Synthesis of trisaccharide 6' SiaLec and its 6-O-Su derivative

Article abstract of DOI:10.1016/j.mencom.2012.06.007

The synthetic approach to 6'SiaLe^c and its 6-O-Su derivative comprised α-sialylation of a protected Le^c derivative, 2,3-di-OAcGalβ1-3(3-OAc-6-OBn)GlcNAcβ1-Osp (68% yield) as the key stage. Deprotection of the obtained trisaccharide (three stages, 79% overall yield) led to Neu5Acα2-6Galβ1-3GlcNAcβ1-Osp; partial deprotection followed by selective sulfation and total deprotection - to Neu5Acα2-6Galβ1-3(6-O-Su)GlcNAcβ1-Osp (four stages, 72% overall yield).

Full text of DOI:10.1016/j.mencom.2012.06.007

Mendeleev
Communications
Mendeleev Commun., 2012, 22, 194–195
Synthesis of trisaccharide 6'SiaLec and its 6-O-Su derivative
Galina V. Pazynina, Inna S. Popova, Ivan M. Belyanchikov,
Alexander B. Tuzikov 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: bovin@carb.ibch.ru
DOI: 10.1016/j.mencom.2012.06.007
The synthetic approach to 6'SiaLec and its 6-O-Su derivative comprised a-sialylation of a protected Lec derivative, 2,3-di-OAcGalb1-
3(3-OAc-6-OBn)GlcNAcb1-Osp (68% yield) as the key stage. Deprotection of the obtained trisaccharide (three stages, 79% overall
yield) led to Neu5Aca2-6Galb1-3GlcNAcb1-Osp; partial deprotection followed by selective sulfation and total deprotection – to
Neu5Aca2-6Galb1-3(6-O-Su)GlcNAcb1-Osp (four stages, 72% overall yield).
OBn
Mammals have two enzymes capable of synthesizing Neu5-
Aca2-6Gal motif in glycoproteins and glycolipids, ST6Gal-I and
ST6Gal-II. Both sialyltransferases specifically elongate Galb1-
4GlcNAc terminated substrates, whereas isomeric substrate, Galb1-
3GlcNAc (Lec) motif was found to be ~100 times less potent in
in vitro assays.1 Therefore, question about possibility of in vivo
synthesis of Neu5Aca2-6Galb1-3GlcNAc terminated glycans
remains unclear. Classical works on structures and glycomics also
give no certain information about abundance of this and related
Neu5Aca2-6Galb1-3(6-O-Su)GlcNAc motifs (Su = SO3H),
mass spectrometry data cannot differentiate Neu5Aca2-6Galb1-
3GlcNAc vs. common Neu5Aca2-6Galb1-4GlcNAc without
standards suitable for MS/MS experimentation. Here, we describe
the synthesis of trisaccharides Neu5Aca2-6Galb1-3GlcNAc and
Neu5Aca2-6Galb1-3(6-O-Su)GlcNAc capable of solving this
problem. Additionally, these trisaccharides in a spacer-armed
form are convenient substrates for the study of a1-4 fucosyl-
transferases – enzymes synthesizing glycans with terminations
Neu5Aca2-6Galb1-3(Fuca1-4)GlcNAc and Neu5Aca2-6Galb1-
3(Fuca1-4)(6-O-Su)GlcNAc – analogues of SiaLea, well known
as cancer-associated antigen CA-19-9. Particular fucosyltrans-
ferase(s) responsible in mammalian cells for biosynthesis of these
glycans have not been identified yet.
O
HO
HO
O(CH2)3NHCOCF3
NHAc
1
i
OBn
OAc
AcO
AcO
O
O
HO
O(CH2)3NHCOCF3
NHAc
2 (68%)
O
AcO
ii, iii, iv, v
OBn
OH
HO
AcO
O
AcO
O
O(CH2)3NHCOCF3
NHAc
O
AcO
3 (67%)
OAc
Cl
AcO
vi, iv (68%),
vii (88%)
O
AcO
AcHN
COOMe
Hgii-catalysed glycosylation of 3,4-diol 1 with acetobromo-
galactose proceeded preferably with formation of Lec-derivative
2 (68%). Subsequent deacetylation, benzylidenation, acetylation,
and debenzylidenation led to 4',6'-diol 3 in 67% total yield. Gly-
cosylation of the latter compound with neuraminic acid chloride 4
under conditions described earlier2,3 (catalysis with Ag2CO3)
afforded protected 6'-sialylated Lec derivative (68%). Hydro-
genolysis of benzyl ether moiety in this compound led to
trisaccharide 5 (88%). The presence of one OH group in GlcNAc
fragment allowed us to perform efficient sulfation with Py∙SO3.4, 5
Further deprotection (treatment with MeONa/MeOH, NaOH/H2O,
neutralization with AcOH), purification on Sephadex LH-20
(MeCN–0.01 m aq. Py∙AcOH, 1:1, pH 6), and ion-exchange to
Na+ on Dowex gave target 6-O-Su derivative 7 (82%).
AcO
4
OAc
AcO
COOMe
AcO
AcO
AcHN
O
OH
O
AcO
O
AcO
O
O
O(CH2)3NHCOCF3
NHAc
AcO
OAc
5
ii, ix
viii, ii, ix
Neu5Ac 2-6Gal 1-3GlcNAc O(CH2)3NH2
6 (90%)
Similar deprotection of 5 gave the second target compound 6
(90%) isolated using cation-exchange chromatography on Dowex
H+ resin (elution with 1 m aqueous pyridine).
Neu5Ac 2-6Gal 1-3(6-O-Su)GlcNAc O(CH2)3NH2
7 (82%)
The structure of all synthesized compounds was confirmed by
high resolution 1H NMR spectroscopy.†
Scheme 1 Reagents and conditions: i, Ac4GalBr, Hg(CN)2/HgBr2, MeNO2/
MePh, 15 h; ii, 0.1 m NaOMe/MeOH, 0.5 h; iii, PhCH(OMe)2, TsOH, MeCN,
2 h; iv, Ac2O, Py, 12 h; v, 80% aq. AcOH, 70°C, 2 h; vi, Ag2CO3, CH2Cl2,
70 h; vii, Pd/C, H2, MeOH, 2 h; viii, Py∙SO3/Py, room temperature, 1 h;
ix, 0.1 m NaOH/H2O, 15 h.
This work was supported by the Russian State Contract
no. 16.512.11.2039 (code 2011-1.2-512-013-008).
© 2012 Mendeleev Communications. All rights reserved.
– 194 –

Mendeleev Commun., 2012, 22, 194–195
4 G. V. Pazynina, V. V. Severov, M. L. Maisel, I. M. Belyanchikov and N. V.
References
Bovin, Mendeleev Commun., 2008, 18, 238.
5 G. Pazynina, M. Sablina, M. Mayzel, V. Nasonov, A. Tuzikov and N. Bovin,
Glycobiology, 2009, 19, 1078.
1 J. Weinstein, U. de Souza-e-Silva and J. C. Paulson, J. Biol. Chem., 1982,
257, 13845.
2 G. Pazynina, A. Tuzikov, A. Chinarev, P. Obukhova and N. Bovin, Tetra-
hedron Lett., 2002, 43, 8011.
3 G. Pazynina, V. Nasonov, I. Belyanchikov, R. Brossmer, M. Maisel,
A. Tuzikov and N. Bovin, Int. J. Carbohydr. Chem., 2010,Article ID 594247,
doi:10.1155/2010/594247.
Received: 7th March 2012; Com. 12/3890
For 5: 1H NMR (CDCl3, 800 MHz) d: 1.825–1.922 (m, 2H, CspH2), 1.938
(dd ≈ t, 1H, H-3''', J 12.6 Hz), 1.906, 1.971, 2.007, 2.036, 2.059, 2.067,
†
1H NMR spectra were recorded on a Bruker AVANCE 600, 700 and
ax
2,080, 2.144, 2.169, 2.210 (10s, 10×3H, COMe), 2.569 (dd, 1H, H-3''',
eq
800 MHz spectrometer at 303 K. Chemical shifts d for characteristic
protons are given in ppm with the use of HOD (d 4.750), CHCl3 (d 7.270),
or CHD2OD (d 3.500) as reference. The signals in 1H NMR spectra were
assigned using a technique of spin–spin decoupling (double resonance)
J3ax,3eq 12.9 Hz, J3eq,4 4.6 Hz), 3.272–3.311 (m, 1H, NCspH), 3.373 (dd,
1H, H-6''a, J5,6a 7.8 Hz, J6a,6b 10.8 Hz), 3.512 (ddd, 1H, H-5', J4,5 10.0 Hz,
J5,6a 4.5 Hz, J5,6b 2.3 Hz), 3.522–3.584 (m, 2H, NCspH, OCspH), 3.609
(dd, 1H, H-6'a, J5,6a 4.5 Hz, J6a,6b 12.6 Hz), 3.753 (dd, 1H, H-6'b, J5,6b 2.3 Hz
,
and 2D H,1H COSY experiments. The values of optical rotation were
1
J6a,6b 12.6 Hz), 3.754 (dd, 1H, H-6''b, J5,6b 6.0 Hz, J6a,6b 10.8 Hz), 3.821
(s, 3H, OMe'''), 3.851 (br.q, 1H, H-2', J 9.0 Hz), 3.905 (br.t, 1H, H-5'',
J 7.0 Hz), 3.976–4.014 (m, 2H, OCspH, H-5''', J4,5 10.5 Hz, J5,NH 9.9 Hz,
J5,6 10.8 Hz), 4.056 (dd, 1H, H-9'''b, J8,9b 5.4 Hz, J9a,9b 12.1 Hz), 4.109 (dd,
1H, H-6''', J5,6 10.8 Hz, J6,7 1.4 Hz), 4.181 (dd ≈ t, 1H, H-3', J2,3 9.1 Hz,
J3,4 9.3 Hz), 4.373 (dd, 1H, H-9'''a, J8,9a 2.8 Hz, J9a,9b 12.1 Hz), 4.667 (d,
1H, H-1', J1,2 7.9 Hz), 4.689 (d, 1H, H-1'', J1,2 7.6 Hz), 4.903 (ddd, 1H,
H-4''', J3eq,4 4.6 Hz, J3ax,4 12.2 Hz, J4,5 10.5 Hz), 4.918 (dd, 1H, H-4',
J3,4 9.3 Hz, J4,5 10.0 Hz), 4.992 (dd, 1H, H-3'', J2,3 10.4 Hz, J3,4 3.4 Hz),
5.024 (dd, 1H, H-2'', J2,3 10.4 Hz, J1,2 7.6 Hz), 5.115 (d, 1H, NH''',
JNH,5 9.9 Hz), 5.305 (dd, 1H, H-7''', J6,7 1.7 Hz, J7,8 9.4 Hz), 5.323 (ddd,
1H, H-8''', J7,8 9.4 Hz, J8,9a 2.8 Hz, J8,9b 5.4 Hz), 5.409 (dd ≈ d, 1H, H-4'',
J 3.0 Hz), 6.755 (br.s, 1H, NH'), 7.649 (m ≈ s, 1H, NHCOCspF3).
For 6: 1H NMR (D2O, 700 MHz) d: 1.721 (dd ≈ t, 1H, H-3a''x', J 12.1 Hz);
1.966–2.005 (m, 2H, CspH2), 2.060 (s, 2×3H, NCOMe), 2.729 (dd, 1H,
H-3e''q', J3ax,3eq 12.4 Hz, J3ex,4 4.7 Hz), 3.123 (m ≈ t, 2H, NCspH2, J 7.0 Hz);
3.525–3.925 (m, 17H), 3.942 (dd ≈ d, 1H, H-4'', J 3.3 Hz), 3.985–4.035
(m, 2H), 4.040–4.072 (m, 1H, OCspH), 4.416 (d, 1H, H-1'', J1,2 7.8 Hz),
4.581 (d, 1H, H-1', J1,2 8.5 Hz). [a]D –36.0 (c 0.45; MeCN–H2O, 1:1).
MS, m/z: 732 [M]+ (calc. for C28H49N3O19, m/z: 731.71 [M]+).
measured on a Jasco DIP-360 digital polarimeter at 25°C. Mass spectra
were recorded on a MALDI-TOF Vision-2000 spectrometer using dihydroxy-
benzoic acid as a matrix.
1
For 2: H NMR (CDCl3, 600 MHz) d: 1.757–1.922 (m, 2H, CspH2),
1.992, 2.014, 2.025, 2.091, 2.166 (5s, 5×3H, COMe), 3.165 (ddd, 1H,
H-2', J1,2 8.3 Hz, J2,3 10.2 Hz, JNH,2 7.2 Hz), 3.40–3.51 (m, 3H, NCspH2,
H-4'), 3.529 (ddd, 1H, H-5', J4,5 9.6 Hz, J5,6a 1.9 Hz, J5,6b 6.3 Hz), 3.636
(dd, 1H, H-6'b, J5,6b 6.3 Hz, J6a,6b 10.8 Hz), 3.656–3.692 (m, 1H, OCspH),
3.815 (br.s, 1H, 4-OH), 3.844 (dd, 1H, H-6'a, J5,6a 1.9 Hz, J6a,6b 10.8 Hz),
3.888–3.923 (m, 1H, OCspH), 4.010 (ddd ≈ t, 1H, H-5'', J 6.6 Hz), 4.130
(m ≈ d, 2H, H-6''a, H-6''b, J 6.6 Hz), 4.208 (dd, 1H, H-3', J2,3 10.2 Hz,
J3,4 8.3 Hz), 4.558–4.601 (m, 3H, H-1'', CH2Ph), 4.889 (d, 1H, H-1',
J1,2 8.3 Hz), 5.013 (dd, 1H, H-3'', J2,3 10.5 Hz, J3,4 3.4 Hz), 5.232 (dd, 1H,
H-2'', J1,2 8.0 Hz, J2,3 10.5 Hz), 5.387 (dd ≈ d, 1H, H-4'', J 2.9 Hz), 5.795
(d, 1H, NH', JNH,2 7.2 Hz), 7.27–7.36 (m, 6H, Ph, NHCOCF3).
1
For 3: H NMR (CDCl3–CD3OD 3:1, 700 MHz) d: 1.886–2.015 (m,
2H, CspH2), 2.149, 2.154, 2.201, 2.212 (4s, 4×3H, COMe), 3.360–3.398
and 3.590–3.628 (2m, 2×1H, NCspH2), 3.64–3.68 (m, 2H, H-5', H-5''),
3.684 (dd, 1H, H-6'a, J5,6a 5.8 Hz, J6a,6b 10.8 Hz), 3.718 (dd, 1H, H-6'b,
J5,6b 2.8 Hz, J6a,6b 10.8 Hz), 3.778–3.804 (m, 1H, OCspH), 3.879 (dd, 1H,
H-6''a, J5,6a 5.2 Hz, J6a,6b 11.4 Hz), 3.917 (dd, 1H, H-6''b, J5,6b 6.6 Hz,
J6a,6b 11.4 Hz), 3.973 (dd, 1H, H-3', J2,3 10.0 Hz, J3,4 8.6 Hz), 4.011-4.041
(m, 1H, OCspH), 4.149–4.177 (m, 2H, H-2', H-4''), 4.536 (d, 1H, H-1',
J1,2 8.3 Hz), 4.654 and 4.685 (2d, 2×1H, CH2Ph, Jhem 11.8 Hz), 4.699 (d,
1H, H-1'', J1,2 7.9 Hz), 4.943 (dd, 1H, H-3'', J3,4 3.4 Hz, J2,3 10.4 Hz),
4.990 (dd ≈ t, 1H, H-4', J 9.4 Hz), 5.285 (dd, 1H, H-2'', J2,3 10.4 Hz,
J1,2 7.9 Hz), 7.41–7.50 (m, 5H, Ph).
For 7: 1H NMR (D2O, 700 MHz) d: 1.723 (dd ≈ t, 1H, H-3’’’, J 12.1 Hz);
ax
1.952–2.032 (m, 2H, CspH2), 2.058 (s, 2×3H, NCOMe), 2.750 (dd, 1H,
H-3e''q', J3ax,3eq 12.4 Hz, J3eq,4 4.7 Hz), 3.144 (m ≈ t, 2H, NCspH2, J 6.8 Hz),
3.546 (dd, 1H, H-2'', J1,2 7.9 Hz, J2,3 9.8 Hz), 3.58–3.99 (m, 17H), 4.006–4.037
(m, 1H, OCspH), 4.266 (dd, 1H, H-6'a, J6a,6b 11.3 Hz, J5,6a 6.1 Hz), 4.438
(d, 1H, H-1'', J1,2 7.9 Hz), 4.442 (dd, 1H, H-6'b, J6a,6b 11.3 Hz, J5,6b 1.9 Hz),
4.597 (d, 1H, H-1', J1,2 8.5 Hz). [a]D –22.5 (c 0.4; MeCN–H2O, 1:1). MS,
m/z: 834 [M]+ (calc. for C28H49N3NaO22S, m/z: 833.76 [M]+).
– 195 –