in EtOH with heating to 200–300°C. The eluents were C H –i-PrOH (10:1, 1); CHCl –i-PrOH (15:1, 2; 5:1, 3); and
6
6
3
n-BuOH–H O–AcOH (3:3:1, 4). Column chromatography (CC) was performed over silica gel 60 (63–200 μm, Merck).
2
Phenyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranoside (2). Amixture of 1 (1.0 g, 2.57 mmol) [16],
phenol (2.0 g, 21.27 mmol), and freshly calcined ZnCl (0.25 g, 1.84 mmol) were fused at 150°C for 1.5 h (TLC monitoring
2
by system 1). The reaction mixture was extracted with CHCl (30 mL). The organic layer was washed with KOH solution
3
(
2 M, 30 mL), dried over anhydrous Na SO , and evaporated to dryness. The solid was crystallized by adding Et O to afford
2
4
2
2
(0.92 g, 83%), mp 113–114°Ñ; [α]546 +169° (ñ 1.0, ÑÍCl ). Lit. [10]: mp 119.5–120°Ñ (pentane), [α] +150° (ÑÍCl ).
3 D 3
1
Í NMR spectrum (300 MHz, ÑDCl , δ, ppm, J/Hz): 1.96 (3Í, s, NAc), 2.02, 2.04, 2.06 (9Í, s, 3 OÀc), 4.03 (1Í, dd, J = 2.4,
3
1
2.9, Í-6a), 4.52 (1Í, ddd, J = 9.6, 2.4, 4.8, H-5), 4.07 (1Í, m, Í-2), 4.21 (1Í, dd, J = 4.8, 12.9, Í-6b), 5.57 (1Í, d, J = 3.6,
H-1), 5.22 (1Í, dd, J = 9.6, 9.6, H-4), 5.44 (1Í, dd, J = 10.5, 9.6, H-3), 5.82 (1Í, d, J = 9.3, NH), 7.04–7.38 (5Í, m, Ph).
+
+
+
ESI -MS, m/z 446.34 [M + Na] , calcd for Ñ H NO Na, 446.41; 462.43 [M + K] , calcd for Ñ H NO K, 462.52.
2
0
25
9
20 25
9
Phenyl-2-acetamido-2-deoxy-α-D-glucopyranoside (3). Peracetate 2 (1.99 g, 4.69 mmol) was dissolved in anhydrous
MeOH (10 mL) and treated with NaOMe in MeOH (0.5 mL, 0.1 M). When the reaction was finished (TLC monitoring by
systems 1 and 2), the precipitate was filtered off and rinsed with cold MeOH. The mother liquor was neutralized with KU-2
+
cation exchanger (H ). The resin was rinsed with MeOH. The filtrate was evaporated. The solid was again evaporated with
+
+
C H . The total yield of 3 was 1.25 g (90%), mp 187–188°Ñ, [α] +17° (ñ 1.0, C H OH). ESI -MS, m/z 320.10 [M + Na] ,
6
6
546
2 5
calcd for Ñ H NO Na, 320.30.
1
4
19
6
Phenyl-2-acetamido-2-deoxy-4,6-O-isopropylidene-α-D-glucopyranoside (4). A suspension of 3 (1.25 g,
.21 mmol) in anhydrous THF (15 mL) was heated with stirring to 50–55°C and treated with 2,2-dimethoxypropane (2.0 mL,
1.05 mmol) and anhydrous p-toluenesulfonic acid (15 mg). The reaction mixture was cooled after 1 h (TLC monitoring by
4
2
system 3), neutralized with Py (~50 μL), and evaporated. The solid was purified by CC (gradient elution, C H –i-PrOH,
6
6
+
+
5
0:1→10:1). Yield of 4, 0.56 g (40%), glassy solid; [α]546 +163° (ñ 1.03, CH Cl ). ESI -MS, m/z 338.19 [M + Í] , calcd for
2 2
+
Ñ H NO , 338.39; 360.18 [M + Na] , calcd for Ñ H NO Na, 360.37.
1
7
24
6
17 23
6
O-(Phenyl-2-acetamido-2,3-dideoxy-4,6-O-isotpropylidene-α-D-glucopyranosid-3-yl)-D-lactyl-L-alanyl-D-
isoglutamine Benzyl Ester (6). A suspension of 4 (0.56 g, 1.66 mmol) in anhydrous dioxane (20 mL) was stirred and treated
in portions with NaH (270 mg, 6.64 mmol) as a suspension (60%) in oil. The reaction mixture was heated at 95°C for 1 h,
cooled to 65°C, treated with (S)-2-bromopropanoic acid (0.22 mL, 2.49 mmol), heated at 65°C for another 3 h (TLC monitoring
by system 3), and cooled. The excess of NaH was decomposed by EtOH. The mixture was concentrated, poured into cold
H O (50 mL), acidified with HCl (2 M) to pH 3–4, and extracted with CHCl (3 × 20 mL) to afford 5. The extract was washed
2
3
with cold H O (10 mL). The organic layer was dried over anhydrous Na SO and evaporated.
2
2
4
Resulting acid 5 (650 mg, 1.59 mmol, 97%) was dissolved without further purification in anhydrous THF (15 mL),
stirred, treated with N-hydroxysuccinimide (205 mg, 1.75 mmol) and DCC (375 mg, 1.75 mmol), and left for 5 h.
The resulting precipitate of dicyclohexylurea was filtered off and rinsed with solvent. The filtrate was treated with
L-alanyl-D-isoglutamine benzyl ester trifluoroacetate [prepared by treatment of the corresponding Boc-derivative with
trifluoroacetic acid (675 mg, 1.66 mmol) followed by evaporation to dryness] and Et N to pH 8. When the reaction was
3
finished (TLC monitoring by system 3), the reaction mixture was evaporated. The solid was dissolved in CHCl (60 mL) and
3
washed with HCl (20 mL, 1 M), saturated NaHCO solution (20 mL), and H O (20 mL). The organic layer was dried over
3
2
anhydrous Na SO and evaporated. The solid was purified by CC (gradient elution, CHCl –i-PrOH, 50:1→10:1) to afford
2
4
3
+
+
amorphous 6 (0.95 g, 86%); [α] +111° (ñ 0.47, CH Cl ). ESI -MS, m/z 699.53 [M + Í] , calcd for Ñ H N O , 699.79;
21.59 [M + Na] , calcd for Ñ H N O Na, 721.79.
5
46
2
2
35 47 4 11
+
7
35 46 4 11
O-(Phenyl-2-acetamino-2,3-dideoxy-α-D-glucopyranosid-3-yl)-D-lactyl-L-alanyl-D-isoglutamine Benzyl Ester
7). Glycopeptide 6 (900 mg, 1.29 mmol) was dissolved in AcOH (70%, 3 mL) with heating on a boiling-water bath, held at
(
that temperature for 20 min (TLC monitoring by system 3), and evaporated to dryness. The solid was evaporated together with
toluene. The solid was purified by CC (gradient elution, CHCl –i-PrOH, 50:1→5:1). Yield of amorphous diol 7 (360 mg,
3
1
4
2%); [α]546 +119° (ñ 1.0, C H OH). Í NMR spectrum (400 MHz, DMSO-d , δ, ppm, J/Hz): 1.23, 1.28 (3Í each, d, J = 6.8,
2
5
6
ÌåÑÍ), 1.76, 2.00 (2Í, m, β-ÑÍ -iGln), 1.80 (3Í, s, NAc), 2.35 (2H, t, γ-ÑÍ -iGln), 4.58 (1Í, t, J = 6.4, 6-ÎÍ), 5.07 (2Í,
2
2
s, ÑÎ ÑH ), 5.40 (1Í, d, J = 6.0, 4-ÎÍ), 5.41 (1Í, d, J = 4.0, H-1), 6.99–7.10, 7.27–7.36 (12Í, m, 2 Ph, ÑÎNH ), 7.76 (1Í,
2
2
2
+
+
d, J = 7.2, NH-Ala), 8.18 (1Í, d, J = 8.0, NH-GlcN), 8.30 (1Í, d, J = 7.6, NH-iGln). ESI -MS, m/z 659.46 [M + Í] , calcd for
+
Ñ H N O , 659.72; 681.39 [M + Na] , calcd for Ñ H N O Na, 681.70.
3
2
43
4
11
32 42 4 11
O-(Phenyl-2-acetamido-2,3-dideoxy-α-D-glucopyranosid-3-yl)-D-lactyl-L-alanyl-D-isoglutamine (8). Benzyl
ester 7 (290 mg, 0.44 mmol) was dissolved in THF–H O (15 mL, 9:1) and subjected to hydrogenolysis over Pd/C (50 mg,
2
195