Hexafluoroacetone as protecting and activating reagent: A new approach to O-glycosides

Article abstract of DOI:10.3987/COM-04-S(P)6

O-Glycosylated hexafluoroacetone-protected amino acid derivatives have been synthesized starting from serine, threonine, 4-hydroxyproline and tyrosine. They represent a new class of building blocks suitable for a divergent approach to O-glycopeptides.

Full text of DOI:10.3987/COM-04-S(P)6

HETEROCYCLES, Vol. 64, 2004
143
HETEROCYCLES, Vol. 64, 2004, pp. 143 - 152
Received, 15th March, 2004, Accepted, 18th May, 2004, Published online, 18th May, 2004
HEXAFLUOROACETONE AS PROTECTING AND ACTIVATING
REAGENT: A NEW APPROACH TO O-GLYCOSIDES
Klaus Burger*, Michael Kluge, Beate Koksch, Susanna Fehn, Christoph Böttcher,
Lothar Hennig, and Gerhard Müller
Department of Organic Chemistry, University of Leipzig, Johannisallee 29, D-
04103 Leipzig, Germany
Abstract - O-Glycosylated hexafluoroacetone-protected amino acid derivatives
have been synthesized starting from serine, threonine, 4-hydroxyproline and
tyrosine. They represent a new class of building blocks suitable for a divergent
approach to O-glycopeptides.
INTRODUCTION
Carbohydrates are building blocks i.a. of ribonucleic acids, glycoproteins, glycolipids, and
glycophospholipids.1 In many cases the carbohydrate moiety is the carrier of informations.2 As a
constituent of the cell membrane, glycoconjugates are responsible for important functions3-5 in cell-cell
recognition, in cell-matrix interactions, and cell growth regulation, consequently also in the
development of tumors.6 Furthermore, they play a significant role in the interaction with biologically
active factors as enzymes, hormones, bacteriotoxines, and viruses.3
The carbohydrate units undergo permanent changes7 during the life cycle of a cell, influencing the
biological properties of peptides and proteins.1 In general, glycosylation increases proteolytic stability,8
enhances solubility, improves transmembrane transport properties, and decreases excretion rate,9 thus
enhancing bioavailability. Since glycosylations can influence conformational flexibility of peptides,
they play a significant role in peptide folding processes.10
______________
* Dedicated to Prof. Dr. Pierre Potier on the occasion of his 70th birthday
** Corresponding author. E-mail: burger@organik.chemie.uni-leipzig.de

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HETEROCYCLES, Vol. 64, 2004
In many cases, the glycosylation of naturally occurring and non-natural peptides leads to a change in
their activity profile. For instance, the analgesic activity of enkephalines11 was increased by
glycosylation which was mainly attributed to an improved passage of the glycosylated species across
the blood/brain barrier.12
The convergent synthesis of O-glycosides by coupling reactions at the carbohydrate/peptide interface is
problematic because of the generally poor solubility of peptides under glycosylation conditions and also
because of regio- and stereochemical aspects.13,14 Therefore, the method of choice is an alternative
strategy that involves the stepwise construction starting from O-glycosylated amino acid derivatives and
small O-glycosylated peptide fragments, which can be assembled using solid phase techniques.15
Conventional syntheses of glycosylamino acids and peptides require orthogonal protection strategies for
the α-amino and the α-carboxylic groups. After glycosylation of the unprotected hydroxy group, the
carboxylic group is deprotected, activated, and finally the C-terminus is coupled with an amino acid
derivative.
RESULTS AND DISCUSSION
The new protecting / activating strategy described herein offers some significant advantages:
Introduction and cleavage of the protecting group occur under mild conditions. This resolves the issue
of acid lability of the O-glycoside bond as well as the tendency of the O-glycosylated serine and
threonine derivatives to undergo β-elimination in the presence of strong base. As an additional result,
the new strategy offers the possibility to save steps. Moreover, the reaction can be monitored easily and
quickly by 19F NMR spectroscopy without any loss of material.
Multifunctional α-amino acids such as serine, threonine, 4-hydroxyproline, and tyrosine react with
hexafluoroacetone (HFA) in good yields to form 2,2-bis(trifluoromethyl)-1,3-oxazolidin-5-ones.16 The
regioselective heterocyclization process allows a simultaneous protection of the α-amino and α-
carboxylic groups, concomitantly an activation of the α-carboxylic group is achieved. Since an excess
of hexafluoroacetone is normally used, the hydroxy groups of serine, threonine, and 4-hydroxyproline
in the crude product are partially protected as hemiketals. However, the hexafluoroacetone can be
readily cleaved by stirring a solution of the respective hemiketal in dichloromethane (DCM) in the
presence of silica gel at room temperature. The progress of the deprotection process can be monitored
efficiently by 19F NMR spectrocopy. The 2,2-bis(trifluoromethyl)-1,3-oxazolidin-5-ones (2) can be
stored for several months when kept below 0 oC in moisture-free environment.

HETEROCYCLES, Vol. 64, 2004
145
R
O
H
R
R
OAc
O
H
H
O
i)
i)
i)
ii)
AcO
AcO
HO
CO2H
O
O
HO
O
HN
F3C
HN
F3C
OAc
NH2
CF
3
CF
3
1a R=H
3a, A:57%, B:61%, C:78%
b, A:86%, B:70%, C:82%
2a, 61%
b, 83%
b R=CH3
O
O
H
H
OAc
CO2H
NH
O
ii)
AcO
AcO
O
O
O
N
HO
HO
N
OAc
CF
F3C
CF
3
F3C
3
1c
3c, A:21%, B:40%, C:27%
2c, 65%
O
O
OAc
CO2H
NH2
O
O
ii)
AcO
AcO
HN
O
O
HN
CF
F3C
3
HO
HO
OAc
CF
F3C
3
1d
2d, 84%
3d, B:83%, C:93%
Scheme 1: i) HFA, DMSO, rt; ii) method A: Ac4-α-D-Glc-Br, Hg(CN)2, CH3CN; method B: Ac4-β-D-
Glc-OAc, BF3·Et2O, CH2Cl2; method C: Ac4-β-D-Glc-O-C(=NH)CCl3; BF3·Et2O, CH2Cl2.
The HFA-protected amino acids (2a-d) were reacted with 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl
bromide / Hg(CN)2 according to Helferich,17 with penta-O-acetyl-β-D-glucopyranose / BF3·Et2O
according to Paulsen18 and with 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl trichloroacetimidate /
BF3·Et2O according to Schmidt.19 Good to excellent yields of HFA-protected O-glycosylated amino
acids (3a-d) indicate that the new protective group strategy can be applied successfully for all standard
O-glycosylation procedures.20
Compared to the glycosylation of HFA-protected Ser (2a) and Thr (2b), the yield of HFA-protected
Hyp derivatives (3c) was surprisingly low (27%) even on using the trichloroacetimidate method. The
main product in this case (55%) was 4-Ac-(HFA)-Hyp which indicates that an orthoester is formed as
an intermediate.13
To date, only a few glycopeptides containing glycosyl-Tyr have been isolated from natural sources. β-
D-Glucosyl-O-tyrosine itself was found as a tyrosine metabolite in Lepidoptera.21 The moderate yields
(40%) in the glycosylation of 9-fluorenylmethoxycarbonyltyrosine-O-pentafluorophenyl ester (9-Fmoc-

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HETEROCYCLES, Vol. 64, 2004
Tyr-OPfp) have been attributed to the low nucleophilicity of the phenolic OH-group.22 O-Glycosylation
of HFA-Tyr (2d → 3d) via trichloroacetimidate method gives a yield of 93%.
The O-glycosylated oxazolidin-5-ones (3a-d) can be used directly as activated esters in peptide
syntheses. When the lactone ring is opened with amino acid esters or amides, glycosylated dipeptide
amides crystallize from the reaction mixture in good yields. The formation of the amido bond is coupled
with the deprotection of the amino group, therefore an extention of the peptide chain at the N-terminus
can be performed without any additional activation and deprotection steps.
R
O
R
O
OAc
OAc
O
O
i)
ii)
AcO
AcO
NH2
AcO
AcO
NH2
3a
b
O
N
O
N
H
O
H
OAc
NH2
O
OAc
O
HN
Z
N
H
4a, 79%
b, 68%
5b, 73%
Scheme 2: i) H-Phe-NH2, EtOAc; ii) Z-Ala-OH, DIC, HOAt, CH2Cl2/DMF.
The glycosylated tripeptide Z-Ala-Thr(β-D-Ac4-Glc)-Phe-NH2 (5b) was obtained from Z-Ala-OH and
4b on activation with diisopropylcarbodiimide (DIC) / 7-aza-1-hydroxy-1H-benzotriazole (HOAt) in
73% yield.
The protecting / activation concept disclosed herein allows the construction of O-glycosylated dipeptide
(1 → 2 → 3 → 4) and tripeptide fragments (1 → 2 → 3 → 4 → 5) starting from hydroxy amino acids
in only three or four synthetic steps, and therefore can be considered as a useful alternative to the
pentafluorophenyl ester method.23
EXPERIMENTAL
1
IR spectra were obtained on a Genesis ATI Mattson/Unicam FTIR spectrometer. H NMR spectra were
recorded at 300 and 400 MHz. Chemical shifts were reported in ppm relative to tetramethylsilane
13
(TMS, δ = 0 ppm; J values are given in Hz (Hertz). C NMR spectroscopy was performed at 75 and
19
100 MHz. F NMR spectra were recorded at 282 MHz with trifluoroacetic acid (TFA, δ = 0 ppm) as
external standard. Melting points were determined on a Boëtius heating table. For C,H,N analyses a
CHNO-Rapid-Elemental Analyzer (Hereaus) was used. For flash chromatography, silica gel (32 – 63

HETEROCYCLES, Vol. 64, 2004
147
µm) was used with solvent systems given in the text. Organic solvents were dried and distilled prior to
use.
Protocol 1 (Helferich variant of the Koenigs/Knorr method)17: To a stirred solution of the HFA-
protected amino acid (2) (5.0 mmol) and 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide (2.06 g,
5.0 mmol) in acetonitrile (25 mL), Hg(CN)2 (1.26 g, 5.0 mmol) was added slowly under inertgas. After
24 h dichloromethane (DCM) (50 mL) was added, the undissolved material was filtered off, the organic
layer was washed with saturated NaHCO3 solution and water. The organic layer was dried (MgSO4), the
solvent evaporated in vacuo and the residue purified by flash chromatography.
Protocol 2 (Paulsen method)18: To a stirred mixture of the HFA-protected amino acid (2) (5.0 mmol) and
penta-O-acetyl-β-D-glucopyranose (1.95 g, 5.0 mmol) in dry DCM (20 mL) BF3·Et2O (635 µL, 5.0
mmol) was added. The mixture was stirred for 24 h at rt, diluted with DCM (50 mL) and extracted with
conc NaHCO3 solution and water. The organic layer was dried (MgSO4) and evaporated. The remaining
residue was purified by flash chromatography.
Protocol 3 (R.R. Schmidt method)19: To a stirred mixture of the HFA-protected amino acid (2) (5.0
mmol) and 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyltrichloroacetimidate (2.46 g, 5.0 mmol) in DCM
(20 mL), BF3·Et2O (127 µL, 1.0 mmol) was added and the mixture stirred for 1 h at rt. After addition of
DCM (20 mL) and water (10 mL) the mixture was stirred vigorously for 5 min. After separation, the
organic layer was dried (MgSO4) and evaporated under reduced pressure. The residue was purified by
flash chromatography.
(4S)-4-(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyloxymethyl)-2,2-bis(trifluoromethyl)-1,3-oxazo-
lidin-5-one (3a). 2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl trichloroacetimidate (2.46 g, 5.0 mmol),
2a (1.27 g 5.0 mmol) and BF3·Et2O (127 µL, 1.0 mmol) were reacted due to protocol 3. Purification by
o
flash chromatography (petroleum ether / ethyl acetate, 2:1). Yield: 78% (2.28 g) 3a, mp 173 – 174 C.
1H NMR (CDCl3, 300 MHz): δ = 2.01 (3H, s), 2.03 (3H, s), 2.06 (3H, s), 2.09 (3H, s), 3.69 – 3.75 (2H,
m), 3.98 – 4.11 (3H, m), 4.20 – 4.21 (2H, m), 4.59 (1H, d, J = 8.0 Hz,), 4.99 (1H, dd, J = 9.5 Hz, J =
8.0 Hz), 5.08 (1H, dd, J = 9.5 Hz, J = 9.5 Hz), 5.20 (1H, dd, J = 9.5 Hz, J = 9.5 Hz). 13C NMR (acetone-
d6): δ = 20.5, 20.6, 55.9, 62.6, 68.5, 69.2, 71.7, 72.7, 73.6, 89.3 (sept, J = 34 Hz), 101.0, 121.4 (q, J =
19
286 Hz), 122.5 (q, J = 287 Hz), 169.7, 170.0, 170.2, 170.3, 170.8. F NMR (acetone-d6): δ = -1.85 (3F,

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HETEROCYCLES, Vol. 64, 2004
q, J = 8.7 Hz), - 2.38 (3F, q, J = 8.7 Hz). IR (KBr): ν = 3320, 1840, 1750 cm-1. MS (EI, 70 eV): m/z =
584 [M+H]+, 583 [M]+, 540 [M – CH3CO]+, 332 [M – C6H3NO3F6]+, 331 [M – C6H4NO3F6]+, 271, 169,
109, 81. Anal. Calcd for C20H23NO12F6: C, 41.18; H, 3.97; N, 2.40. Found: C, 41.24; H, 3.84; N, 2.35.
(4S)-4-[(1R)-1-(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyloxy)ethyl]-2,2-bis(trifluoromethyl)-1,3-
oxazolidin-5-one (3b). 2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl trichloroacetimidate (2.46 g, 5.0
.
mmol), 2b (1.34 g, 5.0 mmol) and BF3 Et2O (127 µL, 1.0 mmol) were reacted due to protocol 3.
Purification by flash chromatography (toluene / ethyl acetate, 5:1). Yield: 82% (2.44 g) 3b, mp 112 –
1
113 °C. H NMR (CDCl3, 300 MHz): δ = 1.38 (3H, d, J = 6.5 Hz), 2.01 (3H, s), 2.02 (3H, s), 2.06 (3H,
s), 2.07 (3H, s), 3.66 – 3.76 (3H, m), 4.06 – 4.13 (2H, m), 4.22 (1H, dd, J = 12.5 Hz, J = 4.2 Hz), 4.57
(1H, d, J = 8.0 Hz), 4.95 (1H, dd, J = 9.6 Hz, J = 8.0 Hz), 5.09 (1H, dd, J = 9.7 Hz, J = 9.7 Hz), 5.20
13
(1H, dd, J = 9.7 Hz, J = 9.6 Hz). C NMR (CDCl3): δ = 18.1, 20.4, 20.5, 20.8, 59.9, 61.3, 68.0, 71.2,
72.2, 72.6, 75.7, 89.3 (sept, J = 34 Hz), 100.2, 120.2 (q, J = 286 Hz), 121.1 (q, J = 287 Hz), 169.2,
19
169.3, 169.4, 170.2, 170.7. F NMR (CDCl3, 282 MHz): δ = -0.94 (3F, q, J = 8.6 Hz, CF3), -2.89 (3F,
q, J = 8.6 Hz, CF3). IR (KBr): ν = 3000 – 2940, 1830, 1750 cm-1. MS (EI, 70 eV): m/z = 597 [M]+, 554
[M-CH3CO]+, 331 [M-C7H6NO3F6]+, 271, 169, 109. Anal. Calcd for C21H25NO12F6: C, 42.22; H, 4.22;
N, 2.34. Found: C, 42.22; H, 4.08; N, 2.36.
(5S,7R)-7-[2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyloxy]-2,2-bis(trifluoromethyl)-1-aza-3-oxabi-
cyclo[3.3.0]octan-4-one (3c). 2,3,4,6-Tetra-O-acetyl-α-D-glucopyranosyl bromide (1.03 g, 2.5 mmol),
2c (0.70 g, 2.5 mmol) and Hg(CN)2 (0.63 g, 2.5 mmol) were reacted due to protocol 1. Purification by
o
flash chromatography (petroleum ether / ethyl acetate, 2:1). Yield: 21% (0.32 g) 3c, mp 106 – 107 C.
1H NMR (CD3CN, 400 MHz): δ = 1.92 (3H, s), 1.96 (3H, s), 1.98 (3H, s), 1.99 (3H, s), 2.11 (1H, ddd, J
= 13.8 Hz, J = 9.1 Hz, J = 4.8 Hz), 2.31 (1H, ddd, J = 13.8 Hz, J = 7.9 Hz, J = 1.6 Hz), 3.31 (1H, dm, J
= 11.1 Hz), 3.51 (1H, ddq, J = 11.1 Hz, J = 4.2 Hz, J = 2.1 Hz), 3.81 (1H, ddd, J = 10.0 Hz, J = 5.3 Hz,
J = 2.5 Hz), 4.06 (1H, dd, J = 12.3 Hz, J = 2.5 Hz), 4.19 (1H, dd, J = 12.3 Hz, J = 5.3 Hz), 4.29 (1H,
dd, J = 9.1 Hz, J = 7.9 Hz), 4.58 (1H, m), 4.72 (1H, d, J = 8.1 Hz), 4.86 (1H, dd, J = 9.7 Hz, J = 8.1
13
Hz), 5.00 (1H, dd, J = 10.0 Hz, J = 9.8 Hz), 5.21 (1H, dd, J = 9.8 Hz, J = 9.7 Hz). C NMR (CD3CN,
100 MHz): δ = 20.8, 20.9, 34.8, 55.7, 61.2, 62.7, 69.3, 71.8, 72.7, 73.3, 81.4, 92.3 (m), 100.6, 121.0 (q,
19
J = 289 Hz), 122.2 (q, J = 287 Hz), 170.3, 170.5, 170.9, 171.3, 172.1. F NMR (CD3CN, 282 MHz): δ
= -3.09 (3F, q, J = 9.6 Hz, CF3), 5.48 (3F, q, J = 9.6 Hz, CF3). IR (KBr): ν = 3454, 1833, 1756 cm-1.
Anal. Calcd for C22H25NO12F6: C, 42.11; H, 4.02; N, 2.23. Found: C, 41.91; H, 3.90; N, 2.26.

HETEROCYCLES, Vol. 64, 2004
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(4S)-4-[4-(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyloxy)benzyl]-2,2-bis(trifluoromethyl)-1,3-ox-
azolidin-5-one (3d). 1,2,3,4,6-Penta-O-acetyl-β-D-glucopyranose (1.95 g, 5.0 mmol), 2d (1.64 g, 5.0
mmol) and BF3·Et2O (635 µL, 5.0 mmol) were reacted due to protocol 2. Purification by flash
chromatography (petroleum ether / ethyl acetate, 1:1). Yield: 83 % (2.74 g) 3d, mp 155 -158 oC (ethanol
1
/ petroleum ether). H NMR (DMSO-d6, 300 MHz): δ = 1.97 (3H, s), 2.01 (9H, s), 2.79 (1H, dd, J =
14.0 Hz, J = 8.0 Hz), 3.02 (1H, dd, J = 14.0 Hz, J = 4.5 Hz), 4.07 (1H, m), 4.20 (1H, m), 4.25 (1H, m),
4.43 (1H, m), 5.00 (1H, m), 5.05 (1H, m), 5.42 (1H, dd, J = 9.6 Hz, J = 9.6 Hz), 5.51 (1H, d, J = 8.0
Hz), 6.07 (1H, d, J = 6.7 Hz), 6.98 (2H, d, J = 8.7 Hz), 7.21 (2H, d, J = 8.7 Hz). 13C NMR (DMSO-d6,
75 MHz): δ = 20.2, 20.2, 20.3, 55.1, 61.6, 68.1, 70.7, 71.9, 88.1 (sept, J = 33 Hz), 97.1, 116.0, 120.2 (q,
19
J = 286 Hz), 120.9 (q, J = 289 Hz), 130.2, 130.9, 155.4, 169.0, 169.3, 169.5, 169.9, 171.3. F NMR
(DMSO-d6, 188 MHz): δ = -0.54 (3F, q, J = 9 Hz, CF3), -2.12 (3F, q, J = 9 Hz, CF3). IR (KBr) ν : 1830,
1732 cm-1. Anal. Calcd for C26H27NO12F6: C, 47.35; H, 4.13; N, 2.12. Found: C, 47.00; H, 4.20; N,
1.99.
O-(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl)serylphenylalanine amide (4a). A solution of 3a
(584 mg, 1.0 mmol) and phenylalanine amide (164 mg, 1.0 mmol) in ethyl acetate (20 mL) is stirred at
19
rt until the starting material was consumed (3 d, F NMR reaction control). The precipitate was filtered
o
off and carefully washed with ethyl acetate and ether. Yield: 79% (0.46 g) 4a, mp 183 C (methanol).
1H NMR (CDCl3): 2.00 (3H, s), 2.03 (3H, s), 2.04 (3H, s), 2.09 (3H, s), 3.07 (1H, dm; J = 13.8 Hz),
3.16 (1H, dm, J = 13.8 Hz), 3.53 (1H, dd, J = 7.4 Hz, J = 4.8 Hz), 3.63 (1H, dd, J = 10.0 Hz, J = 7.4
Hz), 3.70 (1H, ddd, J = 9.8 Hz, J = 4.8 Hz, J = 2.4 Hz), 3.79 (1H, dd, J = 10.0 Hz, J = 4.8 Hz), 4.14
(1H, dd, J = 12.5 Hz, J = 2.4 Hz), 4.25 (1H, dd, J = 12.5 Hz, J = 4.8 Hz), 4.50 (1H, d, J = 7.7 Hz), 4.65
(1H, m), 4.92 (1H, dd, J = 9.7 Hz, J = 7.7 Hz), 5.04 (1H, dd, J = 9.8 Hz, J = 9.7 Hz), 5.19 (1H, dd, J =
13
9.7 Hz, J = 9.7 Hz), 5.64 (1H, s), 6.15 (1H, s), 7.25 (5H, m), 7.65 (1H, d, J = 8.1 Hz). C NMR
(CD3OD): δ = 20.5, 20.6, 39.0, 55.4, 56.0, 63.1, 69.8, 72.8, 73.0, 73.2, 74.2, 101.9. 127.9, 129.5, 130.5,
138.3, 171.2, 171.3, 171.6, 172.4, 174.5, 175.8. IR (KBr): ν = 3400, 3020, 1726, 1688 cm-1. MS (FAB):
m/z = 604 [M + Na]+. Anal. Calcd for C26H35N3O12: C, 53.70; H, 6.07; N, 7.23. Found: C, 53.68; H,
6.09; N, 7.10.
O-(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl)threonylphenylalanine amide (4b). A solution of
3b (597 mg, 1 mmol) and phenylalanine amide (164 mg, 1 mmol) in ethyl acetate is stirred until the
19
starting material was consumed (3 d, F NMR reaction control). After evaporation of the solvent, the

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HETEROCYCLES, Vol. 64, 2004
residue was purified by flash chromatography (CHCl3 / CH3OH, 8:1). Yield: 68% (403 mg) 4b, amorph,
o
1
mp 75 -78 C. H NMR (CD3OD): δ = 1.12 (3H, d, J = 6.5 Hz), 1.96 (3H, s), 1.97 (3H, s), 1.99 (3H, s),
2.02 (3H, s), 3.00 (1H, J = 14.0 Hz, J = 7.6 Hz), 3.13 (1H, dd, J = 14.0 Hz, J = 5.8 Hz), 3.28 (1H, d, J =
6.5 Hz), 3.86 (1H, m), 3.99 (1H, dq, J = 5.0 Hz, J = 6.5 Hz), 4.09 (1H, dd, J = 12.3 Hz, J = 4.8 Hz),
4.28 (1H, dd, J = 12.3 Hz, J = 2.2 Hz), 4.61 (1H, dd, J = 6.0 Hz, J = 8.0 Hz), 4.73 (1H, d, J = 8.0 Hz),
4.84 (1H, dd, J = 8.0 Hz, J = 9.6 Hz), 4.98 (1H, dd, J = 9.6 Hz, J = 9.9 Hz), 5.26 (1H, dd, J = 9.6 Hz, J
13
= 9.6 Hz), 7.21-7.33 (5H, m). C NMR (CD3OD): δ = 16.8, 20.5, 20.6, 20.7, 38.9, 55.5, 60.3, 63.0,
69.8, 72.8, 74.1, 78.9, 100.6, 127.9, 129.5, 130.6, 138.2, 171.2, 171.3, 171.6, 172.4, 174.2, 175.7. MS
(MALDI): m/z = 635.3[M + K]+, 618.2 [M + Na]+.
N-Benzyloxycarbonylalanyl-O-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)threonylphenylalanine
amide (5b). To a stirred solution of Z-Ala-OH (76.7 mg, 0.34 mmol) in DCM (20 mL) HOAt (46.8 mg,
0.34 mmol) and DIC (53.3 µL, 0.34 mmol) were added at rt. Then a solution of 4b (100 mg, 0.17 mmol)
in DMF (1 mL) was added. After 2 h the reaction mixture was concentrated in vacuo, the residue was
redissolved in ethyl acetate (25 mL) and carefully washed with citric acid (10% solution), conc
NaHCO3 solution and water. The organic phase was dried (MgSO4) and evaporated; the residue was
purified by flash chromatography (CHCl3 / CH3OH, 10:1). Yield: 73% (101 mg) 5b, crystalline solid,
o
1
mp 190 C. H NMR (CD3OD): δ = 1.12 (3H, d, J = 6.2 Hz), 1.30 (3H, d, J = 7.2 Hz), 1.93 (3H, s),
1.98 (3H, s), 2.00 (3H, s), 2.03 (3H, s), 3.04 (1H, dd, J = 14.0 Hz, J = 7.7 Hz), 3.17 (1H, dd, J = 14.0
Hz, J = 5.5 Hz), 3.90 (1H, m), 4.07 (1H, dd, J = 12.3 Hz, J = 5.3 Hz), 4.14 (1H, m), 4.28 (1H, dd, J =
12.3 Hz, J = 5.3 Hz), 4.45 (1H, d, J = 6.2 Hz), 4.60 (1H, m), 4.77 (1H, d, J = 8.0 Hz), 4.85 (1H, dd, J =
9.6 Hz, J = 9.6 Hz), 5.01 (1H, dd, J = 9.6 Hz, J = 9.6 Hz), 5.05/5.10 (2H, dd, J = 12.5 Hz, J = 12.5 Hz),
13
5.27 (1H, dd, J = 9.6 Hz, J = 9.6 Hz), 7.15-7.35 (10H, m). C NMR (CD3OD): δ = 16.5, 17.8, 20.5,
20.6, 20.7, 38.6, 52.3, 55.8, 58.6, 63.1, 67.8, 69.8, 72.8, 73.2, 74.1, 76.4, 100.8, 127.9, 128.9, 129.0,
129.5, 129.6, 130.5, 138.3, 138.3, 158.5, 171.3, 171.4, 171.6, 172.4, 175.5, 175.9, 176.9. MS (MALDI):
m/z = 839.1 [M + K]+, 823.2 [M + Na]+.
ACKNOWLEDGEMENTS
The authors are grateful to Deutsche Forschungsgemeinschaft and to the Fonds of the Chemical Industry
for financial support.

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151
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