A novel and efficient route towards α-GalNAc-Ser and α-GalNAc-Thr building blocks for glycopeptide synthesis

Article abstract of DOI:10.1002/(SICI)1099-0690(199905)1999:5<1167::AID-EJOC1167>3.0.CO;2-2

Michael addition of serine and threonine derivatives 4a-4c to 3,4,6-tri- O-benzyl-2-nitro-D-galactal (1) afforded the corresponding 2-deoxy-2-nitro- α-D-galactopyranosides 5a-5c in good yield and stereoselectivity. 2-deoxy-2- nitroglycosides 5a and 5b were reduced to the 2-acetamido compounds by platinized Raney nickel T4. Manipulation of the protecting groups afforded known N-Fmoc-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D- galactopyranosyl)-L-serine (8a) and -threonine (8b), valuable building blocks for O-glycopeptide synthesis.

Full text of DOI:10.1002/(SICI)1099-0690(199905)1999:5<1167::AID-EJOC1167>3.0.CO;2-2

FULL PAPER
A Novel and Efficient Route towards α-GalNAc-Ser and α-GalNAc-Thr
Building Blocks for Glycopeptide Synthesis
Gottfried A. Winterfeld,[a,b] Yukishige Ito,[b] Tomoya Ogawa,[b] and Richard R. Schmidt*[a]
Keywords: Carbohydrates / Glycals, nitro / Michael additions / Glycosylations / Glycosides, galactosamine / Reduction,
nitro group / Glycopeptides
Michael addition of serine and threonine derivatives 4a–4c
to 3,4,6-tri-O-benzyl-2-nitro- -galactal (1) afforded the
corresponding 2-deoxy-2-nitro-α- -galactopyranosides 5a–
compounds by platinized Raney nickel T4. Manipulation of
the protecting groups afforded known N-Fmoc-O-(2-
D
D
acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-
D-galactopyranos-
5c in good yield and stereoselectivity. 2-deoxy-2-
nitroglycosides 5a and 5b were reduced to the 2-acetamido
yl)- -serine (8a) and -threonine (8b), valuable building
blocks for O-glycopeptide synthesis.
L
The α-glycosidic linkage between 2-acetamido-2-deoxy-
-galactopyranose and the side-chain hydroxy groups of -
serine or -threonine is a common motif in numerous glyco-
proteins.[1] It is found in mucins, cell-membrane glyco-
proteins, blood-group determinants, immunoglobulins,
anti-freeze glycoproteins and glycoprotein hormones.[1Ϫ3]
Chemical synthesis of this 1,2-cis-glycosidic bond of 2-acet-
amido-2-deoxy--galactopyranosides proved to be difficult,
since it necessitates a non-participating latent amino func-
tion at the C-2 atom of the glycosyl donor.[4] Lemieux et
al.[5] addressed this problem using addition reactions to 2-
nitrosoglycals and observed good α stereoselectivity. The re-
duction of the resulting 2-acetoximino-α--lyxo-hexopy-
ranosides, however, afforded mixtures of epimeric galacto
and talo products.[6] After the introduction of 2-azido-2-
deoxyaldoses to glycoside synthesis by Paulsen et al.[7Ϫ9]
and the azidonitration protocol by Lemieux,[10] derivatives
of 2-azido-2-deoxygalactose remained the only possible gly-
cosyl donors for the construction of the α-glycosidic bond
of N-acetylgalactosamine to serine and threonine.[11Ϫ17] Re-
cently, Michael-type addition to 3,4,6-tri-O-benzyl-2-nitro-
-galactal (1)[18] was shown to be a convenient glycosyl-
ation method for the synthesis of α-glycosides of galactos-
amine,[18] avoiding the often lengthy preparation of 2-azido-
2-deoxygalactosyl donors. We present a new and efficient
procedure based on this concept for the preparation of α-
GalNAc-Ser and α-GalNAc-Thr building blocks of type A
for glycopeptide synthesis (Scheme 1).
Scheme 1
required N-Boc/OtBu amino acids 4a and 4b were obtained
from commercial Boc-serine/threonine-(O-benzyl)-OH (3a
and 3b) in two steps using tert-butyltrichloroacetimidate[20]
and subsequent debenzylation (Scheme 2).
Scheme 2. Reagents: i) tert-butyltrichloroacetimidate, BF3·Et2O,
CH2Cl2, cyclohexane; ii) Pd(OH)2/C, H2, EtOH
First, the glycosylation of Fmoc--Ser-tBu (4c)[19,21] with
1 was established (Scheme 3). Since strong bases were re-
ported to favour addition of the aglycon from the α side,[18]
sterically hindered potassium tert-butoxide was chosen as
base and found to be most efficient in catalytic quantities
(0.1 equiv.) in combination with dry toluene as solvent. The
reaction proceeds reasonably fast at room temperature and
all of 1 is consumed after 3 h. As products the α-glycoside
5c (83%) and the corresponding β-glycoside 5cβ (14%) were
isolated, showing a very good overall yield of glycosides
and good α stereoselectivity. No loss of Fmoc protection
was observed in the reaction medium. To ascertain that no
racemization occurs during the base-catalysed glycosyl-
As glycosyl donor known 3,4,6-tri-O-benzyl-2-nitro--
galactal (1) was used, which was prepared according to a
slight modification of the reported procedure from 1-O-
acetyl-3,4,6-tri-O-benzyl-2-deoxy-2-nitro-α--galactopyra-
nose (2).[18] Glycosylation was performed with serine
and threonine acceptors carrying the protecting-group
patterns N-fluoren-9-ylmethoxycarbonyl (Fmoc)/O-tert-bu-
tyl (tBu)[19] and N-tert-butyloxycarbonyl (Boc)/OtBu. The
[a]
Fakultät für Chemie, Universität Konstanz, Fach M 725,
D-78457 Konstanz, Germany
[b]
RIKEN (The Institute of Physical and Chemical Research),
Wako-shi, Saitama, 351Ϫ01, Japan
Eur. J. Org. Chem. 1999, 1167Ϫ1171
WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
1434Ϫ193X/99/0505Ϫ1167 $ 17.50ϩ.50/0
1167

G. A. Winterfeld, Y. Ito, T. Ogawa, R. R. Schmidt
FULL PAPER
ation, the corresponding -Ser derivative of 5c, -5c, was tives 6a (89%) and 6b (84%, Scheme 3) were thus obtained
synthesized. TLC analysis (toluene/ethyl acetate, 9:1) clearly after reduction at ambient pressure and temperature and
separated - and -serine glycosides and confirmed that no subsequent N-acetylation. Exchange of O-benzyl protection
-serine glycoside is formed during the glycosylation of 4c. on the carbohydrate moiety by O-acetyl groups afforded 7a
For Fmoc protection of the amino function does not resist and 7b. Cleavage of Boc and tBu protection with the help
hydrogenolytic conditions, the protecting-group pattern of trifluoroacetic acid and attachment of the Fmoc group
Boc/tBu was then envisaged to extend the synthesis to to the amino function gave known building blocks 8a[19][23]
known 2-acetamido-3,4,6-tri-O-acetylgalactose derivatives. and 8b[19][23] in good overall yield. The importance of these
Glycosylation with Boc--Ser-tBu (4a) works equally ef- building blocks for O-glycopeptide synthesis is well docu-
ficiently and affords 5a (80%) and 5aβ (13%). The addition mented.[4]
of the secondary hydroxy group of threonine derivative 4b
to 2-nitrogalactal 1 proceeds highly selectively from the α
side yielding glycoside 5b in excellent yield (98% based on
consumed 1) with some glycosyl donor (20%) remaining un-
changed.
Experimental Section
General: Dry solvents were purchased from Kanto Chemicals Inc.
Ϫ Column chromatography: Silica gel 60 N 40Ϫ100µm (Kanto
Chemicals) and silica gel for flash chromatography 40 µm (J. T.
Baker). Ϫ Analytical TLC: HPTLC plates, silica gel 60 F254
(Merck), and TLC plastic sheets, silica gel 60 F254 (Merck); detec-
tion with UV light (254 nm) and with 5% (NH4)2MoO4, 0.1%
Ce(SO4)2 in 10% H2SO4 and heating to 160°C. Ϫ Melting points:
Büchi 510 melting-point apparatus, values uncorrected. Ϫ Optical
rotations: JASCO DIP 370 polarimeter or PerkinϪElmer polar-
imeter 241 MC in 1-dm cells at 22°C. Ϫ IR spectra: Shimadzu FT-
IR 8100 M, solutions in CHCl3 on NaCl cells. Ϫ 1H-NMR spectra:
Bruker AC 250 (250 MHz) Cryospec, JEOL EX 270 MHz spec-
trometer, JEOL EX 400 MHz spectrometer, JEOL EX 500-MHz
spectrometer or Bruker DRX 600 (600 MHz); tetramethylsilane as
internal standard. Ϫ 13C-NMR spectra: JEOL EX 270-MHz spec-
trometer (68 MHz); tetramethylsilane as internal standard. Ϫ FAB
mass spectra: JEOL HX 110 from an m-nitrobenzyl alcohol matrix
with NaI as additive. Ϫ MALDI mass spectra: Kratos Analytical
Kompact Maldi from 2,5-dihydroxybenzoic acid.
3,4,6-Tri-O-benzyl-2-nitro-D-galactal (1): 1-O-Acetyl-3,4,6-tri-O-
benzyl-2-deoxy-2-nitro-α--galactopyranose (2)[18] (2.0 g, 3.96
mmol) was dissolved in CH2Cl2 (5 mL) and slowly added to an ice-
cold, stirred solution of NEt3 (0.66 mL, 4.75 mmol) in CH2Cl2 (5
mL). After complete addition, the cooling bath was removed and
stirring continued for 20 min. The organic phase was washed with
2  HCl solution and dried with Na2SO4. Removal of the volatiles
and column-chromatographic purification (toluene/ethyl acetate,
98:2) of the residue furnished 1 (1.45 g, 79%). Analytical data are
in accordance with the literature.[18]
N-(tert-Butyloxycarbonyl)-L-serine tert-Butyl Ester (4a): A solution
of tert-butyltrichloroacetimidate (1.48 g, 6.77 mmol) in cyclohex-
ane (6.8 mL) was added to a stirred solution of commercial 3a (1.0
g, 3.39 mmol) in CH2Cl2 (3.4 mL) followed by boron trifluorideϪ
diethyl ether (68 µL, 0.55 mmol). Stirring was continued for 14 h
before solid NaHCO3 was used to neutralize the acid. Evaporation
of the solvents and purification on a short silica-gel column (tolu-
ene/ethyl acetate, 9:1) afforded the intermediate N-Boc--serine-
(OBn) tert-butyl ester (1.14 g, 96%). This material was dissolved in
ethanol/acetic acid (5:1, 12 mL) and stirred together with Pd(OH)2/
C (0.11 g, 20% Pd) under H2 for 36 h. Filtration through Celite
and evaporation of all volatiles gave 4a (0.80 g, 90%), which crys-
tallized upon standing, m.p. 80°C. Ϫ TLC (toluene/ethyl acetate,
Scheme 3. Reagents: i) tBuOK, toluene; ii) Ra-Ni T4-Pt, H2 EtOH;
iii)Pd/C, H2, MeOH, AcOH; iv) Ac2O, pyridine; v) TFA, CH2Cl2;
vi) Fmoc-ON-Su, NaHCO3, MeCN, H2O
22
1
4:1): Rf ϭ 0.33. Ϫ [α]D ϭ Ϫ22.5 (c ϭ 1.8, ethanol). Ϫ H NMR
(270 MHz, CDCl3): δ ϭ 5.40 (br. s, 1 H, NH), 4.25 (br. s, 1 H, α-
CH), 3.90 (br. s, 2 H, CH2), 2.35 (br. s, 1 H, OH), 1.49 (s, 9 H,
C4H9), 1.46 (s, 9 H, C4H9). Ϫ MS (FAB): calcd. 261.16 ϩ 22.99
For the reduction of the nitro group the system platinized
[22]
(Na) ϭ 284.15; found 284.16 (M ϩ Na)ϩ. Ϫ Ref.[24] m.p. 76Ϫ78°C,
Ra-Ni T4/H2
was found to work very efficiently under
22
convenient experimental conditions. 2-Acetamido deriva- [α]D ϭ Ϫ20.0 (c ϭ 1.8, ethanol).
1168
Eur. J. Org. Chem. 1999, 1167Ϫ1171

A Novel and Efficient Route towards α-GalNAc-Ser and α-GalNAc-Thr Building Blocks
FULL PAPER
N-(tert-Butyloxycarbonyl)-L-threonine tert-Butyl Ester (4b): Com-
4.50Ϫ4.40 (m, 4 H, 3 benzyl. H, H3), 4.32 (br. dd, Jβ-CH,γ-CH3 ϭ
mercial 3b (1.0 g, 3.23 mmol) was treated as described for 4a to 6.15, 1 H, β-CH), 4.10 (d, Jα-CH,NH ϭ 10.00, 1 H, α-CH),
afford intermediate N-Boc-threonine-(OBn) tert-butyl ester (1.10 g,
93%). Hydrogenation as for 4a gave 4b (0.82 g, 92%), which crys-
4.05Ϫ4.03 (m, 2 H, H4, H5), 3.58Ϫ3.51 (m, 2 H, H6, H6Ј), 1.47 (s,
9 H, C4H9), 1.46 (s, 9 H, C4H9), 1.28 (d, Jγ-CH3,β-CH ϭ 6.41, 3 H,
tallized upon standing, m.p. 70°C. Ϫ TLC (toluene/ethyl acetate, γ-CH3). Ϫ 13C NMR (68 MHz, CDCl3): δ ϭ 169.19 (1 C, COOR),
22
1
4:1): Rf ϭ 0.38. Ϫ [α]D ϭ Ϫ23.4 (c ϭ 1.0, ethanol). Ϫ H NMR
(270 MHz, CDCl3): δ ϭ 5.24 (br. d, 1 H, NH), 4.23 (br. d, 1 H, β-
156.19 (1 C, CONHR), 137.86, 137.64, 137.25 (3 C, subst. arom.
C), 129.02Ϫ127.72 (15 C, arom. C), 96.43 (1 C, C1), 84.26 (1 C,
CH), 4.13 (br. d, 1 H, α-CH), 1.97 (br. s, 1 H, OH), 1.49 (s, 9 H, C2), 82.67 (1 C, tert-Bu ester quat. C), 79.90 (1 C, Boc quat. C),
C4H9), 1.46 (s, 9 H, C4H9), 1.24 (d, 3 H, CH3). Ϫ C13H25NO5 75.76 (1 C, β-C), 75.15 (1 C, benzyl. C), 75.04 (1 C, C3), 73.55 (1
(275.2): calcd. C 56.71, H 9.15, N 5.09; found C 56.35, H 9.16, N
5.02. Ϫ MS (FAB): calcd. 275.17 ϩ 22.99 (Na) ϭ 298.16; found
298.14 (M ϩ Na)ϩ.
C, benzyl. C), 73.01 (2 C, C4, benzyl. C), 69.94 (1 C, C5), 68.19 (1
C, C6), 58.57 (1 C, α-C), 28.34 (3 C, Boc prim. C), 27.95 (3 C, tert-
Bu ester prim. C), 18.67 (1 C, γ-C). Ϫ C40H52N2O11 (736.4): calcd.
C 65.20, H 7.11, N 3.80; found C 65.27, H 7.13, N 3.55. Ϫ MS
(FAB): calcd. 736.36 ϩ 22.99 (Na) ϭ 759.4; found 759.2 (M ϩ
Na)ϩ.
O-(3,4,6-Tri-O-benzyl-2-deoxy-2-nitro-α-
D-galactopyranosyl)-N-
(tert-butyloxycarbonyl)- -serine tert-Butyl Ester (5a): 1 (0.25 g, 0.54
L
mmol) and 4c (0.15 g, 0.57 mmol) were dried under high vacuum
and dissolved in dry toluene (25 mL) under argon. Freshly acti-
vated molecular sieve (3 A, 0.3 g) was introduced and the mixture
O-(3,4,6-Tri-O-benzyl-2-deoxy-2-nitro-α--galactopyranosyl)-N-
˚
(fluorenylmethoxycarbonyl)-L-serine tert-Butyl Ester (5c): 1 (46 mg,
0.10 mmol) and 4c[21] (42 mg, 0.11 mmol) were dried under high
stirred for 1 h. Then 1  potassium tert-butoxide solution in THF
(53 µL, 0.05 mmol) was added and stirring continued for 75 min.
Acetic acid (50 mL) was used to acidify the reaction mixture, the
molecular sieve was filtered off and all solvents were removed. The
residue was purified by column chromatography (toluene/ethyl
acetate, 9:1) to furnish 5a (0.31 g, 80%) and the corresponding β-
glycoside 5aβ (0.05 g, 13%). Ϫ 5a: TLC (toluene/ethyl acetate, 9:1):
vacuum and dissolved in dry toluene (5 mL) under argon. Freshly
˚
activated molecular sieve (3 A, 0.1 g) was introduced and the mix-
ture stirred for 1 h. Then 1  potassium tert-butoxide solution in
THF (10 µL, 0.01 mmol) was added and stirring continued for 3
h. Acetic acid (10 µL) was used to acidify the reaction mixture, the
molecular sieve was filtered off and all solvents were removed. The
residue was purified by column chromatography (toluene/ethyl
acetate, 9:1) to furnish 5c (70 mg, 83%) and the corresponding β-
glycoside 5cβ (12 mg, 14%). Ϫ 5c: TLC (toluene/ethyl acetate, 9:1):
22
Rf ϭ 0.48. Ϫ [α]D ϭ 67.0 (c ϭ 1.0, chloroform). Ϫ IR: ν˜ ϭ
1
1561cmϪ1 (NO2). Ϫ H NMR (400 MHz, CDCl3): δ ϭ 7.37Ϫ7.19
(m, 15 H, arom. H), 5.44 (d, JNH,α-CH ϭ 7.81 Hz, 1 H, NH), 5.27
22
(d, JH1,H2 ϭ 3.91, 1 H, H1), 4.96 (dd, JH2,H1 ϭ 4.39, JH2,H3
ϭ
Rf ϭ 0.48. Ϫ [α]D ϭ 58.5 (c ϭ 1.0, chloroform). Ϫ IR: ν˜ ϭ 1561
1
cmϪ1 (NO2). Ϫ H NMR (500 MHz, CDCl3): δ ϭ 7.76Ϫ7.19 (m,
10.73, 1 H, H2), 4.82 (d, Jgem H ϭ 10.73, 1 H, benzyl. H), 4.72 (s,
2 H, benzyl. H), 4.53Ϫ4.40 (m, 3 H, benzyl. H), 4.38 (dd, JH3,H2 ϭ
10.73, JH3,H4 ϭ 3.90, 1 H, H3), 4.31 (br. d, Jα-CH,NH ϭ 7.81, 1 H,
α-CH), 4.04Ϫ3.99 (m, 2 H, H4, H5), 3.88 (d, Jβ-CH2,α-CH ϭ 2.93, 2
H, β-CH2), 3.58Ϫ3.55 (m, 2 H, H6, H6Ј), 1.46 (s, 9 H, C4H9), 1.44
(s, 9 H, C4H9). Ϫ 13C NMR (68 MHz, CDCl3): δ ϭ 168.65 (1 C,
COOR), 155.38 (1 C, CONHR), 137.87, 137.62, 137.23 (3 C, subst.
arom. C), 128.47Ϫ127.83 (15 C, arom. C), 96.71 (1 C, C1), 83.94
(1 C, C2), 82.79 (1 C, tert-Bu ester quat. C), 79.93 (1 C, Boc quat.
C), 75.10 (1 C, benzyl. C), 74.93 (1 C, C3), 73.51 (1 C, benzyl. C),
72.98 (1 C, C4), 72.90 (1 C, benzyl. C), 69.71 (2 C, C5, β-C), 67.94
(1 C, C6), 54.24 (1 C, α-C), 28.29 (3 C, Boc prim. C), 27.86 (3 C,
tert-Bu ester prim. C). Ϫ C39H50N2O11 (722.3): calcd. C 64.80, H
6.97, N 3.88; found C 64.91, H 6.97, N 3.76. Ϫ MS (FAB): calcd.
722.34 ϩ 22.99 (Na) ϭ 745.3; found 745.4 (M ϩ Na)ϩ. Ϫ 5aβ:
TLC (toluene/ethyl acetate, 9:1): Rf ϭ 0.30. Ϫ 1H NMR (270 MHz,
CDCl3): δ ϭ 5.23 (d, JH1,H2 ϭ 7.42, 1 H, H1).
23 H, arom. H), 5.89 (d, JNH,α-CH ϭ 7.62, 1 H, NH), 5.27 (d,
JH1,H2 ϭ 4.10, 1 H, H1), 4.98 (dd, JH2,H1 ϭ 4.10, JH2,H3 ϭ 10.55,
1 H, H2), 4.82 (d, Jgem H ϭ 11.43, 1 H, benzyl. H), 4.75Ϫ4.70 (m,
2 H, benzyl. H), 4.46Ϫ4.31 (m, 7 H, 3 benzyl. H, H3, 2 Fmoc-CH2,
α-CH), 4.21Ϫ4.19 (m, 1 H, Fmoc-CH), 4.01Ϫ3.92 (m, 4 H, H4,
H5, β-CH2), 3.55Ϫ3.52 (m, 1 H, H6), 3.48Ϫ3.44 (m, 1 H, H6Ј), 1.48
(s, 9 H, C4H9). Ϫ 13C NMR (68 MHz, CDCl3): δ ϭ 168.36 (1 C,
COOR), 143.85Ϫ119.96 (30 C, arom. C), 97.00 (1 C, C1), 83.99 (1
C, C2), 83.07 (1 C, tert-Bu ester quat. C), 75.04 (2 C, benzyl. C,
C3), 73.42, 73.07, 72.99 (3 C, 2 benzyl. C, C4), 70.08, 69.81 (2 C,
C5, β-C), 68.32 (1 C, C6), 67.03 (1 C, Fmoc-CH2), 54.72 (1 C, α-
C), 47.14 (1 C, Fmoc-CH), 27.89 (3 C, tert-Bu ester prim. C). Ϫ
C49H52N2O11 (844.4): calcd. C 69.65, H 6.20, N 3.32; found C
69.46, H 6.21, N 3.21. Ϫ MS (FAB): calcd. 844.4 ϩ 22.99 (Na) ϭ
867.4; found 867.4 (M ϩ Na)ϩ. Ϫ 5cβ: TLC (toluene/ethyl acetate
9:1): Rf ϭ 0.33. Ϫ1H NMR (270 MHz, CDCl3): δ ϭ 5.60 (d,
JH1,H2 ϭ 7.92, 1 H, H1).
O-(3,4,6-Tri-O-benzyl-2-deoxy-2-nitro-α-
D-galactopyranosyl)-N-
(tert-butyloxycarbonyl)- -threonine tert-Butyl Ester (5b): 1 (46 mg,
0.10 mmol) and 4b (30 mg, 0.11 mmol) were dried under high vac-
L
O-(3,4,6-Tri-O-benzyl-2-deoxy-2-nitro-α-
D
-galactopyranosyl)-N-
-5c): -5c
(fluorenylmethoxycarbonyl)- -serine tert-Butyl Ester (
D
D
uum and dissolved in dry toluene (5 mL) under argon. Freshly
was synthesized exactly as 5c starting from Fmoc--Ser-tBu (-
˚
activated molecular sieve (3 A, 0.1 g) was introduced and the mix-
4c).[21] Ϫ TLC (toluene/ethyl acetate, 9:1): Rf ϭ 0.41. Ϫ H NMR
1
ture stirred for 1 h. Then 1  potassium tert-butoxide solution in
THF (10 µL, 0.05 mmol) was added and stirring continued for 4
h. Acetic acid (10 µL) was used to acidify the reaction mixture, the
molecular sieve was filtered off and all solvents were removed. The
residue was purified by column chromatography (toluene/ethyl
acetate, 9:1) to furnish recovered 1 (9 mg, 20%) and 5b (58 mg,
98% based on consumed 1). No corresponding β-glycoside could
(270 MHz, CDCl3): δ ϭ 7.77Ϫ7.15 (m, 23 H, arom. H), 5.52 (d,
JNH,α-CH ϭ 8.44, 1 H, NH), 5.31 (d, JH1,H2 ϭ 3.96, 1 H, H1), 5.01
(dd, JH2,H1 ϭ 4.12, JH2,H3 ϭ 10.72, 1 H, H2), 4.83 (d, Jgem H
ϭ
11.21, 1 H, benzyl. H), 4.71 (s, 2 H, benzyl. H), 4.53Ϫ4.22 (m, 9
H), 4.15Ϫ4.12 (m, 1 H), 3.97Ϫ3.93 (m, 2 H), 3.66Ϫ3.63 (m, 1 H),
3.53 (d, 2 H), 1.48 (s, 9 H, C4H9).
be detected. Ϫ TLC (toluene/ethyl acetate, 9:1): Rf ϭ 0.45. Ϫ [α]
O-(2-Acetamido-3,4,6-tri-O-benzyl-2-deoxy-α-
N-(tert-butyloxycarbonyl)- -serine tert-Butyl Ester (6a): 5a (0.40 g,
0.55 mmol) was dissolved in ethanol (7 mL) and transferred to a
D-galactopyranosyl)-
22
ϭ 71.2 (c ϭ 1.0, chloroform). Ϫ IR: ν˜ ϭ 1561cmϪ1 (NO2). Ϫ
L
D
1H NMR (500 MHz, CDCl3): δ ϭ 7.40Ϫ7.16 (m, 15 H, arom. H),
5.39 (d, JH1,H2 ϭ 4.10, 1 H, H1), 5.02 (d, JNH,α-CH ϭ 10.00 Hz, 1 hydrogenation vessel. Platinized Raney nickel T4 catalyst was
H, NH), 4.96 (dd, JH2,H1 ϭ 4.10, JH2,H3 ϭ 10.77, 1 H, H2), 4.83
freshly prepared as described in ref.[22] and the material obtained
(d, Jgem H ϭ 11.28, 1 H, benzyl. H), 4.72 (s, 2 H, benzyl. H), from 2 g of Raney nickel/aluminium alloy was suspended in ethanol
Eur. J. Org. Chem. 1999, 1167Ϫ1171
1169

G. A. Winterfeld, Y. Ito, T. Ogawa, R. R. Schmidt
FULL PAPER
(15 mL). From a homogeneous suspension of this catalyst 7 mL
was added to the reaction vessel and the suspension shaken under
H2 for 48 h at ambient temperature and pressure. The catalyst was
filtered off and the solvent evaporated. The residue was dissolved
in pyridine/acetic anhydride (2:1, 9 mL) and stirred for 3 h. Re-
give 7b (78 mg, 96%). Ϫ TLC (toluene/ethyl acetate, 1:1): Rf ϭ
22
0.17. Ϫ [α]D ϭ 83.1 (c ϭ 1.0, chloroform). Ϫ 1H NMR (600
MHz, CDCl3): δ ϭ 5.99 (d, JNH,CH2 ϭ 9.96, 1 H, AcNH), 5.38 (d,
JH4,H3 ϭ 3.36, 1 H, H4), 5.20 (d, JNH,α-CH ϭ 9.4, 1 H, BocNH),
5.08 (dd, JH3,H2 ϭ 11.32, JH3,H4 ϭ 3.21, 1 H, H3), 4.87 (d, JH1,H2 ϭ
moval of the volatiles and column-chromatographic purification 3.65, 1 H, H1), 4.60 (ddd, JH2,H1 ϭ 3.69, JH2,H3 ϭ 10.76, JH2,NH ϭ
(toluene/acetone, 3:1) gave 6a (0.36g, 89%). Ϫ TLC (toluene/ace-
tone, 3:1): Rf ϭ 0.54. Ϫ [α]D ϭ 77.5 (c ϭ 2.0, chloroform). Ϫ H
10.25, 1 H, H2), 4.23 (m, 1 H, H5), 4.20Ϫ4.16 (m, Jα-CH,NH ϭ 9.7,
Jβ-CH,γ-CH ϭ 6.4, 2 H, α-CH, β-CH), 4.10Ϫ4.06 (m, 2 H, H6, H6Ј),
22
1
NMR (270 MHz, CDCl3): δ ϭ 7.40Ϫ7.16 (m, 15 H, arom. H), 5.32 2.16, 2.04, 2.01, 2.00 (4 s, 12 H, CH3CO), 1.49, 1.46 (2 s, 18 H, 2
(br. d, 1 H, NH), 4.94 (d, Jgem H ϭ 11.54, 1 H, benzyl. H), 4.80 (d, C4H9), 1.33 (d, Jγ-CH3,β-CH ϭ 6.08, 3 H, γ-CH3). Ϫ MS (MALDI):
JH1,H2 ϭ 3.63, 1 H, H1), 4.73Ϫ4.62 (m, 2 H, H2, benzyl. H),
4.58Ϫ4.39 (m, 4 H, benzyl. H), 4.32 (br. s, 1 H, α-CH), 4.00 (s, 1
H, H4), 3.89Ϫ3.74 (m, 3 H, H5, β-CH2), 3.64Ϫ2.51 (m, 3 H, H3,
H6, H6Ј), 1.88 (s, 3 H, CH3CO), 1.45 (s, 9 H, C4H9), 1.42 (s, 9 H,
C4H9). Ϫ MS (FAB): calcd. 734.88 ϩ 22.99 (Na) ϭ 757.87; found
757.4 (M ϩ Na)ϩ.
calcd. 605 ϩ 23 (Na) ϭ 628; found 629 (M ϩ Na)ϩ.
O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-
N-(fluorenylmethoxycarbonyl)- -serine (8a): 7a (10 mg, 0.02 mmol)
was dissolved in a mixture of trifluoroacetic acid and CH2Cl2 (2
mL, 1:1) and the solution stirred at room temperature for 12 h.
Then all solvents were evaporated. The residue was redissolved to-
gether with Fmoc-ONSu (10 mg, 0.03 mmol) and NaHCO3 (25
mg, 0.30 mmol) in acetonitrile/water (1:1, 4 mL) and the mixture
stirred for 12 h. The solution was acidified with 2  HCl solution
(5 mL) and the aqueous layer extracted with CH2Cl2 (3 ϫ 5 mL).
The combined organic extracts were dried with Na2SO4 and the
volatiles evaporated. Column-chromatographic separation (tolu-
D-galactopyranosyl)-
L
O-(2-Acetamido-3,4,6-tri-O-benzyl-2-deoxy-α-
D-galactopyranosyl)-
N-(tert-butyloxycarbonyl)- -threonine tert-Butyl Ester (6b): 5b (0.62
L
g, 0.85 mmol) was dissolved in ethanol (10 mL) and transferred to
a hydrogenation vessel. The platinized Raney nickel T4 suspension
(8 mL, see preparation of 6a) was added to the reaction vessel and
the mixture shaken under H2 for 12 h at ambient temperature and
pressure. The catalyst was filtered off and the solvent evaporated.
The residue was dissolved in pyridine/acetic anhydride (2:1, 6 mL)
and stirred for 3 h. Removal of the volatiles and column-chromato-
graphic purification (toluene/ethyl acetate, 6:1Ϫ4:1) gave 6b (0.53g,
ene/ethanol/acetic acid, 14:1:1) of the residue afforded 8a (10 mg,
22
90%). Ϫ TLC (chloroform/methanol, 4:1): Rf ϭ 0.45. Ϫ [α]D
ϭ
20
88.0 (c ϭ 0.3, chloroform); ref.[19] [α]D ϭ 89.9 (c ϭ 1.0, chloro-
20
form), ref.[23] [α]D ϭ 87.5 (c ϭ 2.0, chloroform).
22
84%). Ϫ TLC (toluene/acetone, 3:1): Rf ϭ 0.48. Ϫ [α]D ϭ 78.0
O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-
D-galactopyranosyl)-
(c ϭ 1.0, chloroform). Ϫ 1H NMR (600 MHz, CDCl3): δ ϭ
7.37Ϫ7.24 (m, 15 H, arom H), 5.69 (s, 1 H, NH), 4.99Ϫ4.97 (m, 2
H, NH, benzyl. H), 4.79Ϫ4.72 (m, JH1,H2 ϭ 3.84, 3 H, H1, H2,
N-(fluorenylmethoxycarbonyl)- -threonine (8b): 7b (10 mg, 0.02
L
mmol) was treated as described for the preparation of 8a. Column-
chromatographic separation (toluene/ethanol/acetic acid, 14:1:1) of
the residue afforded 8b (10 mg, 88%). Ϫ TLC (chloroform/meth-
anol, 4:1): Rf ϭ 0.55. Ϫ [α]D22 ϭ 61.5 (c ϭ 0.2, chloroform); ref.[19]
benzyl. H), 4.57Ϫ4.40 (m, 4 H, benzyl. H), 4.12 (d, Jα-CH,β-CH
ϭ
9.13, 1 H, α-CH),4.09Ϫ4.06 (m, 1 H, β-CH), 3.98 (s, 1 H, H4), 3.93
(m, 1 H, H5), 3.59Ϫ3.52 (m, 3 H, H3, H6, H6Ј), 1.99 (s, 3 H,
CH3CO), 1.47 (s, 9 H, C4H9), 1.43 (s, 9 H, C4H9), 1.27 (d, Jγ-
20
20
[α]D ϭ 65.0 (c ϭ 1.45, chloroform), ref.[23] [α]D ϭ 59.0 (c ϭ
0.50, chloroform).
ϭ 5.82, 3 H, γ-CH3). Ϫ MS (MALDI): calcd. 749 ϩ 23
CH3,βCH
(Na) ϭ 772; found 773 (M ϩ Na)ϩ.
Acknowledgments
O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-
D-galactopyranosyl)-
N-(tert-butyloxycarbonyl)- -serine tert-Butyl Ester (7a): 6a (0.10 g,
L
This work was supported by the Fonds der Chemischen Industrie
and the European Community (grant no. FAIR-CT97Ϫ3142). G.
A. W. is grateful for a RIKEN/Studienstiftung des Deutschen
Volkes fellowship. We thank Dr. S. Manabe for helpful discussions.
0.14 mmol) was dissolved in methanol/acetic acid (1:1, 4 mL) and
Pd/C (0.05 g, 10% Pd) was suspended in the solution. This mixture
was stirred for 3 h under H2. After complete disappearance of the
starting material (TLC: CHCl3/CH3OH, 9:1), the catalyst was fil-
tered off and all solvents were removed. The residue was treated
with pyridine/acetic anhydride (2:1, 6 mL) and stirred at room tem-
perature for 12 h. All volatiles were evaporated and the product
purified by column chromatography (toluene/ethyl acetate, 1:1) to
give 7a (75 mg, 93%). Ϫ TLC (toluene/ethyl acetate, 1:1): Rf ϭ
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3.80, Jβ-CH,βЈ-CH ϭ 10.09, 2 H, α-CH, β-CH), 4.08Ϫ4.06 (m, JβЈ-
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H. Paulsen, H. Koebernick, W. Stenzel, P. Köll, Tetrahedron
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CH,α-CH
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[8]
JH6,H5 ϭ 3.35, JH6,H6Ј ϭ 10.51, 1 H, H6), 3.83 (dd, JH6Ј,H5 ϭ 3.17,
JH6Ј,H6 ϭ 10.51, 1 H, H6Ј), 2.51, 2.06, 2.00, 1.95 (4 s, 12 H,
CH3CO), 1.48, 1.47 (2 s, 18 H, 2 C4H9). Ϫ MS (MALDI): calcd.
591 ϩ 23 ϭ 614; found 615 (M ϩ Na)ϩ.
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G. Grundler, R. R. Schmidt, Liebigs Ann. Chem. 1984,
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1826Ϫ1847.
1170
Eur. J. Org. Chem. 1999, 1167Ϫ1171

A Novel and Efficient Route towards α-GalNAc-Ser and α-GalNAc-Thr Building Blocks
FULL PAPER
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Received November 26, 1998
[O98537]
Eur. J. Org. Chem. 1999, 1167Ϫ1171
1171