Chemoselectively template-assembled glycopeptide presenting clustered cancer related T-antigen

Article abstract of DOI:10.1016/j.tetlet.2003.10.126

The first synthesis of the tumor-associated α-aminooxy T-antigen 1, a relevant recognition motif for the direct construction of multitopic carbohydrate architecture of biological interest is described. The usefulness of this building block is emphasized with the efficient preparation through oxime ligation of a neoglycopeptide cluster, which is readily suitable for evaluating the role of multivalency in antigen presentation to the immune system from an anticancer vaccine perspective.

Full text of DOI:10.1016/j.tetlet.2003.10.126

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 65–68
Chemoselectively template-assembled glycopeptide
presenting clustered cancer related T-antigen
Olivier Renaudet and Pascal Dumy*
LEDSS, UMR-CNRS 5616 & ICGM FR 2607, Universiteꢀ JosephFourier, F-38041 Grenoble Cedex 9, France
Received 17 September 2003; revised 15 October 2003; accepted 22 October 2003
Abstract—The first synthesis of the tumor-associated a-aminooxy T-antigen 1, a relevant recognition motif for the direct con-
struction of multitopic carbohydrate architecture of biological interest is described. The usefulness of this building block is
emphasized with the efficient preparation through oxime ligation of a neoglycopeptide cluster, which is readily suitable for eval-
uating the role of multivalency in antigen presentation to the immune system from an anticancer vaccine perspective.
ꢀ 2003 Elsevier Ltd. All rights reserved.
Mucins comprise a large family of heavily glycosylated
proteins expressed on the cell surface of epithelial tis-
molecules, the control of the alpha anomeric configu-
ration of the O-glycosidic linkage between the peptide
and the carbohydrate moiety remains the major problem
of this methodology, involving many tedious manipu-
lations of orthogonal protecting groups and activation
steps.7 As an attractive alternative, we and others have
shown more recently that chemoselective ligation fol-
lowing an oxime-based strategy is of major interest for
the convergent assembly of complex macromolecules.8
In this context, we developed an efficient route providing
aminooxy carbohydrate recognition motifs at both the
alpha and beta anomeric carbon positions9 (comprising
the mucin-related TN antigen), which we assembled
chemoselectively into oligonucleotides10 and the cyclo-
decapeptide RAFT template.11 Surprisingly, whereas
complex aminooxylated cancer-related antigens (sialyl
LeX or sialyl TN) have been obtained by chemical or
chemoenzymatic methods,8c;12 the preparation of
T-antigen is still not described so far (Fig. 1).
sues.1 They are commonly composed of the a-
D-Gal-
NAc residue as the core fragment attached in a clustered
mode to the hydroxyl side chain of serine or threonine of
glycoproteins. These typical structural units are involved
in various biological processes and also represent one of
the most important class of antigenic markers related to
autoimmune or infectious diseases and cancers, making
them attractive targets for carbohydrate-based tumor
therapy.2 In particular, molecular constructions pre-
senting clustered cancer-related carbohydrate antigens
mimicking the cell surface such as glycopeptide-based
vaccines3 have promising potential for clinical trials and
their synthetic access remains a crucial challenge for the
future.
Among all the identified mucin-associated antigens, the
disaccharide b-
D
-Gal(1 fi 3)-a-D-GalNAc (or T-anti-
gen) represents one of the most prevalent structures to
be abundantly expressed on carcinoma malignancies of
the colon and the prostate.4 Thus, many synthetic
methods regarding the preparation and the incorpora-
tion of sucha moiety into peptidic backbone have been
described,5 including the powerful glycal assembly and
To this end, we herein report the first synthesis of the
a-aminooxy mucin-related T-antigen and subsequent
2b;6
OH
OH
HO
O
HO
HO
ꢀcassetteꢁ approachdeveloped by Danishefsky et al.
However, due to the polyfunctionality of the target
O
O
OH
AcHN
O
NH2
Keywords: T-antigen; glycopeptide; multivalency; oxime ligation.
* Corresponding author. Tel.: +33-476-635-545; fax: +33-476-514-382;
e-mail: pascal.dumy@ujf-grenoble.fr
1
Figure 1. Alpha aminooxy T-antigen.
0040-4039/$ - see front matter ꢀ 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.10.126

66
O. Renaudet, P. Dumy / Tetrahedron Letters 45 (2004) 65–68
Ph
OAc
O
OH
OAc
AcO
AcO
HO
HO
O
AcO
AcO
a
b
c
O
O
O
O
HO
N3
ONO2
N3 OMe
N3
OMe
4
2
3
5 (α), 6 (β)
OAc
AcO
AcO
O
7
NH
1
d
AcO
O
CCl3
Ph
k
O
OAc
OAc
O
OAc
AcO
AcO
AcO
O
OAc
AcO
AcO
OAc
AcO
O
AcO
AcO
O
O
e
O
O
i
O
O
O
N3
O
OAc
N3
OAc
R
R
OMe
OAc
O
N
10, R = αOMe or 11, R = βOMe
12, R = α/βOAc
8 (α), 9 (β)
f
O
15, R = N3
16, R = NHAc
g
h
j
13, R = α/βOH
14, R = α/βF
Scheme 1. Reagents and conditions: (a) CAN, NaN3, CH3CN, )15 °C, 77%; (b) MeONa/MeOH, 100% (a=b, 0.3/1); (c) PhCH(OMe)2, p-TsOH,
CH3NO2, 85%; (d) TMSOTf, CH2Cl2, )30 °C, 47% for 8 (from 5) or 50% for 9 (from 6); (e) (i) aq AcOH 80%, 45 °C; (ii) Ac2O, pyridine, 85–87% for
10 (from 8) or for 11 (from 9); (f) Ac2O/H2SO4 40/1, 0 °C, 95%; (g) ethylenediamine, AcOH, THF, 100% (a=b ratio, 1/1, determined by integration of
the anomeric protons in the 1H spectrum); (h) DAST, THF, 80% (a=b ratio, 1/1, determined by integration of the anomeric protons in the 1H
spectrum); (i) BF3ÆEt2O, PhtOH, TEA, 40% (+30% of b anomer); (j) H2, Pd/C, MeOH/Ac2O, 45%; (k) MeNHNH2, 70%.
glycopeptide cluster formation through oxime ligation
as a preliminary study for designing synthetic anti-
tumoral vaccines.
We decided to follow a synthetic route based on the
strategy described initially by Paulsen et al. (Scheme
1).5f Two protected methyl galactosyl acceptors 5 or 6
were prepared from triacetyl galactal 2 according to
the classical azidonitration reaction using cerium
ammonium nitrate and sodium azide in acetonitrile.13
Subsequent treatment of the azido-nitrate mixture 3
with sodium methoxide in methanol14 followed by the
protection of the resulting anomeric mixture of 4 affor-
ded the 4,6-O-benzylidene protected methyl galactosides
5 and 6, which were separated by silica gel chromato-
graphy and crystallized from diethyl ether/pentane
mixture (Fig. 2).15
Figure 2. ORTEP drawings of glycosyl acceptors 5 (C14H17N3O5,
FW ¼ 307.31, orthorhombic, it P212121, a ¼ 5:085ð5Þ, b ¼ 11:820ð2Þ,
3
c ¼ 23:990ð4Þ A, V ¼ 1441ð3Þ A , Z ¼ 4, Dcalcd ¼ 1:416 g cmÀ3, R ¼
ꢁ
ꢁ
0:045, Rw ¼ 0:041) and 6 (C14H17N3O5, FW ¼ 307.31, orthorhombic,
3
ꢁ
ꢁ
P212121, a ¼ 5:055ð4Þ, b ¼ 12:790ð3Þ, c ¼ 22:568ð5Þ A, V ¼ 1456ð4Þ A ,
Z ¼ 4, Dcalcd ¼ 1:401 g cmÀ3, R ¼ 0:059, Rw ¼ 0:027).15
Interestingly, bothglycosyl acceptors were suitable for
the preparation of the desired compound. Indeed, the
Koenigs–Knorr-like coupling reaction between 5 or 6
and the trichloroacetimidate glycosyl donor 716 by
treatment withtrimetyhlsilyl triflate as Lewis acid
catalyst in dichloromethane gave the corresponding
disaccharides 8 or 9 withcomparable moderate yields
(47–50%). Further mild acidic deprotection with aque-
ous acetic acid and O-acetylation at positions 4 and 6 of
8 and 9 provided the methyl glycosides 10 and 11 in high
yields. These two compounds were then converted into
the corresponding peracetylated derivatives 12, using a
solution of sulfuric acid and acetic anhydride (1/40),5f as
an anomeric mixture, which was used without further
separation. The regioselective anomeric deacetylation
was subsequently achieved with a solution of ethylene-
diamine and acetic acid (1/1, v/v) in THF at room
temperature17 to obtain the anomerically free derivative
13.
We previously reported that N-hydroxyphthalamide
(PhtOH) provides the most suitable precursor for in-
troducing an aminooxy function at the anomeric posi-
tion of carbohydrates.9 After activation of 13 witha
fluoride atom using DAST, the glycosylation reaction
was realized in dichloromethane in the presence of tri-
ethylamine and PhtOH by treatment with BF3ÆEt2O as
promotor. The reaction occurred within 15 min in 70%
yield and the corresponding alpha and beta phthalam-
ido derivatives (a=b ratio: 4/3) were separated by silica
gel chromatography. The azido function of the desired

O. Renaudet, P. Dumy / Tetrahedron Letters 45 (2004) 65–68
67
alpha compound 15 was then converted into the N-
acetylated amide group by a controlled catalytic
hydrogenation in methanol/acetic anhydride at room
temperature to prevent any N–O cleavage under the
reductive conditions.9 The complete deprotection and
aminooxy formation of the acetylated derivative 16 was
finally achieved in methylhydrazine as solvent to obtain
the stable alpha aminooxylated T-antigen 1 in 70% yield
after chromatography and lyophilization. Multidimen-
sional NMR and mass spectrometry analysis confirmed
the purity and the structure of the desired compound
1.18
To assess the ability of 1 to form a multitopic carbo-
hydrate architecture, we used the RAFT template whose
utility as a polyvalent presentation scaffold for sugar
recognition motifs has been shown previously with
relevant lectins.11 Thus, the RAFT 17 (Scheme 2)
presenting clustered aldehydes was prepared following
the classical procedure using serine as precursor of the
glyoxo-aldehyde functions, which were liberated by
ꢁ
Figure 3. (A) HPLC profile (Nucleosil 100 A 5 lm C18 particles,
250 · 4.6 mm; solvent B: 0.09% TFA in 90% acetonitrile and solvent A:
0.09% TFA, 1 mL minÀ1, linear gradient 95:5 A/B to 60:40 A/B in
30 min detection k ¼ 214 and 250 nm) of crude reaction mixture of
oxime ligation between 1 and 17 after 2 h: RAFT 17 (dotted line):
Rt ¼ 16:9 min; RAFT/T-antigen conjugate 18: Rt ¼ 17:2 min. (B) ESI-
MS (positive mode) of RAFT/T-antigen conjugate 18 (Mcalcd for
C108H174N22O58: 2708.7).
19
oxidative cleavage withsodium periodate.
The chemoselective oxime ligation between 17 and a
slight excess of 1 was finally performed in aqueous
sodium acetate buffer at room temperature and was
monitored by reverse-phase HPLC. After complete
disappearance of 17 (Fig. 3A), semi-preparative HPLC
purification afforded the stable tetravalent RAFT/
T-antigen conjugate 18 (70% yield), which was charac-
terized by mass spectrometry (Fig. 3B). This neoglyco-
conjugate 18 is readily suitable for further biological
recognition studies.
In conclusion, we report in this paper the first synthetic
route to the alpha aminooxy mucin-related T-antigen 1
starting from two glycosyl acceptors 5 or 6 and the tri-
chloroacetimidate galactosyl donor 7. Suchmodified
carbohydrate antigens are potentially useful for the
rapid and direct assembly of multitopic neoglycoconju-
gate architectures of biological interest. Indeed, we have
shown that the subsequent incorporation of 1 into the
cyclodecapeptidic RAFT template 17 presenting alde-
hydes using an oxime-based strategy efficiently gave a
new tetravalent neoglycopeptide T-antigen cluster 18,
which could provide a useful tool for evaluating the role
of multivalency in antigen presentation to the immune
system. Recognition studies withrelevant antibodies
and lectins involved in pathogen processes are currently
being investigated in our laboratory in the context of
synthetic anticancer vaccine research. The results will be
reported in due course.
O
O
H
H
NH
NH
O
O
O
O
H
H
NH K
NH K
O
O
OH
OH
HO
O
HO
HO
G
A
P
O
O
+
K
K
A
G
P
OH
AcHN
O
NH2
1
17
Acknowledgements
oxime ligation
a
This work was supported by the Association pour la
Recherche contre le Cancer (ARC), the Centre National
pour la Recherche Scientifique (CNRS) for specific
action (AIP) and the Institut Universitaire de France
(IUF). We also acknowledge the MENRT for grant no
98-4-23548 to O.R. We also thank C. Philouze for per-
forming the X-ray analyses.
T-antigen
T-antigen
T-antigen
O
OH
OH
HO
O
HO
HO
O
O
N
O
O
N
O
T-antigen
O
N
OH
AcHN
NH
NH
O
O
N
NH K
NH K
A
P
G
References and Notes
K
K
A
G
P
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18
Scheme 2. Reagents and conditions: (a) 0.1 M sodium acetate buffer
pH 4.0, 2 h, 70% after semi-preparative RP-HPLC.

68
O. Renaudet, P. Dumy / Tetrahedron Letters 45 (2004) 65–68
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18. 1H NMR (300 MHz, D2O):
d
ppm 4.95 (d, 1H,
3
3
J1 ;2 ¼ 4:0 Hz, H-10), 4.45 (d, 1H, J1;2 ¼ 7:7 Hz, H-1),
0
0
3
4.39 (d, 1H, J2 ;3 ¼ 11:4 Hz, H-20), 4.24 (bd, 1H,
0
0
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J3 ;4 ¼ 2:8 Hz, H-40), 4.02 (bt, 1H, J5 ;6 ¼ 6:3 Hz, H-50),
3
3
0
0
0
0
3.97 (dd, 1H, J3 ;4 ¼ 2:8 Hz, J2 ;3 ¼ 11:4 Hz, H-30), 3.91
3
3
0
0
0
0
3
(bd, 1H, J3;4 ¼ 3:2 Hz, H-4), 3.80–3.72 (m, 4H, H-6, H-
60), 3.66–3.64 (m, 1H, H-5), 3.61 (dd, 1H, J3;H ¼ 3:2 Hz,
3
3J2;3 ¼ 9:9 Hz, H-3), 3.50 (dd, 1H, J1;2 ¼ 7:7 Hz, J2;3
¼
3
3
9:9 Hz, H-2), 2.03 (s, 3H, CH3); 13C NMR (75 MHz,
D2O): d ppm 175.0 (C@O), 105.0 (C-1), 101.1 (C-10), 77.3
(C-30) 75.3 (C-5), 72.9 (C-3), 71.1, 70.9 (C-2, C-50), 69.1,
68.9 (C-4, C-40), 61.5, 61.3 (C-6, C-60), 48.2 (C-20), 22.3
(CH3); MS (FABþ, glycerol+NaCl): Mcalcd ¼ 398, m=z:
[M+H+K]þ ¼ 438.
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