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2
-Azido-2-deoxy-3,4,6-tri-O-acetyl-d-mannose
imidate (9): 3,4,6-tri-O-acetyl-2-azido-2-deoxy-d-mannopyranose
400 mg, 1.20 mmol, 1 mol equiv) was dissolved in 5 mL CH Cl ,
trichloroacet-
protected product was satisfactory, the crude was used directly in
the following Zemplꢀn-deprotection step.
(
2
2
General procedure for Zemplꢀn deacetylation: 1 mol equiv
acetylated compound was dissolved in distilled MeOH and 1m
freshly prepared NaOMe in MeOH was added to the solution
cooled to 08C and trichloroacetonitrile (1.33 mL, 13.29 mmol,
1 mol equiv) was added to the solution. Then, 1,8-diazabicy-
clo[5.4.0]undec-7-ene (DBU; 45 mL, 0.30 mmol, 0.25 molequiv) was
1
(1.5 mol equiv NaOMe) to 0.1m final concentration of the sub-
added and the reaction was stirred at RT under N overnight. After
completion, the mixture was concentrated and the crude was puri-
fied by flash chromatography (R : 0.38 in hexane/EtOAc=75:25) to
yield the pure product as a white foam (439 mg, 77% yield, exclu-
2
strate. After completion, the reaction was neutralized with Amber-
liteꢄ IR120 hydrogen form ion-exchange resin, filtered and concen-
trated in vacuo. The crude was purified by direct (CH Cl /MeOH) or
f
2
2
reversed-phase flash chromatography (water/MeOH/MeCN, for am-
monium salts 0.01% TFA was added), yielding the pure product.
Compounds 5a–c, 12a–c and 16 were isolated as ammonium tri-
fluoroacetate salts.
2
D
0
1
sively a-product). [a] : +88.98 (c=0.6 in CH Cl ). H NMR
2
2
(
400 MHz, CDCl ): d=8.77 (s, 1H, NH), 6.29 (d, 1H, H , J =1.8 Hz),
3
1
1
-
2
5
.45 (t, 1H, H , J =J4-5 =10.0 Hz), 5.43 (m, 1H, H ), 4.28 (dd, 1H,
4
4-3
3
H , J =3.3 Hz), 4.25 (dd, 1H, H , J =12.7 Hz, J6a-5 =4.8 Hz,),
6a-6b
2
2-3
6a
Compound 16: Obtained in 76% yield over two steps (CuAAC and
4
2
.18–4.12 (m, 2H, H , H ), 2.12 (s, 3H, OAc), 2.09 (s, 3H, OAc),
.07 ppm (s, 3H, OAc); C NMR (100 MHz, CDCl ): d=170.8 (C=O
1
5
6b
deacetylation). R : 0.38 in water/MeOH=1:1 +0.01% TFA. H NMR
13
f
3
(
400 MHz, CD OD): d=8.29 (s, 1H, HTrCH), 7.30–7.18 (m, 8H, H12,
3
(
AcO)); 170.1 (C=O (AcO)); 169.6 (C=O (AcO)); 160.0 (C=NH); 95.5
H ), 5.28 (s, 1H, H ), 5.22 (dd, 1H, H , J =1.0 Hz, J2-3 =5.1 Hz),
13
1
2
1-2
(
C ); 90.42 (CCl ); 71.4 (C ); 70.8 (C ); 65.2 (C ); 61.8 (C ); 60.0 (C );
1
3
5
3
4
6
2
4
.55 (d, 4H, H , J=2.6 Hz), 4.32–4.20 (m, 7H, H , H , H ), 4.12–
10 3 15 16
2
0.9 (OAc); 20.8 (OAc); 20.7 ppm (OAc); MS (ESI): m/z calculated for
4
.08 (m, 1H, C ), 3.92–3.82 (m, 3H, H , H ), 3.79–3.72 (m, 3H, H ,
+
+
2
6a,b
7a
5
[
C H Cl N O Na] : 497.01 [M+Na] ; found: 496.88.
14 17 3 4 8
H , C ), 3.69–3.60 (m, 3H, H , H ), 3.01–2.83 (m, 2H, C , C ), 2.03–
7
b
1
8a,b
3
4
4
5
1
1
1
1
7
.89 ppm (m, 4H, C , C ); C NMR (100 MHz, D O): d=177.9 (C );
3 6 2 9
1
,2-Cyclohexanedicarboxylic acid, 4-(2-chloroethoxy)-5-((3,4,6-O-
77.6 (C ); 142.5, 142.4 (C ); 140.7 (C ); 139.8 (C ); 129.3 (C );
9
14
Trq
11
13
triacetyl)-2-azido-a-d-2-deoxymannopyranosyloxy)-1,2-di-
methylester (1S,2S,4S,5S) (11): A mixture of the acceptor 10
29.2 (C ); 126.8 (C ); 98.3 (C ); 77.1 (C or C ); 76.7 (C or C );
[3a]
12
TrCH
1
C1
5
5
C1
3.2 (C ); 71.6 (C ); 71.1 (C ); 69.0 (C ); 65.8 (C ); 65.5 (C ); 63.0
C2
7
3
4
10
2
(
124 mg, 0.42 mmol, 1 mol equiv) and the donor 9 (200 mg,
(
2
7
C ); 45.2 (C ); 44.6, 44.5 (C ); 42.5 (C , C ); 36.3 (C ); 30.8,
6 8 15 C4 C5 16
0
.42 mmol, 1 molequiv) was co-evaporated with toluene three
+
9.3 ppm (C , C ). MS (HRMS): m/z calculated for [C H ClN O ] :
C3
C6
+
35 48
6
10
times. Powdered and activated 4 ꢃ molecular sieves (acid washed)
were added. The mixture was kept under vacuum for 3–4 hours
and then dissolved in dry CH Cl (10 mL). After cooling to À308C,
47.3120 [M+H] , found: 747.3123.
2
2
trimethylsilyl
trifluoromethanesulfonate
(TMSOTf;
15.2 mL,
0
.084 mmol, 0.2 mol equiv) was added to the stirred mixture. The
Sample preparation for ITC
solution was stirred at À308C for 1 hour and upon completion, the
reaction was quenched with triethylamine (TEA). The mixture was
warmed to room temperature and filtered over a Celite pad. The
filtrate was evaporated at reduced pressure and the crude product
was purified by flash chromatography (hexane/EtOAc=72:28) to
yield the pure product as a white foam (240 mg, 94% yield, exclu-
ITC experiments were performed at 258C by using a TA Instrument
Nano Isothermal Titration Calorimeter Low Volume (Nano ITC LV)
with a 190 mL cell volume. Compound 16 and DC-SIGN ECD were
prepared in 25 mm Tris-HCl at pH 8, 150 mm NaCl, 4 mm CaCl and
2
4
% DMSO. 100 mm of DC-SIGN ECD and 2.5 mm compound con-
centrations were used. The compound was stepwise injected
1.03 mL) into DC-SIGN solution by using 5 min intervals between
20
1
sively a-product). [a] : +73.48 (c=0.68 in CH Cl ); H NMR
D
2
2
(
(
400 MHz, CDCl ): d=5.35 (dd, 1H, H , J =3.6 Hz, J3-4 =9.6 Hz),
3 3 3-2
injections. A blank titration (compound to buffer) was done for
subtraction of dilution heat from the integrated data. A one-site
binding model was fitted to the data (nanoAnalyse 2.20 TA), yield-
5
.29 (dd, 1H, H , J = 9.4 Hz), 4.98 (d, 1H, H , J = 1.5 Hz), 4.21
4 4-5 1 1-2
(
dd, 1H, H , J = 5.4 Hz, J6-6b = 12.2 Hz), 4.08 (dd, 1H, H , J
=
6
a
6-5
6b 6b-5
2
.3 Hz), 4.03 (m, 1H, H ), 3.98–3.94 (m, 1H, H ), 3.94–3.90 (m, 1H,
2 5
ing dissociation constants (K ) and binding enthalpies (DH).
D
C ), 3.87–3.80 (m, 1H, H ), 3.70 (s, 3H, H ), 3.69 (s, 3H, H ), 3.68–
2
7a
10
10
3
3
1
.64 (m, 1H, H ), 3.62–3.59 (m, 2H, H8a,b), 3.59–3.58 (m, 1H, C1),
7b
.02 (dt, 1H, C , J
=4.0 Hz, JC4-C3ax =JC4-C5 =12.0 Hz), 2.88 (dt,
C4-C3eq
4
H, H , J
= 4.0 Hz, JC5-C6ax =12.0 Hz), 2.10 (s, 3H, OAc), 2.08
C5-C6eq
C5
Surface plasmon resonance (SPR) analysis
(
s, 3H, OAc), 2.04 (s, 3H, OAc), 2.10–1.80 ppm (m, 4H, HC3ax, HC3eq,
1
3
HC6eq, HC6ax); C NMR (100 MHz, CDCl ): d=174.9 (C ); 174.6 (C );
The extracellular domain (ECD) of DC-SIGN (residues 66–404) was
overexpressed and purified as previously described. The SPR ex-
periments were performed on a BIAcore T200 using a CM3 sensor
chip. Flow cells were activated as previously described. Flow cell
one was functionalized with BSA and blocked with ethanolamine
and subsequently used as a control surface. Flow cells 2 and 3
3
9
9
1
7
70.8 (C=O (AcO)); 170.0 (C=O (AcO)); 169.6 (C=O (AcO)); 95.9 (C1);
5.3 (C ); 71.7 (C ); 71.0 (C ); 69.6 (C ); 69.2 (C ); 66.3 (C ); 62.6
C1
C2
3
7
5
4
[18]
(
(
C ); 61.9 (C ); 52.2 (C ); 43.2 (C ); 39.2 (C ); 39.0 (C ); 28.0, 26.8
C , C ); 20.9 (Me (AcO)); 20.8 (Me (AcO)); 20.7 ppm (Me (AcO));
6
2
10
8
C5
C4
C3
C6
+
+
MS (ESI): m/z calculated for [C H ClN O Na] : 630.17 [M+Na] ,
24
34
3
13
found: 630.51
General procedure for CuAAC reactions. 1m CuSO ·5H O and Na
were
treated
with
BSA-Mana1-3[Mana1-6]Man
(Dextra)
À1
(60 mgmL ) in 10 mm NaOAc pH 4 to reach different binding den-
sities and blocked with ethanolamine. The final densities on flow
cells 2 and 3 were 2579 and 2923 RU, respectively. The affinity of
the various compounds for DC-SIGN ECD were evaluated via an es-
tablished inhibition assay in which DC-SIGN ECD was injected at
20 mm alone or in the presence of increasing concentration of in-
hibitors (ranging from 0 to 5 mm). Injections were performed at
4
2
ascorbate solutions were prepared in degassed water. The alkyne
1 molequiv) was dissolved in degassed THF and 0.1 equiv
CuSO ·5H O solution and 0.4 molequiv Na ascorbate solutions
(
[18]
4
2
were added, under nitrogen atmosphere. The azide was also dis-
solved in degassed THF and added to the mixture. The reaction
TM
À1
was stirred under nitrogen, then, upon completion, QuadraSil MP
5 mLmin using 25 mm Tris-HCl pH 8, 150 mm NaCl, 4 mm CaCl2,
metal scavenger was added to remove the copper, the mixture
was filtered and concentrated in vacuo. When the purity of the
0,05% P20 surfactant as running buffer. The surface was regenerat-
ed by the injection of 50 mm EDTA.
&
&
Chem. Eur. J. 2019, 25, 1 – 11
8
ꢂ 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!