glycopeptide fragments were
linked to the target glycopeptide with two S-glycosyl resi-
dues mimicking a part of Tamm–Horsfall glycoprotein.
unclear.11 Structurally, THP, built up from a polypeptide back-
bone of 616 amino acids, contains approximately 30% carb-
ohydrate (w/w), comprising seven N-glycosidically bound sugar
chains,9 which are specific ligands for lectins and cytokines and
are probably responsible for most of THP functions.12
In order to contribute to the unraveling of the biological
function of human THP, a small S-linked glycopeptide carrying
two sugar residues 1 was designed (Fig. 1) and synthesized,
which corresponds to the sequence Ala484–Ala490 in THP,
containing Thr485 as potential O-glycosylation site and
Asn489 as N-glycosylation site.
Glycosylated proteins are ubiquitous components of extra-
cellular matrices and cellular surfaces where their oligo-
saccharide moieties are implicated in a wide range of cell–cell
and cell–matrix recognition events.1 Abnormalities in post-
translational glycosylation are often intimately associated with
cell transformation, malignancy and other pathological condi-
tions, including immunodeficiency syndromes, cancer and
inflammation.2 However, homogenous glycopeptides for bio-
logical investigation are difficult to obtain from biological
sources due to the significant levels of microheterogeneity in
naturally produced glycopeptides.3 Therefore, the synthesis of
structurally defined glycopeptides for the study of glycoprotein
binding interactions, cell–cell adhesion and receptor binding
specificity is of significant current interest.4
Naturally occurring glycopeptides most commonly incor-
porate an O-glycosidic or an N-glycosidic linkage between the
carbohydrate moiety and the side-chain of an appropriate
amino acid residue.4a However, the use of these substances as
therapeutics is hampered in many cases due to their inherent
in vivo instability, i.e. glycosidase involved cleavage of the glycan
from the peptide backbone. This has stimulated the develop-
ment of catabolically stable glycopeptide mimetics as fund-
amental tools for biological research and as potential agents for
therapeutic intervention.5 As a result, S-linked glycopeptides
which contain sulfur in the glycosidic linkage have attracted
much attention,6,7 because of their enhanced chemical stability
and enzymatic resistance.8 In this context, we have initiated a
program to synthesize S-linked glycopeptides6 that may prove
useful in biological studies and as potential therapeutic agents.
Herein we wish to report the synthesis of an S-linked glyco-
peptide analog derived from Tamm–Horsfall glycoprotein
(THP),9 the most abundant glycoprotein present in normal
human urine. Over the past 50 years, considerable investi-
gations have been centered on THP, owing to its various bio-
logical activities.10 THP was shown to block hemagglutination
induced by influenza, mumps, and New Castle disease viruses.
THP has been assumed essential for protecting the kidneys
from bacterial infections, also it has been implicated as a poten-
tial component necessary for maintenance of the electrolyte
balance in the nephron. In addition, THP may also play a
significant role in several pathological conditions involving the
kidney, including acute renal failure, urinary tract infection,
stone formation and interstitial nephritis. However, the bio-
logical role of THP, although extensively studied, remains
Fig.
1
Structures of THP fragment Ala484–Ala490 and target
molecule 1.
As is evident from Fig. 1, Thr485 and Asn489 in THP were
replaced by the corresponding cysteine (Cys) and homocysteine
(Hcy) residues in 1, respectively, which was based on the view-
point that the distance between sugar residue and cysteine is
similar to O-glycan linkage to threonine and ligation of sugar
residue to homocysteine mimics N-glycan linkage to aspara-
gine. Thus, a catabolically stable S-linked glycopeptide was then
resultant from the above substitution, providing the opportun-
ity to probe biological properties of glycopeptides at the
molecular level.
Very recently, we reported a new protocol for the synthesis of
S-linked glycopeptides in aqueous solution by direct S-
glycosylation of cysteine or homocysteine containing peptides
with O-acetyl protected glycosyl halides,6a wherein chemical
ligation techniques13 were utilized to prepare the required pep-
tides. This procedure is a significant advance in the convergent
assembly of S-linked glycopeptides because it demonstrates
that under mild basic, aqueous conditions peptides can be S-
glycosylated effectively. Based on this approach, the corre-
sponding cysteine and homocysteine containing peptides
should be firstly prepared in order to construct the target struc-
ture 1. For this purpose, the coupling reaction between com-
mercially available N-Boc protected cystine (Boc-Cys-OH)2 and
† This work was supported by the Deutsche Forschungsgemeinschaft,
the Fonds der Chemischen Industrie, and the European Community
(Grant No. HRPN-CT-2000-0001/GLYCOTRAIN).
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 3 1 – 3 3
31

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H-Pro-OBzlؒHCl was performed in DMF using PyBOP as
condensing agent,14 as shown in Scheme 1, leading to the desired
peptide 2 in almost quantitative yield, which was then treated
with 20% TFA in CH2Cl2 to give the free amine 3. The disulfide
bond of 3 was subsequently cleaved by DTT15 in Tris buffer
(pH 8) at room temperature, and ensuing addition of the
alanine thioester 416 to the reaction mixture gave rise to the
desired tripeptide Boc-Ala-Cys-Pro-OBzl 5 by chemical ligation
strategy in 68% overall yield. At the same time, for the sake of
mimicking Asn489 in THP, homocysteine containing dipeptide
8 (Scheme 1) was prepared in excellent yield by coupling of
N-Boc protected homocystine 66a with H-Ala-OBzlؒHCl in the
presence of PyBOP and DIPEA in DMF followed by reduction
of its disulfide bond with PBu3 17 in CHCl3–MeOH–H2O.
Scheme 2 Reagents and conditions: (a) Na2CO3, DMF–H2O, 65%; (b)
H2, 10% Pd/C, EtOAc–EtOH, 94% for 11, 85% for 13, 89% for 15; (c)
H-Ser-OBzlؒHCl, PyBOP, DIPEA, DMF, 88% for 12, 85% for 14; (d)
Na2CO3, TBAHS, EtOAc–H2O, 90%; (e) Zn, Ac2O, Et3N, ultrasonic
bath, 3 h, 87%; (f ) TFA, TESH, CH2Cl2.
Coupling between acid 15 and amine 19 was conducted in the
presence of PyBOP and DIPEA in DMF, as shown in Scheme 3,
and fortunately, S-linked glycopetide 20 was produced in pleas-
ing yield (69%). Subsequently, deprotection of 20 to convert
into final product 1 was achieved in three steps: firstly, deben-
zylation by catalytic hydrogenation; secondly, deacetylation by
treatment with 6 mM NaOMe in MeOH;20 finally, removal of
the Boc protecting group by the action of TFA in the presence
of triethylsilane and water. The synthetic target 1 was purified
by reversed-phase HPLC and characterized by MALDI-MS
and 600 MHz NMR.21
Scheme 1 Reagents and conditions: (a) H-Pro-OBzlؒHCl, PyBOP,
DIPEA, DMF, quant.; (b) TFA, CH2Cl2; (c) DTT, Tris buffer (pH 8.0),
guanidinium hydrochloride, 68% from 3; (d) H-Ala-OBzlؒHCl, PyBOP,
DIPEA, DMF, quant.; (e) PBu3, CHCl3–MeOH–H2O, 98%.
With the requisite peptides 5 and 8 in hand, introduction of
the sugars by their sulfhydryl groups was then investigated
based on our previous work.6a In practice, tripeptide 5 was
treated with lactosyl bromide 918 in the presence of Na2CO3
in DMF–H2O (Scheme 2) and, as expected, the desired glyco-
tripeptide 10 was smoothly produced in 65% yield. Similarly,
glycosylation of dipeptide 8 with N-Troc protected 2-amino
glucosyl bromide 1618 also proceeded smoothly by the action of
Na2CO3, providing the desired glycodipeptide 17 in very high
yield (90%). To access the target molecule 1, the peptide chain
of 10 was then elongated at the C-terminus as shown in Scheme
2. Debenzylation of 10 by catalytic hydrogenation gave the
acid 11 in almost quantitative yield, which was subsequently
coupled with H-Ser-OBzlؒHCl by the action of PyBOP to
produce the desired glycotetrapeptide 12 in high yield (86%).
The removal of the benzyl group from 12 was then conducted
again by catalytic hydrogenation to give rise to the free acid 13,
which was converted into glycopentapeptide 15 in two steps
by treatment with H-Ser-OBzlؒHCl in the presence of PyBOP
and DIPEA in DMF followed by debenzylation with catalytic
hydrogenation (Scheme 2). On the other hand, using the pro-
cedure developed recently,19 the N-Troc protecting group in
glycodipeptide 17 was selectively transformed to an N-Ac group
in compound 1819 in the presence of N-Boc protecting group by
treatment with Zn–Ac2O–Et3N. Then 18 was exposed to 20%
TFA in CH2Cl2 to lead smoothly to the required building block
19, which was used directly in the next reaction.
Scheme 3 Reagents and conditions: (a) PyBOP, DIPEA, DMF, 69%
from 18; (b) H2, 10% Pd/C, EtOAc–EtOH–HOAc; (c) 6 mM NaOMe,
MeOH; (d) TFA, TESH, H2O, 0 ЊC, then RP–HPLC, 37% (3 steps).
In conclusion, as an extension of our previous project, in this
report, an S-linked glycopeptide (1) carrying two sugar moieties
was synthesized in solution phase, which mimics the peptide
sequence Ala484–Ala490 in the Tamm–Horsfall protein (THP)
by replacing of the glycosylation sites Thr485 and Asn489 in
THP with Cys and Hcy respectively. In view of the enhanced
catabolic stability of S-linked glycopeptides, the resultant struc-
ture of 1 is of particular interest for studying its biological
properties, also because in terms of distance between the
sugar and the peptide backbone ligation of sugar to Cys mimics
O-glycan linkage to Thr and linkage to Hcy mimics N-glycan
linkage to Asn, respectively.
Notes and references
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2 S. Tsuboi and M. Fukuda, BioEssays, 2001, 23, 46.
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Rohde and V. Katta, Anal. Chem., 1995, 67, 1442.
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4 For some recent reviews on glycopeptide synthesis, see: (a) A. M.
Jansson, P. M. S. Hilaire and M. Meldal, in Synthesis of peptides
and peptidomemetics, M. Goodman, A. Felix, L. Moroder and
C. Toniolo, Eds., Houben-Weyl, Stuttgart, 2003, 235–332; (b) B. G.
Davis, Chem. Rev., 2002, 102, 579; (c) V. Wittmann, in Glycoscience:
Glycoproteins, B. O. Fraser-Reid, K. Tatsuta and J. Thiem,
Eds., Springer-Verlag, Berlin, 2001, 2253–2352; (d ) H. Herzner,
T. Reipen, M. Schultz and H. Kunz, Chem. Rev., 2000, 100, 4495;
(e) G. Arsequell and G. Valencia, Tetrahedron: Asymmetry, 1999, 10,
3045; ( f ) C. M. Taylor, Tetrahedron, 1998, 54, 11317.
5 L. A. Marcaurelle and C. R. Bertozzi, Chem. Eur. J., 1999, 5,
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6 (a) X. Zhu, K. Pachamuthu and R. R. Schmidt, J. Org. Chem., 2003,
68, 5641; (b) X. Zhu and R. R. Schmidt, Tetrahedron Lett., 2003, 44,
6063; (c) X. Zhu, R. R. Schmidt, Chem. Eur. J., in press.
7 For more examples of S-glycopeptide synthesis, see ref. 4 cited in
ref. 6b.
8 (a) D. Horton and J. D. Wander, Carbohydrates: Chemistry and
Biochemistry, Vol. 4B, W. W. Pigman and D. Horton, Eds.,
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R. R. Schmidt, Liebigs Ann., 1997, 865–872, 1303; (c) M. J. Kiefel,
R. J. Thomson, M. Radovanovic and M. V. Itzstein, J. Carbohydr.
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Tam, Y. A. Lu, C. F. Liu and J. Shao, Proc. Natl. Acad. Sci. USA,
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21 Analytical data for compound 1: 1H NMR (600 MHz, HMQC,
COSY, ROESY, DMSO-d6) δ 8.72–7.77 (NH), 5.43–4.82 (OH), 4.70
(m, 1H, Cysα), 4.41 (d, J = 9.6 Hz, 1H, HGlc-1), 4.35 (m, 3H, Proα,
Hcyα, HGlcNAc-1), 4.29 (m, 1H, Serα), 4.25 (m, 1H, Serα), 4.20 (d,
J = 6.9 Hz, 1H, HGal-1), 4.09 (m, 1H, Alaα), 3.82 (m, 1H, Alaα), 3.75
(dd br, J ≈ 11.5 Hz, 2H, Serβ), 3.68–3.27 (overlapped with water
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peak, 19H, Serβ, Proδ, HGal-4, HGal-2, HGal-5, 2 HGal-6, HGlc-3, HGlc-4
,
HGlc-5, 2 HGlc-6, HGlcNAc-3, HGlcNAc-4, 2 HGlcNAc-6), 3.10 (m, 5H, Hcyγ),
HGlc-2, HGal-3, HGlcNAc-5), 2.71 (s-like, 2H, Cysβ), 2.65 (m, 1H, Proγ),
2.42 (overlapped m, 1H, Proγ), 1.98 and 1.73 (m, 2H, Proβ), 1.83 (m,
2H, Hcyβ), 1.79 (s, 3H, Ac), 1.30 (d, J = 6.9 Hz, 3H, Alaβ), 1.25 (d,
J = 7.3 Hz, 3H, Alaβ). C44H74N8O25S2 (1179.2); MALDI-MS m/z
1200 [M Ϫ 1 ϩ Naϩ], 1216 [M Ϫ 1 ϩ Kϩ].
10 R. L. Easton, M. S. Patankar, G. F. Clark, H. R. Morris and A. Dell,
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O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 3 1 – 3 3
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