Synthesis of H (type 4) trisaccharide, key structural fragment of globo-H and fucosyl-GM1 cancer-associated antigens

Article abstract of DOI:10.1016/j.mencom.2018.07.027

A convenient synthesis of Fucα1-2Galβ1-3GalNAcβ-OCH₂CH₂CH₂NH₂ (trisaccharide H type 4) is described. This glycan is the terminal part of glycosphingolipids globo-H and fucosyl-GM1 known as cancer-associated carb

Full text of DOI:10.1016/j.mencom.2018.07.027

Mendeleev
Communications
Mendeleev Commun., 2018, 28, 421–422
Synthesis of H (type 4) trisaccharide, key structural fragment
of globo-H and fucosyl-GM1 cancer-associated antigens
Galina V. Pazynina, Svetlana V. Tsygankova, Ivan M. Ryzhov,
Alexander S. Paramonov and Nicolai V. Bovin*
M. M. Shemyakin–Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
117997 Moscow, Russian Federation. Fax: +7 495 330 5592; e-mail: professorbovin@yandex.ru
DOI: 10.1016/j.mencom.2018.07.027
OH
OH
HO
HO
HO
A convenient synthesis of Fuca1-2Galb1-3GalNAcb-OCH2-
CH2CH2NH2 (trisaccharide H type 4) is described. This
glycan is the terminal part of glycosphingolipids globo-H
and fucosyl-GM1 known as cancer-associated carbohydrate
antigens.
O
O
O
O
O(CH2)3NH2
NHAc
Me
HO
O
OH
OH
H (type 4) trisaccharide
According to Landsteiner’s blood group rules, all humans regard-
less of blood group do not have antibodies to H-antigen. The
biosynthetic precursors of blood group A and B glycans are
trisaccharide terminations Fuca1-2Galb1-4GlcNAcb known as
H type 2, and Fuca1-2Galb1-3GlcNAcb known as H type 1.
They are mandatory (with the exception of so-called Bombey
phenotype) components of endothelial, epithelial and red blood
cells,1 found in the composition of fairly long chains of glyco-
proteins and glycolipids. Type 3 form of H glycan, Fuca1-
2Galb1-3GalNAca, is also a normal component of mucin-type
glycoproteins, this trisaccharide is attached directly to Ser or
Thr of polypeptide backbone. Type 4 glycan, Fuca1-2Galb1-
3GalNAcb, is known only as the terminal part of glycosphingo-
lipids; two of them, globo-H and fucosyl-GM1 (Figure 1), are
cancer-associated carbohydrate antigens.2
regarding theranostics strategies when the treatment is corrected
in accordance with the permanent diagnostic monitoring. This
means that the reagents will be in demand for the detection and
affinity isolation of anti-H antibodies. Taking this into account,
we propose a simple synthesis of H type 4 trisaccharide in the
spacer-armed (with a primary NH2 group convenient for bio-
conjugation) form.
Glycosylation10 of the glycosyl acceptor 2 (Scheme 1)11
bearing a hydroxyl group at C-3 with 6-O-benzyl galactosyl
donor 1 in the presence of silver triflate,12 tetramethylurea and
molecular sieves 4 Å in dry dichloromethane at room temperature
for 20 h with two-fold excess of the donor related to the acceptor
led to disaccharide 3 (64%). The Zemplen de-O-acetylation and
following acetonation gave 3',4'-O-isopropylidene derivative 4
(42%) with one hydroxyl group at C-2 galactose unit.
Both globo-H and fucosyl-GM1 antigens have been the objects
of attention in the development of antitumor vaccines and
therapeutic monoclonal antibodies2–6 over the past decade. The
blood of healthy individuals contains a big variety of anti-glycan
antibodies,7 including natural (which are not the result of an
immune response) antibodies against H type 4 trisaccharide and
globo-H hexasaccharide.8,9 The presence of natural antibodies
against the motif of H trisaccharide makes it difficult to interpret
the results when developing the therapeutic vaccines, as well as
Glycosylation of disaccharide 4 with benzylated l-fucose
bromide (it was prepared from the corresponding thioglycoside13
by treatment with bromine in anhydrous dichloromethane10) was
carried out in a manner similar to the synthesis of disaccharide 3,
the yield of trisaccharide 5 was 72%. The isopropylidene and
benzylidene groups were removed under acidic conditions (80%
aq. AcOH, 70°C, 2 h), subsequent hydrogenolysis followed by
acetylation and chromatographic purification led to peracetate 6
(54%). In both glycosylations, the side anomers were not detected.
OH
OH
HO
HO
HO
O
O
O
OH
O
O
OH
AcHN
O
O
O
O
OH
Me
HO
HOOC
O
HO
OH
OH
OH
OH
HO
HO
HO
HO
HO
O
OH
OH
OH
O
O
O
O
O
HO
O
OH
NHAc
Fucosyl GM1
HO
HO
AcHN
OH
O
HO
Me
HO
O
O
O
O
OH
HO
HO
OH
OH
OH
Globo-H
Figure 1 Trisaccharide motif Fuca1-2Galb1-3GalNAcb is shared by two cancer-associated glycosphingolipids, globo-H and fucosyl-GM1.
© 2018 Mendeleev Communications. Published by ELSEVIER B.V.
on behalf of the N. D. Zelinsky Institute of Organic Chemistry of the
Russian Academy of Sciences.
– 421 –

Mendeleev Commun., 2018, 28, 421–422
Me
Me
Ph
Ph
Ph
O
O
O
OBn
OBn
OBn
O
O
AcO
AcO
O
AcO
AcO
O
O
O
O
O
O
O
i
ii, iii
+
O
O
Osp
HO
Osp
O
Osp
OH
AcO
OAc
N3
N3
N3
Br
1
2
3 (64%)
4 (42%)
Me
Me
Ph
O
OAc
OAc
OBn
AcO
AcO
AcO
O
O
O
O
O
O
O
O
O
iv
Osp
NHAc
O
Osp
v, vi
vii
Fuc 1-2Gal 1-3GalNAc -O(CH2)3NH2
O
N3
7 (89%)
Me
AcO
Me
BnO
O
O
OAc
6 (54%)
OBn
5 (72%)
OAc
OBn
sp = (CH2)3NHCOCF3
Scheme 1 Reagents and conditions: i, AgOTf, TMM, CH2Cl2, 16 h; ii, 0.05 m MeONa/MeOH, 1 h; iii, Me2C(OMe)2, TsOH; iv, ‘Bn3FucBr’, AgOTf, TMM,
CH2Cl2, 16 h; v, 80% aq. AcOH, 70°C, 2 h; vi, H2, Pd/C, MeOH, then Ac2O/Py, 24 h; vii, 0.1 m MeONa/MeOH, 1 h, then 0.1 m aq. NaOH, 16 h.
Deacetylation of the peracetate 6 and removal of the N-tri-
fluoroacetyl group followed by purification and isolation on
Dowex H+ resin (elution with 1 m aq. NH3) gave fucosylated
3-aminopropyl glycoside 7 (89%). High-resolution 1H NMR
spectroscopy using COSY experiment proved the structure of
peracetylated trisaccharide 6, in particular, J1,2 8.3 Hz for H-1a,
J1,2 7.7 Hz for H-1b in b-configuration and J1,2 3.5 Hz for H-1c
in a-configuration; upfield shifts of proton H-3a (d 4.91 ppm)
and H-2b (d 3.85 ppm) at glycosidic bonds. The structure of target
4 Z. Zhou, G. Liao, S. S. Mandal, S. Suryawanshi and Z. Guo, Chem. Sci.,
2015, 6, 7112.
5 L. M. Krug, G. Ragupathi, C. Hood, M. G. Kris, V. A. Miller, J. R. Allen,
S. J. Keding, S. J. Danishefsky, J. Gomez, L. Tyson, B. Pizzo, V. Baez
and P. O. Livingston, Clin. Cancer Res., 2004, 10, 6094.
6 P. O. Livingston, C. Hood, L. M. Krug, N. Warren, M. G. Kris, T. Brezicka
and G. Ragupathi, Cancer Immunol. Immunother., 2005, 54, 1018.
7 M. E. Huflejt, M. Vuskovic, D. Vasiliu, H. Xu, P. Obukhova, N. Shilova,
A. Tuzikov, O. Galanina, B. Arun, K. Lu and N. Bovin, Mol. Immunol.,
2009, 46, 3037.
8 T. Pochechueva, S. Alam, A. Schötzau, A. Chinarev, N. V. Bovin, N. F.
Hacker, F. Jacob and V. Heinzelmann-Schwarz, J. Ovarian Res., 2017,
10, 8.
1
compound 7 was confirmed by high-resolution H and 13C NMR
spectroscopy and mass spectrometry.†
Trisaccharide 7 in composition of the 600-component glyco-
array14 allowed us to profile human antibodies9 and characterize
the specificity of galectins.15
9 N. Bovin, P. Obukhova, N. Shilova, E. Rapoport, I. Popova, M. Navakouski
C. Unverzagt, M.Vuskovic and M. Huflejt, Biochim. Biophys. Acta, 2012,
1820, 1373.
,
10 G. V. Pazynina, V. V. Severov and N. V. Bovin, Russ. J. Bioorg. Chem.,
2008, 34, 625 (Bioorg. Khim., 2008, 34, 696).
This work was supported by the Russian Science Foundation
(project no. 14-50-00131).
Experiments were partially carried out using the equip-
ment provided by the Institute of Bioorganic Chemistry core
facility (CKP IBCH, supported by the Russian Ministry of
Education and Science, grant no. RFMEFI62117X0018).
11 T. V. Ovchinnikova, A. G. Ter-Grigoryan, G. V. Pazynina and N. V. Bovin.
Russ. J. Bioorg. Chem., 1997, 23, 55 (Bioorg. Khim., 1997, 23, 61).
12 S. Hanessian and J. Banoub, in Methods of Carbohydrate Chemistry,
eds. R. L. Whistler and J. N. BeMiller, Academic Press, NewYork, 1980,
vol. 8, pp. 247–250.
13 H. Lönn, Carbohydr. Res., 1985, 139, 105.
14 G. Pazynina, M. Sablina, T. Ovchinnikova, T. Tyrtysh, S. Tsygankova,
A. Tuzikov, K. Dobrochaeva, N. Shilova, N. Khasbiullina and N. Bovin,
Carbohydr. Res., 2017, 445, 23.
References
15 Y. A. Knirel, H.-J. Gabius, O. Blixt, E. M. Rapoport, N. R. Khasbiullina,
1 S. Marionneau, A. Cailleau-Thomas, J. Rocher, B. Le Moullac-Vaidye,
N. Ruvoën, M. Clément and J. Le Pendu, Biochimie, 2001, 83, 565.
2 S. Hakomori, Adv. Exp. Med. Biol., 2001, 491, 369.
3 S. J. Danishefsky, Y.-K. Shue, M. N. Chang and C.-H. Wong, Acc. Chem.
Res., 2015, 48, 643.
N. V. Shilova and N. V. Bovin, Glycoconjugate J., 2014, 31, 7.
Received: 20th December 2017; Com. 17/5439
†
1H and 13C NMR spectra were recorded on a Bruker AVANCE
1H, H-3b, J3,4 3.4 Hz, J2,3 10.0 Hz), 5.254 (dd, 1H, H-4c, J3,4 3.2 Hz,
J4,5 1.0 Hz), 5.269 (dd, 1H, H-4b, J3,4 3.4 Hz, J4,5 1.0 Hz), 5.320 (dd,
1H, H-3c, J3,4 3.2 Hz, J2,3 10.9 Hz), 5.410 (br.d, 1H, H-4a, J 3.4 Hz),
5.474 (d, 1H, H-1c, J1,2 3.5 Hz), 6.637 (d, 1H, NHAc a, J2,NH 6.7 Hz),
7.203–7.265 (m, 1H, NHCOCF3 sp). Rf 0.36 (C6H12–CHCl3–PriOH,
4:2:1). MS, m/z: 1019 (calc. for [C41H57N2F3O24]H+, m/z: 1019.32).
spectrometer (Bruker BioSpin MRI GmbH) at 303 K. Chemical shifts
for characteristic protons are given with the use of HOD (d 4.750), CHCl3
(d 7.270) as reference. The signals in 1H NMR spectra were assigned using
a technique of spin–spin decoupling (double resonance) and 2D-1H,1H-
COSY experiments. The values of optical rotation were measured on a
Perkin Elmer 341 digital polarimeter at 25°C. Mass spectra were recorded
on a MALDI-TOF Vision-2000 spectrometer using dihydroxybenzoic
acid as a matrix.
1
Fuca1-2Galb1-3GalNAcb-O(CH2)3NH2 7. H NMR (700 MHz, D2O)
d: 1.225 (d, 3H, H-6c, J5,6 6.6 Hz), 1.897–2.009 (m, 2H, CH2 sp), 2.071
(s, 3H, NCOMe), 3.103 (m≈t, 2H, NCH2 sp, J 6.9 Hz), 3.631–3.832 (m,
11H), 3.848 (dd, 1H, H-3b, J3,4 3.4 Hz, J2,3 9.7 Hz), 3.910 (br.d, 1H,
H-4b, J 3.4 Hz), 3.956 (dd, 1H, H-3a, J3,4 3.0 Hz, J2,3 11.0 Hz), 3.998 (dd,
1H, H-2a, J1,2 8.2 Hz, J2,3 11.0 Hz), 4.019–4.067 (m, 1H, OCH sp), 4.135
(br.d, 1H, H-4a, J 2.9 Hz), 4.233 (br.q, 1H, H-5c, J5,6 6.6 Hz), 4.352 (d,
1H, H-1a, J1,2 8.2 Hz), 4.633 (d, 1H, H-1b, J1,2 7.7 Hz), 5.261 (d, 1H,
H-1c, J1,2 4.1 Hz). 13C NMR (176 MHz, D2O) d: 102.66, 102.13, 99.21
(C-1a, C-1b, C-1c), 76.60, 76.02, 75.15, 74.91, 73.64, 71.89, 69.62,
69.18, 68.50, 68.10, 68.06, 66.81 (C-3a, C-4a, C-5a, C-2b, C-3b, C-4b,
C-5b, C-2c, C-3c, C-4c, C-5c, CH2O sp), 61.04 (C-6a, C-6b), 51.47
(C-2a), 37.74 (NCH2 sp), 26.74 (CH2 sp), 22.29 (NCOMe), 15.32
(C-6c). Rf 0.48 (MeOH–1 m aq. Py·AcOH, 5:1). MS, m/z: 587 (calc. for
[C23H42N2O15]H+, m/z: 587.26). [a]546 –58 (c 0.6, MeCN–H2O, 1:1).
1
Compound 6: H NMR (700 MHz, CDCl3) d: 1.159 (d, 3H, H-6c,
J5,6 6.5 Hz), 1.889–1.937 (m, 2H, CH2 sp), 1.993 (2), 2.001, 2.040, 2.051,
2.068, 2.104, 2.156, 2.173 (9s, 9×3H, COMe), 3.217 (ddd, 1H, H-2a,
J1,2 8.3 Hz, J2,3 11.1 Hz, J2,NH 6.7 Hz), 3.485–3.573 (m, 2H, NCH2 sp),
3.751–3.795 (m, 1H, OCH sp), 3.848 (dd, 1H, H-2b, J1,2 7.7 Hz,
J2,3 10.0 Hz), 3.875 (br.t, 1H, H-5b, J 7.1 Hz), 3.893–3.913 (br.t, 1H,
H-5a, J 6.2 Hz), 3.916–3.956 (m, 1H, OCH sp), 4.037 (dd, 1H, H-6'a,
J6',6'' 11.7 Hz, J5,6' 7.2 Hz), 4.124 (dd, 1H, H-6'b, J6',6'' 11.2 Hz, J5,6' 7.1 Hz),
4.156 (dd, 1H, H-6''b, J6',6'' 11.2 Hz, J5,6'' 6.2 Hz), 4.175 (dd, 1H, H-6''a,
J6',6'' 11.7 Hz, J5,6'' 5.1 Hz), 4.505–4.537 (m, 2H, H-5c, H-1b, J1,2 7.7 Hz),
4.911 (dd, 1H, H-3a, J3,4 3.4 Hz, J2,3 11.1 Hz), 4.952 (d, 1H, H-1a,
J1,2 8.3 Hz), 4.980 (dd, 1H, H-2c, J1,2 3.5 Hz, J2,3 10.9 Hz), 4.986 (dd,
– 422 –