EFFECT OF SUGAR CHAIN LENGTH ON THE ANTIBACTERIAL ACTIVITY
compound 5 (4.8g, 69%). 1H NMR (300MHz, CDCl3) δ7.77 (d,
J= 7.4Hz, 2H, Ar), 7.63 (d, J = 7.3Hz, 2H, Ar), 7.42–7.27 (m, 9H, Ar),
5.67 (d, J = 9.3Hz, 1H, NH), 5.30 (s, 2H, OCH2Ph), 5.19–5.04 (m, 3H,
H-2’ , H-3, H-3’ , H-4’ ), 4.96–4.81 (m, 2H, H-2, CHThrα), 4.53–4.33 (m,
7H, H-1’ , H-1, FmocCH2, CHThrβ, H-6a, H-6’a), 4.24 (t, 1H, J= 7.2Hz,
FmocCH), 4.07–3.97 (m, 2H, H-6b, H-6’b) 3.74–3.64 (m, 2H, H-4,
H-5’ ), 3.34–3.30 (m, 1H, H-5), 2.10, 2.05, 2.04, 2.03, 2.01, 1.99 (6s,
21H, 7Ac), 1.21 (d, J= 6.2 Hz, 3H, Thr-CH3). 13C NMR (75MHz, CDCl3):
δ 170.65, 170.44, 170.38, 169.99, 169.95, 169.63, 169.48 (COCH3),
169.23, 156.91 (CO Fmoc), 144.13, 143.88, 141.44, 135.53, 128.83,
128.77, 128.35, 127.87, 127.24, 125.39, 120.12 (Ar C), 100.94 (C-1),
98.78 (C-1’ ) 76.36, 75.29, 73.09, 72.73, 72.32, 72.14, 71.74, 71.61,
67.96, 67.48, 67.40, 61.76, 58.59, 47.30, 20.89, 20.83, 20.74, 20.71
(COCH3), 17.46 (CH3 Thr). ESI HRMS: calcd for C52H59NO22[M+H]+
m/z, 1050.3607; found, 1050.2557.
(COCH3), 17.48 (CH3 Thr). ESI HRMS: calcd for C52H59NO22[M+H]+
m/z, 1050.3607; found, 1050.3026.
Nα-Fluoren-9-ylmethoxycarbonyl-O-(2,3,4,6-tetra-O-acetyl-β-D-
galactopyranosyl-(1->4)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl)-
L-threonine (III)
To a solution of 6 (2g) in methanol, 100 ml was added 5% Pd/C
(400 mg). The mixture was stirred under one atmosphere of
hydrogen gas until the reaction was complete. The catalyst was
removed by filtration, and after solvent evaporation, the residue
was purified by flash chromatography using DCM as solvent A
and 10% MeOH-DCM as solvent B (gradient of 0–60% B over
50 min) to afford the titled compound III (1.2g, 65%). 1H NMR
(300 MHz, CD3OD): δ 7.79 (d, J= 7.5 Hz, 2H, Ar), 7.69–7.65 (m, 2H,
Ar), 7.42–7.29 (m, 4H, Ar), 5.36 (d, J= 3.1Hz, 1H, H-4’ ), 5.19 (t,
J = 9.2Hz, 1H, H-3), 5.21 (dd, J = 10.3, 3.2 Hz, 1H, H-3’ ), 5.03 (dd,
J = 10.3, 7.7 Hz, 1H, H-2’ ), 4.86–4.78 (m, 3H, FmocCH2, H-2), 4.66 (d,
J = 7.4Hz, 1H, H-1’ ), 4.65 (d, J = 7.6Hz, 1H, H-1), 4.49–4.32 (m, 3H,
Fmoc CH, CHThrβ, CHThrα), 4.28–4.21 (m, 2H, H-6a, H-6’a),
4.17–4.09 (m, 3H, H-5’ , H-6b, H-6’b), 3.84 (t, J= 9.1Hz, 1H, H-4),
3.72–3.65 (m, 1H, H-5), 2.12, 2.08, 2.06, 2.03, 2.01, 1.94 (6s, 21H,
7Ac), 1.18 (d, J = 6.0Hz, 3H, Thr-CH3); 13C NMR (75 MHz, CD3OD) δ
173.27, 172.63, 172.14, 172.04, 171.79, 171.56, 171.22 (COCH3),
158.77 (CO Fmoc), 145.40, 145.19, 142.64, 128.97, 128.35, 126.44,
121.13 (Ar C), 102.12 (C-1), 100.06 (C-1’ ), 77.63, 76.55, 74.35, 73.85,
73.13, 72.51, 71.86, 70.79, 68.69, 68.21, 63.64, 62.51, 61.64, 59.81,
54.97, 48.49 (CH Fmoc), 21.30, 20.99, 20.96, 20.92, 20.82, 20.68
(COCH3), 17.96 (CH3 Thr). ESI HRMS: calcd for C45H53NO22 [M+H]+
m/z, 960.3137; found, 960.3051.
Nα-Fluoren-9-ylmethoxycarbonyl-O-(2,3,4,6-tetra-O-acetyl-β-D-
glucopyranosyl-(1->4)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl)-
L-threonine (II)
A mixture of 5 (1.6g) and 10%Pd/C (700 mg) in ethyl acetate was
treated as described for the synthesis of I. Purification by flash chro-
matography using DCM as solvent A and 10% MeOH-DCM as sol-
vent B (gradient of 0–50% B over 40 min) to afford II (990 mg,
68%) as a white solid. 1H NMR (300MHz, CD3OD): δ 7.81 (d,
J= 7.4Hz, 2H, Ar), 7.70–7.67 (m, 2H, Ar), 7.42–7.29 (m, 4H, Ar), 5.25
(t, J = 9.4Hz, 1H, H-3 ), 5.18 (t, J= 9.3 Hz, 1H, H-3), 5.01 (t, J = 9.6 Hz,
1H, H-4’ ), 4.85–4.78 (m, 2H, H-2’ , H-2), 4.68 (d, J= 7.9 Hz, 1H, H-1’ ),
4.67 (d, J = 7.9Hz, 1H, H-1), 4.51–4.32 (m, 5H, CHFmocα, FmocCH2,
H-6a, H-6’a), 4.27–4.20 (m, 2H, H-6’b, FmocCH), 4.15–4.02 (m, 2H,
H-4, H-6b), 3.89–3.81 (m, 2H, H-5’, CHThrβ), 3.73–3.68 (m, 1H, H-5),
2.09, 2.05, 2.04, 2.03, 2.01, 1.99, 1.94 (7s, each 3H, 7Ac), 1.19 (d,
J= 6.3Hz, 3H, Thr-CH3); 13C NMR (75 MHz, CD3OD) δ 173.37,
172.70, 172.32, 172.00, 171.76, 171.57, 171.32, 171.08, 158.93
(COCH3 and CO Fmoc), 145.46, 145.27, 142.75, 128.99, 128.37,
126.47, 121.13 (Ar C), 102.06 (C-1), 100.17 (C-1’), 78.07, 76.58,
74.59, 74.23, 73.99, 73.24, 73.07, 69.43, 68.31, 63.64, 62.96, 59.90,
54.95, 21.10, 20.95, 20.90, 20.83, 20.79, 20.69 (COCH3), 17.94 (CH3
Thr). ESI HRMS: calcd for C45H53NO22 [M+H]+ m/z, 960.3137; found,
960.2976.
Broth Microdilution Assay
Minimum inhibitory concentrations (MICs) of peptides against
various bacterial strains such as Escherichia coli ATCC 35218, E. coli
ATCC 25922, E. coli ATCC 11775, Salmonella typhimurium ATCC
14028 and S. typhi Vi+ were determined following the earlier
reported protocol [20]. Each test condition was set up in duplicate,
and the assay was repeated thrice to calculate the average and the
standard error.
Nα-Fluoren-9-ylmethoxycarbonyl-O-(2,3,4,6-tetra-O-acetyl-β-D-
galactopyranosyl-(1->4)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl)-
L-threonine benzyl ester (6)
Binding Kinetics by Surface Plasmon Resonance
Binding studies between substrate binding domain-DnaK
(substrate binding domain of DnaK-E. coli) and glycosylated
analogs of formaecin I and drosocin were performed on
BiacoreTM T-200 (Biacore, Uppsala, Sweden) following the
earlier described protocol of covalent immobilization of His6-
tagged protein on nickel-nitrilotriacetic acid sensor chip
[21,22]. The reference surface was treated in the same way,
but no ligand was immobilized on this surface. For binding
kinetics, an injection of each peptide with concentration rang-
ing from 5 μM–156 nM was passed over immobilized surfaces,
allowing an association time of 180 s and disassociation time
of 900 s followed by two regeneration injections of 15 s each
with 500 mM NaCl. All the experiments were performed at
25 °C. The reference-subtracted response curves obtained for
peptide binding were evaluated using Biacore T-200 evaluation
software (Biacore). The data obtained with serially diluted
peptide concentrations were fitted in Langmuir 1 : 1 interaction
model to obtain rates of association (kass) and dissociation
(kdiss). The equilibrium dissociation constant (KD) was defined
Purification of crude residue by flash chromatography (gradient of
0–60% EtOAc-hexane over 60 min) afforded 6 (2.25 g, 70%) as an
amorphous white solid. 1H NMR (300MHz, CDCl3): δ 7.76 (d,
J= 7.5Hz, 2H, Ar), 7.63 (d, J = 7.5Hz, 2H, Ar), 7.42–7.28 (m, 9H, Ar),
5.64 (d, J = 9.3 Hz, 1H, NH), 5.36 (1H, d, J = 3.1 Hz, H-4’), 5.30 (2H, br
s, OCH2Ph), 5.20–5.08 (m, 3H, H-2’, H-2, H-3), 4.95 (dd, J = 10.4,
3.4 Hz, 1H, H-3’), 4.86–4.81 (m, 1H, CHThrα), 4.46–4.33 (m, 6H, H-1’,
H-1, FmocCH2, H-6b, H-6’b), 4.24 (t, J = 7.3Hz, 1H, Fmoc CH),
4.17–4.05 (m, 2H, H-6’a, CHThrβ), 4.01 (dd, J = 12.1, 4.8 Hz, 1H,
H-6a), 3.87–3.82 (m, 1H, H-5’), 3.73 (t, J = 9.4Hz, 1H, H-4), 3.38–3.34
(m, 1H, H-5), 2.16, 2.07, 2.06, 2.05, 2.04, 1.97 (6s, 21H, 7Ac), 1.21 (d,
J= 6.1Hz, 3H, Thr-CH3); 13C NMR (75MHz, CDCl3): δ 170.57, 170.34,
170.27, 170.03, 169.97, 169.69, 169.28 (COCH3), 156.95 (CO Fmoc),
144.19, 143.94, 141.5, 135.58, 128.88, 128.80, 128.39, 127.92,
127.29, 125.44, 120.17 (Ar C), 101.28 (C-1), 98.78 (C-1’), 77.43,
76.19, 75.31, 72.78, 72.69, 71.77, 71.20, 70.94, 69.29, 67.55, 67.45,
66.85, 61.95, 61.07, 58.68, 53.63, 47.37, 21.02, 20.94, 20.86, 20.72
J. Pept. Sci. 2015; 21: 833–844
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
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