Neighbouring group participation of C-6 substituents of glucose derivatives on the stereoselectivity of the N-glycosidic linkage of glycopeptides

Article abstract of DOI:10.1515/znb-1999-0519

The first example of a glycopeptide with a direct N-α-glycosidic linkage between the trisaccharide and the amino acid residue was found in the glomerular basement membrane of rats. In connection with the total synthesis of nephritogenoside, glycosyl azides with different protecting groups and carbohydrate chain lengths are synthesized, reduced to the corresponding glycosyl amines and coupled with Z-Asp-OBzl. Remarkable differences in the α:β ratio of the condensation products are observed, caused by neighbouring group participation.

Full text of DOI:10.1515/znb-1999-0519

Neighbouring Group Participation of C-6 Substituents of Glucose Derivatives on
the Stereoselectivity of the N-Glycosidic Linkage of Glycopeptides
Hong Zhanga, Yali Wanga, Rene Thiirmera, Khalid Parvez", Iqbal Choudhary”,
Atta-ur-Rahmanb, Wolfgang Voelter3*
a Abteilung für Physikalische Biochemie des Physiologisch-chemischen Instituts der
Universität Tübingen, Hoppe-Seyler-Str. 4. D-72076 Tübingen
b HEJ Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan
Z. Naturforsch. 54b, 692-698 (1999); received February 2. 1999
Oligosaccharide Synthesis, Glycopeptides, Neighbouring Group Participation
The first example of a glycopeptide with a direct /V-a-glycosidic linkage between the trisac-
charide and the amino acid residue was found in the glomerular basement membrane of rats.
In connection with the total synthesis of nephritogenoside, glycosyl azides with different
protecting groups and carbohydrate chain lengths are synthesized, reduced to the corre-
sponding glycosyl amines and coupled with Z-Asp-OBzl. Remarkable differences in the a:ß
ratio of the condensation products are observed, caused by neighbouring group participation.
Introduction
this N-a-glycosydic bond were therefore carried
out by us.
Many secretory and membrane proteins carry
the N-glycosidic type [1 ], in which the side chain
amide function of an asparagine unit is usually
joined to an Af-acetylglucosamine residue via a ß-
N-glycosidic linkage [2], The structure of this type
[3] belongs to serum components such as amylases,
plasma proteins, immunoglobulins, hormones, hor-
mone precursors and membrane glycoproteins.
More recent investigations showed that cell
surface glycoproteins of the halobacteria contain
Af-glucosyl-[4] and N-galactosaminylasparagine
structures [5]. GP 120 of HIV-1 is also extremely
high glycosylated (31-36 AMinked oligosaccha-
rides per molecule) [6 ] and the full pathogenic po-
tential of HIV-1 in vitro is manifested only if its
viral proteins are N-glycosylated [7]. Nephritogen-
oside, isolated from the glomerular basement
membrane of rats is a glycopeptide with activity
for the induction of glomerulonephritis [8 ], The
Results and Discussion
Syntheses of the glycosylated asparagine linkage
have been carried out most often by Jeanloz’s
methodology [1 0 ] in which a glycosyl azide is re-
duced to the glycosylamine and condensed with
aspartic acid or an aspartoyl-containing peptide
under the agency of carbodiimide or other dehy-
drating agents [11]. The amide linkage can also be
achieved by the reaction of a carboxylic acid with
an azide in the presence of tertiary phosphine [1 2 ]
or coupling of glycosyl isothiocyanates with aspar-
tic acid [13]. A procedure whereby the peptidyl
and glycosyl moieties are linked directly to «-pen-
tenyl glycosides or thioglycosides, has been de-
scribed for the synthesis of /V-a-linked glycopep-
tides [14]. More recently, several groups [15] have
taken advantage of the much more simpler ap-
proach introduced by Kochetkov [16], in which the
reducing oligosaccharide is treated longer with sat-
urated aqueous ammonium bicarbonate to afford
the corresponding amine.
structure,
a-Glc-(1^6)-/?-Glc(l^-6)-a-Glc(l-^
Asn-peptide), is a-Af-glycosidically linked to the
peptide chain with the amido nitrogen of an aspar-
agine residue at the W-terminus [9]. This is the first
example of an a-N-glycosidic bond existing be-
tween glucose and the peptide part. Some studies
on the stereochemistry and the acid stabilities of
The difficulty of the synthesis of the N-a-aspara-
gine linkage is the anomerization of the glycosyl-
amine, generating an a/ß mixture. In the following
experiments, the stereochemical outcome of an N-
a-glycosyl bond relying on the neighbouring group
participation of the substituents at C- 6 of glucose
* Reprint requests to Prof. Dr. Dr. h.c. W. Voelter.
0932-0776/99/0500-0692 $06.00 © 1999 Verlag der Zeitschrift für Naturforschung, Tübingen • www.znaturforsch.com
D
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H. Zhang et al. ■N-Glycoside Linkage of Glycopeptides
Table I. Stereoselectivity of the N-glycosidic linkage relying on the neighbouring group participation of substituents
at C-6 of glucose derivatives.
Entry
Substrate
a : ß
Products
OAc
OAc
AcO
AcO
ac0" V £ j ! \
1 : 10
COOBn
yN I
NHOK X
aco-V ^ t V
AcO
Z-HN
1
6
^
COOtBu
H
NHCO^
AJoc-HN
OBn
OBn
BnO
BnO
BnO
BnO
COOBn
H
io
—-V -V
'
BnO
J
U
Z-HN
8
OH
OH
BnO
BnO
BnO
BnO
COOtBu
H
10' T ^ n I n W NHC0'^^X
BnO
Z-HN
Nj
9
OBn
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694
H. Zhang et al. ■N-Glycoside Linkage of Glycopeptides
OH
derivatives, as free hydroxyl-, acetyl-, benzyl-, O-
(2,3,4,6-tetra-0-acetyl-a-D-glucopyranosyl)-(1^6)-
0-2,3,4-tri-0-acetyl-/3-D-glucopyranosyl-, and O-
(2,3,4,6-tetra-0-benzyl-a-D-glucopyranosyl)-(l^<-6)-
0-2,3,4-tri-0-pivaloyl-ß-D-glucopyranosyl groups,
were studied.
BnO
n3
BnO
BnO
RnD I
OBn
1) H2, Lindlar catalyst. Et^N
The reduction of a-azide derivatives (1, 3, 4. 5)
to form glycosylamines was carried out in the pres-
ence of Lindlar catalyst in THF/MeOH/NEt3. Be-
cause of the instability of the glycosyl amine, puri-
fication is undesirable and the crude product was
used directly in the glycosylation reactions. The
condensation of the glycosylamines with TV-pro-
tected aspartic acid a-monoesters in the presence
of DCCI afforded a mixture of a- and /3-amides.
This mixture can be separated by silica gel column
chromatography and the «//^-configuration at C-l
was assignable according to NMR data. The effect
of substituents at position 6 of the glucose deriva-
tives on the stereooutcome is shown in Table I.
As seen from Table I, stereochemical purity at
C-l is partially lost during the catalytic hydrogena-
tion of the a-azide to the corresponding a-amine.
It is necessary to choose starting a-azide deriva-
tives with a non-neighbouring group activating
substituent at C-2 to introduce the N-a-glycosyl
bond. Entry 2 yields more a-stereochemistry at
the anomeric centre than that of 1 , and also entry
5 compared with entry 3, indicating that neigh-
bouring group participation of the 6-0-(2,3,4,6-
tetra-0-acetyl-a-D-glucopyranosyl)-(1^6)-0-2,3,4-
tri-0-acetyl-/?-D-glucopyranosyl- and the 6-0-
(2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl)-
(1—>6)-0-2,3,4-tri-0-pivaloyl-/?-D-glucopyranosyl
group has an important effect on a-stereoselectiv-
ity in the formation of the /V-glycosidic linkage.
Furthermore, the higher a-stereoselectivity of 9a
compared to 8 a may be due to the existence of an
intermediate (Scheme 1) and nucleophilic opening
of the bicyclic structure to 9a would preferentially
be with a-stereoselectivity.
Glycosidic bonds of glycopeptides are always
more or less acid-sensitive. The lability of the O-
glycosidic bond appears to depend on the struc-
ture of the glycopeptide. We have observed anom-
erization of the /V-a-glycosyl- to the /V-/3-glycosyl
bond in 60% trifluoroacetic acid-dichloromethane
after 1 h. indicating the higher acid lability of N-
a-glycosyl- to the /V-/3-glycosyl bond. The decom-
position of the glycosidic bonds of 10« after 1.5 h
2) Z-Asp-O tBu. TBTU, HOBt. DIEA
OH
BnO
ß—isom er
+
BnO
BnO
J
NHCO
Z-HN
Scheme 1.
also showed the sensitivity of the glycosidic bonds
toward acid conditions.
The stability of the /V-a-glycosyl and glycosidic
bonds in TFE/CH2Cl2/AcOH (2:7:1), the final
cleavage reagent of the glycopeptides from the
acid-sensitive 2 -chlorotrityl resin, was also investi-
gated. The glycosidic or glycosylamine bond
proved to be stable under these conditions.
In conclusion, this paper reports the a-stereo-
chemistry of the amination reaction by the neigh-
bouring group participation of the substituents at
C-6 as well as the lability of the /V-a-glycosyl link-
age toward acid conditions.
Experimental
2,3,4,6-Tetra-O-acetyl-a-D-glucopyranosyl azide (1)
A mixture of 2,3,4,6-tetra-O-acetyl-yS-D-gluco-
pyranosyl chloride (3.67 g, 0.01 mol) and sodium
azide ( 6 g, 0.09 mol) in HMPA (30 ml) was stirred
for 4 h at 20 °C, poured into ice-water and ex-
tracted with ethyl acetate-ether. The organic layer
was washed with water, dried over magnesium sul-
fate and evaporated in vacuum. The residue was
recrystallized from methanol to give 2.9 g of 1
(78% yield), m.p.: 98.5-99.5 °C. - FD-MS: m/z =
374.1 [M]+, 331.1 [M -N 3]+. - 'H NMR (250 MHz,
CDC13): (3 = 5.56 (d, J = 4.28 Hz, 1H, 1-H), 5.32
(t, J = 9.8 Hz, 1H. 3-H), 4.99 (t, / = 9.8 Hz, 1H, 4-
H), 4.90 (q, / = 4.3, 10.1 Hz, 1H, 2-H), 4.25-4.04
(m, 3H, 5-H, 6 -H. 6 '-H), 2.03, 2.02, 1.97, 1.95 (4s,
12H, 4 CH3CO). - 13C NMR (63 MHz, CDC13):
ö = 170.48, 169.83, 169.82, 169.42 (4C, 4 CH3CO),
86.19 (1C, 1-C), 70.11, 69.62, 69.57, 67.93, 61.56,
(5C, 2,3.4,5,6-C), 20.61, 20.57, 20.53, 20.49 (4C, 4
CH3CO).
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695
H. Zhang et al. • N-Glycoside Linkage of Glycopeptides
2,3,4,6-Tetra-O-benzyl-a-D-glucopyranosyl azide (3) wise. The mixture was stirred for further 3 h,
poured into ice-water (300 ml) and extracted with
ether (3x50 ml). The combined ether layers were
washed with water (3x50 ml), dried over sodium
A solution of 1 (3.73 g) in MeOH (20 ml) con-
taining 6 ml of NaOMe solution (0.5 g in 100 ml
methanol) was stirred for
2
h at room temper-
sulfate, filtered and evaporated. This crude pro-
duct was dissolved in dichloromethane ( 1 0 ml) and
10 ml of 50% TFA in dichloromethane were
added. The mixture was stirred at 0 °C for 10 min,
poured into 1 0 0 ml water and extracted with di-
chloromethane (3x50 ml). The combined extracts
were washed with 7% K2C 0 3 (2x50 ml), water
(2x50 ml), dried over sodium sulfate and concen-
trated in vacuum. Chromatography with dichloro-
methane-ethyl acetate (15:1) gave 1.25 g of 4 in
54% yield, m.p. 68-69 °C. TLC (dichloromethane/
ethyl acetate, 15:1): Rf = 0.39. - [a]D = +96.87 (c =
ature, than neutralised with 0.5 g Dowex-50 (H+)
and filtered. The filtrate was evaporated in vac-
uum and the residue was crystallized from metha-
nol to give a-D-glucopyranosyl azide. Yield: 1.76 g
(8 6 %). m.p.: 178-179 °C. - FD-MS: m/z = 228.1
[M+Na]+. - lH NMR (250 MHz, D2 0): (3 = 5.32
(d, J = 3.3 Hz, 1H, 1-H). - 13C NMR (63 MHz,
DMSO-d6): <3= 89.92 (1C, 1-C), 75.40, 73.27, 72.04,
69.85, 60.92 (5C, 2,3,4,5,6-C).
The
a - D - g lu c o p y r a n o s y l
azide
(1.00 g,
4.87 mmol) was then dissolved in dry dimethyl sul-
foxide ( 1 0 ml) and added dropwise to a suspension
of oil-free sodium hydride (0 . 8 g, 2 0 . 0 mmol of
60%) in dry dimethyl sulfoxide ( 1 0 ml) under ni-
trogen. The solution was stirred for 1 h at room
temperature. Benzyl bromide (3.45 ml, 5.0 g,
30.0 mmol) in 10 ml DMSO was added dropwise.
The mixture was stirred for a further 3 h. Excess
NaH was decomposed by adding MeOH (0.5 ml).
The reaction mixture was poured into ice-water
(300 ml) and extracted with ether (3x50 ml). The
combined ether layers were washed with water
(3x50 ml), dried over sodium sulfate, filtered and
evaporated. Chromatography with petroleum
ether-ethyl acetate (10:1—>7:1) gave 2.0 g of 3 in
73% yield. TLC (petroleum ether/ethyl acetate,
7:1): Rf = 0.5. - FD-MS: m/z = 565.0 [M]+. - lU
NMR (250 MHz, CDC13): <3 = 7.31-6.92 (m, 20H,
aromatic H), 5.13 (d, / = 4.2 Hz, 1H, 1-H), 4.86-
2.6, chloroform).
- FD-MS: 475.1 [M+], 951.5
[2M+]. - !H NMR (250 MHz, CDC13): (3 - 7.28-
7.12 (m, 15H, aromat. H), 5.07 (d, J = 4.11 Hz, 1H,
1-H), 4.87-4.53 (m, 6 H, 3 C //2Ph), 3.84-3.41 (m,
6 H, 2-H, 3-H, 4-H, 5-H, 6 -H, 6 '-H), 1.65 (s, 1H,
OH). - 13C NMR (63 MHz, CDC13): (5 = 138.54,
138.05, 137.68 (Cq, 3C, aromat. C), 128.70-127.77
(m, aromat. C), 87.98 (1-C), 81.67-73.36 (7C, 2-C,
3-C, 4-C, 5-C, 3 CH2Ph), 61.50 (6 -C).
2,3,4,6-Tetra-0-acetyl-N-(0-benzyl-N-benzyloxy-
carbonyl-L-ß-aspartyl)-a-D-glucopyranosylamine
(6a) and 2,3,4,6-Tetra-0-acetyl-N-(0-benzyl-N-
benzyloxycarbonyl-L-ß-aspartyl)-ß-D-gluco-
pyranosylamine (6ß)
A solution of 1 (373.3 mg, 1 mmol) in THF-
methanol (1 :1 , 1 0 ml) was hydrogenolyzed under
2 atm. in the presence of Lindlar’s catalyst
(186 mg) and triethylamine (1.26 g, 12.5 mmol) for
16 h. The catalyst was filtered off and the filtrate
was evaporated to dryness to give an a/ß mixture
of the glycosylamine. The anomeric mixture, the
amino acid Z-Asp-OBzl (421.7 mg, 1.2 mmol) and
1-hydroxybenzotriazole (229.7 mg, 1.7 mmol) were
dissolved in 5 ml dichloromethane at 0 °C and
4.33 (m, 8 H,
4 C //2Ph), 3.81-3.51 (m, 6 H,
2,3,4,5,6,6'-H). - 13C NMR (63 MHz, CDC13): (3 =
138.61, 138.18, 137.81, 137.75 (Cq, 4C, aromat. C),
128.71-127.02 (m, aromat. C), 88.16 (1C, 1-C),
81.85-65.30 (9C, 2,3,4,5,6-C, 4 CH2Ph).
2,3,4-Tri-O-benzyTcL-D-glucopyranosyl azide (4)
A
s o lu tio n o f a - D - g lu c o p y r a n o s y l a z id e (1.0 g,
4.87 mmol) and trityl chloride (1.36 g, 4.87 mmol) then DCC (247.6 mg, 1.2 mmol) was added. The
in pyridine (10 ml) was stirred at 50 °C for 16 h, mixture was stirred at 0 °C for 1 h and then left at
then the mixture was poured into 1 0 0 ml water room temperature for 12 h. Dicyclohexyl urea was
and extracted with dichloromethane (3x50 ml). filtered and the organic layers were washed with
The combined extracts were washed with water water, 1 0 % citric acid, water, dried and concen-
trated. Chromatography on silica gel with dichlo-
romethane-ethyl acetate (3:1) gave 480 mg of 6 a
(3x50 ml), dried over sodium sulfate and concen-
trated in vacuum. The obtained product was dis-
solved in dry dimethyl sulfoxide ( 1 0 ml) and added and 6ß in 70% yield.
dropwise to a suspension of oil-free sodium hy-
6a: - FD-MS: m/z = 686.0 [M]+. - lH NMR
dride (0.5 g, 12.0 mmol of 60%) in dry dimethyl (400 MHz, CDC13): (3 = 7.31-7.21 (m, 10H, 2Ph),
sulfoxide (10 ml) under nitrogen. The solution was 6.90 (d, J = 6.9 Hz, 1H), 5.75 (t, J = 5.7 Hz, 1H, 1-
stirred for 1 h at room temperature. Benzyl bro- H), 5.67 (d, J = 8.1 Hz, 1H, N//COBn), 5.25-4.93
mide (2.1 ml, 3.0 g, 17.5 mmol) was added drop- (m, 7H, 2,3,4-H, 2C //2Ph), 4.63-4.59 (m, 1H,
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696
H. Zhang et al. ■N-Glycoside Linkage of Glycopeptides
COCH2CH), 4.28-3.98 (m, 2H, 6 -H, 6 '-H), 3.90-
3.85 (m, 1H, 5-H), 2.87-2.65 (m, 2H, CO C//2CH),
2.01, 1.99, 1.95, 1.91 (4s, 12H, 4 CH^CO). - 13C
NMR (100 MHz, CDCI3): 6 = 171.46-156.1 (7C, 7
0 = 0 ), 136.13, 135.08 (Cq, 2C, aromat. C), 128.93-
128.45 (m, aromat. C), 74.45 (1C, 1-C), 70.47-
gel with dichloromethane-ethyl acetate (15:1) gave
6 6 8 mg of 8a and 8ß in 76% yield.
8 «: TLC (dichloromethane/ethyl acetate, 10:1):
R, = 0.43. - FD-MS: m/z = 879.2 [M]+. - ‘H NMR
(400 MHz, CDCI3 ): (3 = 7.23-7.03 (m, 30H. 6 Ph),
6.34 (d, J = 6.1 Hz, 1H), 5.94 (d, J = 7.9 Hz, 1H,
N//COBn), 5.62 (t, J = 6.0 Hz, 1H, 1-H), 5.06-
61.95 (7C, 2,3,4,5,6-C,
2 CH2Ph), 50.67 (1C,
COCH2CH), 37.12 (1C, COCH2CH), 20.95 (4C,
4 CH3 CO).
3.56 (m,
19H, 2,3,4,5,6,6'-H,
6
C //7Ph,
COCH2C//), 2.94-2.68 (m, 2H, COCH2CH). -
13C NMR (63 MHz, CDC13): (3 = 170.75, 170.60,
156.23 (3C, 3 C =0), 138.45-135.29 (Cq, 6 C, aro-
mat. C), 128.55-127.69 (m, aromat. C), 81.88 (1C,
C33H380 14N2 (686.67)
Calcd
C 57.72 H 5.58 N4.08%,
Found C 57.64 H 5.50 N 4.00%.
1-C), 77.58-67.04 (11C, 2,3,4,5,6-C,
6
CH2Ph),
50.95 (1C, COCH2 CH), 38.00 (1C, COCH2CH).
C53H54O 10 N2 (879.0)
6ß: - FD-MS: m/z = 686.0 [M]+. - 'H NMR
(250 MHz, CDCI3 ): (3 = 7.29-7.20 (m, 10H, 2 Ph),
6.63 (d, J = 9.21 Hz, 1H), 5.96 (d, J = 8.82 Hz, 1H,
Calcd
C 72.42 H 6.19 N3.19%,
Found C 72.57 H 6.10 N3.27%.
N//COBn), 5.25-4.79 (m, 8 H, 1,2,3,4-H,
2
C //2Ph), 4.59-4.55 (m, 1H, COCH2 C//), 4.24-
3.94 (m, 2H, 6 -H, 6 '-H), 3.74-3.69 (m, 1H, 5-H),
2.87-2.61 (dd, J = 16.3, 4.3 Hz, 2H, CO C//2CH),
1.98, 1.96, 1.94, 1.86 (4s, 12H, 4 C //3 CO). - ,3C
NMR (63 MHz, CDC13): <5= 171.08-156.15 (7C, 7
C =0), 136.15, 135.31 (Cq, 2C, aromat. C), 128.58-
127.99 (m, aromat. C), 78.06 (1C, 1-C), 73.70-
8ß: TLC (dichloromethane/ethyl acetate, 10:1):
Rf = 0.30. - FD-MS: m/z = 879.2 [M]+. - !H NMR
(250 MHz, CDCI3): (5 = 7.27-6.70 (m, 30H, 6 Ph),
5.92 (d, J = 8.79 Hz, 1H, N//COBn), 5.45 (d, J =
8.95 Hz, 1H), 5.05-4.27 (m, 14H, 1-H, 6 C //2Ph,
COCH2C//), 3.68-3.18 (m, 6 H, 2,3,4,5,6,6'-H),
2.70-2.33 (m, 2H, CO C //2 CH). - 13C NMR (63
MHz, CDCI3): (3 = 170.82, 170.10, 156.24 (3C, 3 C=
O), 138.38-135.52 (Cq, 6 C, aromat. C), 128.99-
127.85 (m, aromat. C), 86.17 (1C, 1-C), 79.30-
61.67 (7C, 2,3,4,5,6-C,
2 CH2Ph), 50.63 (1C,
COCH2CH), 37.69 (1C, COCH2 CH), 20.56 (4C,
4 CH3CO).
67.03 (11C, 2,3,4,5,6-C,
COCH2CH), 37.70 (1C, COCH2 CH).
6
CH2 Ph), 50.58 (1C,
C33H380 14N2(686.6)
Calcd
C 57.72 H 5.58 N 4.08%,
Found C 57.71 H 5.66 N 3.93%.
C53H54O 10 N2 (879.0)
Calcd
C 72.42 H 6.19 N3.19%,
2,3,4,6-Tetra-O-benzyl-N- (O-benzyl-N-benzyloxy-
carbonyl-L-ß-aspartyl)-a-D-glucopyranosyl-amine
(8a) and 2,3,4,6-Tetra-0-benzyl-N-(0-
benzyl-N-benzyloxycarbonyl-L-ß-aspartyl)-ß-D-
glucopyranosylamine (8ß)
Found C 72.50 H 6.26 N3.39%.
2,3,4-Tri-0-benzyl-N-(0-tert-butyl-N-benzyloxy-
carbonyl-L-ß-aspartyl)-a-D-glucopyranosylamine
(9a) and 2,3,4-Tri-0-benzyl-N-(0-tert-butyl-N-
benzyloxycarbonyl-L-ß-aspartyl)-ß -D-gluco-
pyranosylamine (9ß)
A solution of 3 (539.7 mg, 1 mmol) in THF-
methanol (1 :1 , 1 0 ml) was hydrogenolyzed under
2
atm. in the presence of Lindlar’s catalyst
A solution of 4 (475.6 mg, 1 mmol) in THF-
methanol (1 :1 , 1 0 ml) was hydrogenolyzed under
2 atm. in the presence of Lindlar’s catalyst
(200 mg) and triethylamine (1.26 g, 12.5 mmol) for
16 h. The catalyst was filtered off and the filtrate
was evaporated to dryness to give an alß mixture
(250 mg) and triethylamine (1.26 g, 12.5 mmol) for
16 h. The catalyst was filtered off and the filtrate
was evaporated to dryness to give an alß mixture
of the glycosylamine. The anomeric mixture, the
amino acid Z-Asp-OBzl (421.7 mg, 1.2 mmol) and
1-hydroxybenzotriazole (229.7 mg, 1.7 mmol) were of the glycosylamine. The anomeric mixture was
dissolved in 5 ml dichloromethane/DMF (9:1) at dissolved in TV./V-dimethylformamide and then a
0 °C and then DCC (247.6 mg, 1.2 mmol) was solution of Z-Asp-OtBu (605.6 mg, 1.2 mmol),
added. The mixture was stirred at 0 °C for 1 h, DIEA (20.5 /d, 1.2 mmol), HOBt (162.2 mg,
then left at room temperature for 12 h. Dicyclo-
1.2 mmol) and TBTU (385.3 mg, 1.2 mmol) in 5 ml
hexyl urea was filtered off and the organic layer dichloromethane/DMF (9:1) was added. The mix-
was washed with water, 1 0 % citric acid, water,
ture was stirred at 0 °C for 1 h, left at room tem-
dried and concentrated. Chromatography on silica
perature for 1 2 h, diluted with ethyl acetate,
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H. Zhang et al. ■N-Glycoside Linkage of Glycopeptides
697
washed with water, 1 0 % citric acid, water, dried 2 atm. in the presence of Lindlar’s catalyst (70 mg)
and concentrated. Chromatography on silica gel and triethylamine (0.13 g, 1.25 mmol) for 16 h. The
with chloroform-ethyl acetate (2:1) gave 605 mg of catalyst was filtered off and the filtrate was evapo-
9a and 9ß in 80.1% yield.
rated to dryness to give an a/ß mixture of the gly-
9a: TLC (chloroform/ethyl acetate, 1:1): Rf -
cosylamine. The anomeric mixture was dissolved
0.47. - FD-MS: m/z = 755.8 [M]+. - lH NMR (400 in A^TV-dimethylformamide and then a solution of
MHz, CDC13): (3 = 7.25-7.17 (m, 20H, 4 Ph), 6.53 Z-Asp-OtBu (60.6 mg, 0.12 mmol), DIEA (20.5 //l,
(d, J = 5.0 Hz, 1H), 5.85 (d, J = 7.4 Hz, 1H), 5.55 0.12 mmol), HOBt (16.2 mg, 0.12 mmol) and
(m, 1H), 5.05-4.50 (m, 8 H, 4 C //2 Ph), 4.4 (m, 1H, TBTU (38.5 mg, 0.12 mmol) in 5 ml dichlorometh-
COCH2C //), 3.7-3.45 (m, 6 H, 2,3,4,5,6,6'-H), ane/DMF (9:1) was added. The mixture was stirred
2.85-2.67 (m, 2H, C O C //2 CH), 1.44 (s, 9H, at 0 °C for 1 h, left at room temperature for 12 h,
(C //3 )3 CO). - 13C NMR (63 MHz, CDC13): <5 = diluted with ethyl acetate, washed with water, 1 0 %
171.36, 169.99, 156.40 (3C, 3 C =0), 138.56, 138.11, citric acid, water, dried and concentrated. Chro-
137.39, 136.35 (Cq, 4C, aromat. C), 128.80-127.73 matography on silica gel with dichloromethane-
(m, aromat. C), 82.49 (1C, 1-C), 81.75-61.62 [10C, ethyl acetate (15:1) gave 130 mg of 10« and 10ß in
2.3.4.5.6-C,
4 CH2 Ph, (CH3 )3 CO], 51.49 (1C, 76.8% yield.
COCH2 CH), 38.37 (1C, COCH2 CH), 27.93 [3C,
(CH3 )3 CO)].
10a: TLC (dichloromethane/ethyl acetate, 8:1):
Rf = 0.64. - FD-MS: m/z = 1692.4 [M]+. - lH
NMR (400 MHz, CDC13): (5 = 6.34 (d, J = 6 . 8 Hz,
1H), 5.90 (d, J = 7.4 Hz, 1H), 5.64 (d, J = 4.8 Hz,
1H), 2.73-2.55 (m, 2H, COCH2CH), 1.36 [s, 9H,
C43H50O 10 N2 (754.9)
Calcd
C 68.42 H 6 . 6 8 N3.71%,
Found C 68.77 H 6.43 N3.70%.
(C //3 )3 CO], 1.06, 1.04, 1.02 [3s, 27H,
3
(C //3 )3 CCO], - 13C NMR (100 MHz, CD2 C12):
(3 = 176.30, 175.63, 175.46 [3C, 3 (CH3 )3 CCO],
169.77, 168.99, 155.36 (3C, 3 C=0), 138.49-137.58
(Cq, 8 C, aromat. C), 99.62 (1C, lb-C), 96.31 (1C,
lc-C), 80.83 (1C, la-C), 80.47-67.50 (24 C), 50.67
(1C, COCH2 CH), 37.84 (1C, COCH2 CH), 37.79
[3C, 3 (CH3 )3 CCO], 26.90 [3C, (CH3 )3 CO)], 26.19
[9C, 3 (CH3)3CCO)].
9ß: TLC (chloroform/ethyl acetate, 1:1): Rf =
0.36. - FD-MS: m/z = 755.8 [M]+. - ]H NMR (250
MHz, CDCl,): <3= 7.35-7.15 (m, 20H, 4 Ph), 6.30
(d, J = 8.77 Hz, 1H), 5.89 (d, J = 8.4 Hz, 1H),
5.03-4.47 (m, 9H, 4 CH2Ph, 1-H), 4.29 (m, 1H,
COCH2CH), 3.73-3.23 (m, 6 H, 2,3,4,5,6,6'-H),
2.66-2.37 (m, 2H, C O C //2CH), 1.33 [s, 9H,
(C //3 )3 CO]. - 13C NMR (63 MHz, CDC13): (3 =
170.38, 169.90, 156.33 (3C, 3 C =0), 138.43, 138.20,
138.07, 136.29 (Cq, 4C, aromat. C), 128.63-127.74
(m, aromat. C), 85.84 (1C, 1-C), 82.48-61.36 [10C,
C98H 1 1 8 0 23N2 (1692.0)
Calcd
C 69.57 H 7.03 N 1.66%,
Found C 69.44 H 6.95 N 1.88%.
2.3.4.5.6-C,
4 CH2 Ph, (CH3 )3 CO], 51.15 (1C,
COCH2 CH), 38.16 (1C, COCH2CH), 27.90 [3C,
(CH3 )3 CO)].
10)9: TLC (dichloromethane/ethyl acetate, 8:1):
Rf = 0.53. - FD-MS: m/z = 1693.4 [M+H]+. - !H
NMR (400 MHz, CDC13): <5 = 6.19 (d, J = 9.1 Hz,
1H), 5.90 (d, J = 9.0 Hz, 1H), 2.80-2.41 (m, 2H,
CO C//2CH), 1.34 [s, 9H, (C //3 )3 CO], 1.07, 1.05,
1.03 [3s, 27H, 3 (C //3 )3 CCO], - 13C NMR (100
MHz, CDC13): (3 = 177.06, 176.23, 176.34 [3C, 3
(CH3 )3 CCOj, 171.10, 169.52, 155.98 (3C, 3 C=0),
100.56 (1C, lb-C), 97.10 (1C, lc-C), 85.74 (1C, la-
C), 82.50-66.01 (24 C), 51.27 (1C, COCH2CH),
39.90 (1C, COCH2CH), 38.92 [3C, 3 (CH3 )3 CCO],
28.10 [3C, (CH3 )3 CO)], 27.41, 27.37, 27.34 [9C, 3
(CH3 )3 CCO)].
C43H50O 10N2 (754.9)
Calcd
C 68.42 H 6 . 6 8 N3.71%,
Found C 68.40 H 6.41 N3.88%.
O -(2,3,4,6-Tetra-O-benzyl-a-D-glucopyranosyl) -
(l —>6)-0-(2,3,4-tri-0-pivaloyl-ß-D-gluco-
pyranosyl)-(l^-6)-2,3,4-tri-0-benzyl-l-N -(0-tert-
butyl-Nü-benzyloxy-carbonyl-L-ß-aspartyl)-a-D-
glucopyranosylamine (10«) and 0-(2,3,4,6-Tetra-
0-benzyl-a-D-gluco-pyranosyl)-(1^6)-0-(2,3,4-
tri-O-pivaloyl-ß-D-glucopyranosyl)-(l —*6) -2,3,4-
tri-O-benzyl-1 -N-(0-tert-butyl-Na-benzyloxy -
carbonyl-L-ß-aspartyl)-a-D-glucopyranosylamine
(10/?)
C98H 1 1 8 0 23N2 (1692.0)
Calcd
C 69.57 H 7.03 N 1.66%,
Found C 69.54 H 7.33 N 1.60%.
A solution of 5 (141.3 mg, 0.1 mmol) in THF-
methanol (1 :1 , 1 0 ml) was hydrogenolyzed under
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H. Zhang et al. • N-Glycoside Linkage of Glycopeptides
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