Efficient synthesis of two HNK-1 related pentasaccharides

Article abstract of DOI:10.1055/s-2003-40324

Two pentasaccharides, representative of those found on complex N-glycans, were synthesized for use as potential substrates for sulfotransferases. The synthesis was achieved by the addition of a disaccharide donor β-D-GlcA(1→3)α-D-Gal-trichloroacetimidate

Full text of DOI:10.1055/s-2003-40324

LETTER
1315
Efficient Synthesis of Two HNK-1 Related Pentasaccharides
Efficient
S
ynth
r
esis of
T
e
w
oHN
K
-
d
d
Pe
e
ntasacchari
r
a
The Burnham Institute, 10901 North Torrey Pines Rd., La Jolla, CA, USA 92122
b
University of Alberta, Department of Chemistry, Edmonton, Alberta, Canada T6G 2G2
Fax +1(780)4927705; E-mail: ole.hindsgaul@ualberta.ca
Received 23 January 2002
Dedicated to the memory of Professor Raymond U. Lemieux.
OH-4 using trimethylorthobenzoate (quant.) afforded
Abstract: Two pentasaccharides, representative of those found on
complex N-glycans, were synthesized for use as potential substrates
for sulfotransferases. The synthesis was achieved by the addition of
a disaccharide donor b-D-GlcA(1→3)a-D-Gal-trichloroacetimidate
compound 6 following the hydrolysis of the intermediate
1
3,4-orthobenzoate (Scheme 1). H NMR (CDCl3) con-
firmed that O-4 was benzoylated (H-4: 5.7 ppm, J3,4 = 3.5
to the two acceptor trisaccharides b-D-GlcNAc(1→6)a-D- Hz, J4,5<1.0 Hz, H-3: 3.7 ppm, J2,3 = 10.0 Hz). Coupling
of 6 and 72 (1.2 equiv), using TMSOTf, provided the
target disaccharide 8 in 84% yield. The new glycosidic
Man(1→6)b-D-Man-O-octyl (15) and b-D-GlcNAc(1→2)a-D-
Man(1→6)b-D-Man-O-octyl (14). After deprotection, the two pen-
tasaccharides 1 and 2 were characterized by 1H NMR spectroscopy.
1
linkage was confirmed by H NMR (CDCl3)-: H-1¢: 5.4
Key words: pentassacharides, HNK-1, N-glycans
ppm J1¢,2¢ = 7.5 Hz. Four more steps afforded 9: catalytic
hydrogenation [Pd(OH)2, MeOH, 95%], benzoylation
(BzCl, pyridine, 70%) and activation to provide the
Complex carbohydrates, such as the HNK-1 glycan trichloroacetimidate derivative (CAN, toluene, aceto-
(sulfo→3GlcAb1→3Galb1→4GlcNAc→R) can mediate nitrile, water, (75%) then CCl3CN, DBU, CH2Cl2, 80%)
important cellular functions. The HNK-1 epitope is ex- (Scheme 2).
pressed on adhesion molecules in the nervous system
where it seems to play a role in cell-cell and cell-substra-
OAc
OAc
AcO
AcO
AcO
AcO
a
tum interactions.1 This structure can be present on differ-
ent branches of N-linked glycans, and it is not clear
whether there is a branch-specificity for the sulfotrans-
ferase that completes the epitope. We therefore decided to
synthesize pentasaccharides 1 and 2, representing two of
the branches of N-linked oligosaccharides for use in the
study of sulfotransferase specificity.
O
O
OAc
OMP
OAc
OAc
3
OH
OBn
O
O
HO
d, e
b, c
O
O
OMP
OMP
HO
4
OBn
5
OH
OBn
BzO
HO
In our synthetic scheme, we envisaged building the pen-
tasaccharide backbone by using as the key step the glyco-
sylation reaction between the imidate donor 9 and the
acceptor trisaccharides 14 and 15, respectively. Com-
pound 9 was prepared by the condensation between the
glucuronic acid donor 7 and the galacto derivative 6.
Compounds 14 and 15 in turn, were prepared by the addi-
tion of the glucosamine N-trichloroacetyl donor 10 to two
different acceptor disaccharides, with either OH-2 (12) or
OH-6 (13) of the a Man (1→6)b Man sequence groups
free (Figure 1).
f, g
O
OMP
OBn
6
Scheme 1 Synthesis of 6: a) MPOH, CH2Cl2, TfOH, molecular sie-
ves 4 Å, 1 h (90%); b) MeOH, NaOMe, overnight (quant.); c) 2,2-di-
methoxy propane, CSA, DMF, overnight (79%); d) BnBr, NaH,
DMF, 5 h, (77%); e) AcOH, H2O, 2 h, (73%); f) trimethylorthoben-
zoate, p-TsOH, PhCH3, 1 h, g) AcOH, H2O, 10 min (2 steps, quant.).
Compound 11, a GlcNAc derivative having a temporary
protecting group on OH-4 and a thioethyl anomeric-
blocking group, was selected as the b-hexosamine donor.
The use of N-trichloroacetyl group was preferred to the
more commonly used phthalimido group because the N-
trichloroacetyl group can be easily converted to the N-
acetyl group at the end of the synthesis in a single free rad-
ical reduction step.3
The known compound 102 was deacetylated (MeOH,
NaOMe, quant.) and a benzylidene group was introduced
to mask the 4 and 6 positions (PhCHO, TFA, 83%). After
protecting OH-3 as the benzyl ether (NaH, BnBr, DMF,
78%), regioselective reductive opening of the benzylidene
acetal using Et3SiH / BF3·Et2O,4 followed by acetylation,
afforded 11 [partial 1H NMR (CDCl3) d 7.02 (N-H), 5.05
Starting from b-D-galactose pentaacetate, the anomeric
center was protected as the paramethoxyphenylether to af-
ford compound 3 (90%). Removal of the acetate esters
(MeOH, NaOMe, quant.), followed by selective protec-
tion of OH-3 and OH-4 with 2,2-dimethoxy propane and
CSA in DMF gave compound 4 (79%). The hydroxyl
groups at the 2 and 6 positions were protected as benzyl
ethers [BnBr, NaH, DMF (77%)]. Acetal hydrolysis
afforded compound 5 (73%). Selective benzoylation of
Synlett 2003, No. 9, Print: 11 07 2003.
Art Id.1437-2096,E;2003,0,09,1315,1318,ftx,en;S00802ST.pdf.
© Georg Thieme Verlag Stuttgart · New York

1316
F. Belot et al.
LETTER
OR6
OR2
OAc
OBn
BzO
HO
O
BnO
BnO
MeOOC
BzO
O
12: R6 = Bn, R2 = H
13: R6 = H, R2 = Bn
AcO
AcO
O
O
SEt
OMP
O
BzO
NH
OBn
O
O
OBn
NH
BzO
O
CCl3
BnO
BnO
CCl3
OC8H17
6
10
7
scheme 3
scheme 2
OBn
O
OBn
O
HO
BnO
HO
BnO
O
O
OBn
OBz
O
BzO
MeOOC
CCl3COHN
CCl3COHN
BnO
OBn
O
O
O
BzO
BzO
BnO
BnO
O
BnO
NH
OBz
OBz
O
9
O
O
OBn
CCl3
OBn
O
O
BnO
BnO
BnO
BnO
OC8H17
OC8H17
15
14
scheme 5
15
scheme 4
14
OH
NaOOC
OH
OH
OH
O
OH
OH
NaOOC
HO
HO
O
O
O
O
O
HO
HO
O
O
HO
O
OH
O
O
O
HO
OH
OH
OH
OH
AcHN
AcHN
HO
OH
O
O
HO
HO
HO
2
1
O
O
OH
O
OH
O
HO
HO
HO
HO
OC8H17
OC8H17
Figure 1
(1 H, t, J3.4 = J4.5 = 9.5 Hz, H-4), 5.02 (1 H, d, J1.2 = 10.0
Hz), 4.2 ppm (1 H, t, J2.3 = J3.4 = 9.5 Hz, H-3)].
MeOOC
OBn
BzO
HO
O
BzO
BzO
O
+
OMP
Compound 12 was obtained by condensation between 2-
O-acetyl-3,4,6-tri-O-benzyl-a,b-D-mannopyranosyl tri-
chloroacetimidate5 and octyl 2,3,4-tri-O-benzyl-b-D-
mannopyranoside6 using catalytic TMSOTf, followed by
deacetylation (95%, 2 steps). Characteristic proton and
carbon signals in the 1H and 13C NMR (CDCl3) spectra [d
5.04 (s, J1,2 = 1.5 Hz, aMan H-1), 4.38 (s, J1,2<1 Hz, bMan
H-1), 2.22 (s, OH); 13C NMR: d 101.7, 99.6] confirmed
the structure of 12. Condensation of 11 and 12 and subse-
quent deacetylation provided the trisaccharide 14 in 63%
yield (2 steps) (Scheme 3). Following the same procedure,
compounds 11 and 137 were coupled and the trisaccharide
was deacetylated in order to give compound 15 in 75%
yield (2 steps) (Scheme 3). The observed chemical shifts
and coupling constants in CDCl3 [14: d 7.08 (d, J2,NH = 7.2
Hz, GlcNAc N-H), 5.10 (s, J1,2 = 1.5 Hz, aMan H-1), 4.97
(d, J1,2 = 7.5 Hz, GlcNAc H-1), 4.24 (s, J1,2<1 Hz, bMan
H-1); 15: d 5.19 (d, J1,2 = 8.5 Hz, GlcNAc H-1), 4.90 (s,
BzO
NH
O
OBn
7
CCl3
6
a
OBn
BzO
O
MeOOC
O
O
OBz
BzO
BzO
OMP
OBn
8
b, c, d, e
OBz
BzO
MeOOC
O
O
BzO
BzO
O
BzO
BzO
NH
O
9
CCl3
Scheme 2 Synthesis of 9; a) TMSOTf, molecular sieves 4 Å,
CH2Cl2, 0 °C to r.t. (84%); b) C/Pd(OH)2, H2, MeOH, r.t., 20 h,
(95%); c) BzCl, pyridine, r.t., 20 h, (70%); d) CAN, PhCH3, CH3CN,
H2O, r.t., 90 min., (75%); e) CCl3CN, DBU, CH2Cl2, 0 °C, 1 h, (80%).
J
1,2 = 1.5 Hz, a-Man H-1), 4.36 (s, J1,2<1 Hz, b-Man H-1)]
unambiguously established the expected stereochemistry
in both trisaccharides 14 and 15.
Synlett 2003, No. 9, 1315–1318 ISSN 1234-567-89 © Thieme Stuttgart · New York

LETTER
Efficient Synthesis of Two HNK-1 Related Pentasaccharides
1317
OAc
14 + 9
a
OBn
O
a, b, c, d, e
O
AcO
AcO
AcO
BnO
SEt
SEt
NH
NH
O
O
OBz
BzO
CCl3
CCl3
OBn
MeOOC
11
O
10
O
O
BzO
BzO
O
O
O
BnO
OBz
OBz
NH
O
11 + 12
g, h
16
11 + 13
f, h
CCl3
BnO
O
BnO
BnO
OBn
OBn
O
OBn
O
O
HO
HO
BnO
CCl3COHN
O
BnO
O
O
BnO
BnO
OC8H17
CCl3COHN
OBn
OBn
O
b, c, d
O
BnO
BnO
BnO
BnO
14
O
2
O
OBn
O
15
OBn
O
BnO
BnO
Scheme 4 Synthesis of 2: TMSOTf, CH2Cl2, molecular sieves 4 Å,
0 °C, 5 h, (71%); b) Bu3SnH, PhH, AIBN, 90 °C, 90 min., (77%),
c) C/Pd(OH)2, H2, MeOH (quant.); d) LiOH, H2O2, THF/H2O, 20 h,
then NaOH/MeOH, 8 h, (65%).
BnO
BnO
OC8H17
OC8H17
Scheme 3 Synthesis of trisaccharides 14 and 15: a) NaOMe, MeOH,
r.t., 2 h (quant.); b) PhCHO, TFA, r.t., 3 h, 90 min., (83%); c) BnBr,
NaH, DMF, 0°C, 2 h, (78%), d) Et3SiH, BF3·Et2O, CH2Cl2, 0 °C to
r.t., 90 min., (70%); e) Ac2O, pyridine, r.t., overnight (96%); f) NIS,
TMSOTf, molecular sieves 4 Å, 0 °C to r.t., 2 h (75%); g) NIS,
TMSOTf, molecular sieves 4 Å, 0 °C to r.t., 3h (63%); h) MeOH,
NaOMe, overnight, (quant.).
15 + 9
a
OBz
BzO
MeOOC
OBn
O
O
O
BzO
BzO
O
BnO
O
O
OBz
OBz
Trisaccharides 14 and 15 were each condensed with
trichloroacetimidate 9 to give pentasaccharides 16 and 17
in 71% and 96% yield, respectively (Schemes 4 and 5).
Chemical shifts and coupling constants in their 1H and 13C
NMR (CDCl3) spectra [17: d 5.84 (dd, J3,4 = 3.5 Hz,
NH
O
OBn
O
17
CCl3
BnO
BnO
O
OBn
O
BnO
BnO
J
4,5 = 1 Hz, Gal H-4), 5.77 (m, J2,3 = J3,4 = J4,5 = 10 Hz,
OC8H17
b, c, d
GlcA H-3 and H-4), 5.58 (dd, J1,2 = 8.0 Hz, GlcA, H-2),
5.30 (dd, J1,2 = 7.5 Hz, Gal H-2), 5.07 (d, J1,2 = 1.5 Hz, a-
Man H-1), 4.37 (s, J<1.0 Hz, b-Man H-1); 13C: d 101.7,
100.6, 99.9, 99.3, 98.4; 16: d 5.86 (dd, J3,4 = 3.5 Hz,
1
Scheme 5 Synthesis of 1: TMSOTf, CH2Cl2, molecular sieves 4 Å,
0 °C, 2 h, (96%); b) Bu3SnH, PhH, AIBN, 90 °C, 90 min., (80%),
c) C/Pd(OH)2, H2, MeOH (quant.); d) LiOH, H2O2, THF/H2O, 20 h,
then NaOH/MeOH, 8 h, (43%).
J
4,5 = 1 Hz, Gal H-4), 5.70 (m, J2,3 = J3,4 = J4,5 = 10 Hz,
GlcA H-3 and H-4), 5.58 (dd, J1,2 = 8.2 Hz, GlcA, H-2),
5.30 (dd, J1,2 = 7.0 Hz, Gal H-2); 13C: d 101.7, 100.6,
100.0, 98.5, 97.3] confirmed the stereoselective glyco-
sylation.
of the aMan (1→6)bMan reducing disaccharide. This
could in principle affect the rotameric distribution around
C5-C6 of the b-Man residue resulting in a very different
The N-trichloroacetyl protecting groups were readily
transformed into N-acetyl on treatment with tributylstan-
nane and azo-bis-isobutyronitrile (AIBN) in refluxing
benzene (77% and 80%). Hydrogenation [Pd(OH)2, H2,
MeOH] and careful saponification (LiOH, H2O2,8 THF/
H2O overnight, then NaOH 4M, MeOH, 8 h) led to 2 and
1 in 65% and 43% overall yield, respectively (Schemes 4
and 5). In order to confirm the desired stereochemistry a
1
presentation of the HNK-1 epitope. The H NMR cou-
pling constants (J5,6a and J5,6b) for 1 and 2 were however,
almost identical (5.3 and 1.9/2.0) suggesting similar, if not
identical rotameric populations for these two compounds.
This distribution would be similar to the statistical distri-
bution of 60 (GG): 40 (GT) reported by Bock and Duus.12
The two pentasaccharides 1 and 2 are currently being
evaluated in biological assays. The results of these studies
will be reported elsewhere in due course.
total assignment by NMR H was carried out9 (Table 1).
1
The NMR data confirmed the stereochemistry at each in-
terglycosidic linkage as well as the expected regiochemis-
try.10 The molecular weights of 1 and 2 were confirmed
using high resolution mass spectrometry.11
Acknowledgment
Pentasaccharides 1 and 2 differ only in the position of
linkage of the terminal trisaccharide to either O-2 or O-6
This work was supported by National Institutes of Health Grants
PO1 CA 71932 and CA 33895.
Synlett 2003, No. 9, 1315–1318 ISSN 1234-567-89 © Thieme Stuttgart · New York

1318
F. Belot et al.
LETTER
Table 1 Proton-NMR9 Chemical Shifts and Coupling Constants for 1 and 2
Ring
b-GlcA
b-Gal
b-GlcNAc
a-Man
b-Man
Molecule
1
2
1
2
1
2
1
2
1
2
H-1
(J1,2
4.678
(7.8)
4.678
(7.8)
4.533
(7.9)
4.528
(7.9)
4.586
(8.3)
4.610
(7.8)
4.894
(1.5)
4.930
(1.6)
4.666
(<1)
4.666
(<1)
)
)
)
)
H-2
(J2,3
3.417
(9.1)
3.418
(9.3)
3.697
(9.9)
3.700
(9.8)
3.784
(10.5)
3.750c
3.976d
(3.5)
4.129d
(3.5)
3.983
(3.2)
3.988
(3.3)
H-3
(J3,4
3.514b
(ca.9)
3.516b
(ca.9)
3.816
(3.3)
3.818
(3.3)
3.740c
3.750c
3.808
(9.5)
3.854
(9.5)
3.628
(9.4)
3.630
(9.3)
H-4
(J4,5
3.532b
(8.8)
3.534b
(9.0)
4.195
(ca. 1)
4.196
(ca. 1)
3.746
(9.2)
3.750
(9.2)
3.651
(9.5)
3.518
(9.5)
3.720
(9.6)
3.720
(9.6)
H-5
3.725
–
3.725
–
3.730
3.760
3.608
3.581
4.168d
3.652d
3.497
3.495
H-6a
(J5,6a
3.720c
3.740c
4.008
(2.2)
3.990
(2.4)
3.790d,c
3.914d
(1.8)
3.914
(5.4)
3.964
(5.4)
a
a
)
H-6b
(J5,6b
–
–
3.720c
3.740c
3.848
(4.9)
3.854
(5.0)
3.790d,c
3.660d,c
3.785
(1.9)
3.793
(2.0)
)
AcNH (CH3)
2.06
2.06
a When two distinct signals were observed, the greater chemical shift was assigned to H-6a.
b Assignments might be interchanged.
c Unavailable due to extensive spectral overlap.
d Chemical shifts in bold are most affected by the different substitution at the a-Man residue.
reproducible to three decimals, however, within a given
experiment at 600 MHz, signals can be distinguished
accurately to this precision.
References
(1) Voshol, H.; van Zuylen, C. W. E. M.; Orberger, G.;
Vliegenthart, J. F. G.; Schachner, M. J. Biol. Chem. 1996,
271, 22957.
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1995, 1573.
(3) Blatter, G.; Beau, J.-M.; Jacquinet, J.-C. Carbohydr. Res.
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2000, 11, 385.
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(7) Srivastava, G.; Hindsgaul, O. Carbohydr. Res. 1992, 224,
83.
(8) Lucas, H.; Basten, J. E. M.; Van Dinther, T. G.; Meulemenn,
D. G.; Van Aelst, S. F.; Van Boeckel, C. A. A. Tetrahedron
1990, 46, 8207.
(10) Correct glycosidic linkages are confirmed by the following
inter-residue NOEs (s for strong, m for medium and w for
weak interactions). 1: H1bGlcA-H3bGal (s) and H1bGlcA-
H4Gal (m); H1bGal-H4bGlcNAc (likely s, overlaps with
H1/H5bGal); H1bGlcNAc-H6a/baMan (s) and
H1bGlcNAc-H5aMan (w); H1aMan-H6bbMan (s) and
H1aMan-H6abMan (w) and H1aMan-H5bMan (w). 2:
H1bGlcA-H3bGal (s) and H1bGlcA-H4bGal (m); H1bGal-
H4bGlcNAc (likely s, overlaps with H1/H5bGal);
H1bGlcNAc-H2aMan (s) and H1bGlcNAc-H1aMan (s);
H1aMan-H6bbMan (s) and H1aMan-H6abMan (w) and
H1aMan-H5bMan (w).
Furthermore, typical H1-H3 and H1-H5 intra-residue NOEs
were observed for all b-glycosides. Taken together with the
coupling constants, this confirms that all pyranose rings
adopt the normal chair form.
(9) Data recorded in D2O at 600 MHz, and 27.0 0.1 °C;
concentration ca. 8 mM; chemical shifts are relative to
external 0.1% acetone set at 2.225 ppm measured in a
separate sample under identical experimental conditions.
Even under strict temperature control, chemical shifts are not
(11) HRMS calcd for C40H69NNaO27 (1 and 2): 1018.3955; found
1: 1018.3961; 2:1018.3959
(12) Bock, K.; Duus, J. O. J. Carbohydr. Chem. 1994, 13, 513.
Synlett 2003, No. 9, 1315–1318 ISSN 1234-567-89 © Thieme Stuttgart · New York