# Supplementary Material (ESI) for Chemical Communications
# This journal is © The Royal Society of Chemistry 2003
Supplementary information
A general entry to linear, dendritic and branched thiourea-linked glycooligomers as new motifs for phosphate ester recognition in water
José L. Jiménez Blanco,a Purificación Bootello,a Carmen Ortiz Mellet,*a Ricardo Gutiérrez Gallego,b and José M. García Fernández*c
a Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Apartado 553, E-41071 Sevilla, Spain. Fax: +34 954624960; Tel: +34 954557150; E-mail:
b Pharmacology Research Unit, Municipal Institute of Medicinal Research (IMIM), Department of Experimental and Health Sciences, University Pompeu Fabra, Doctor Aiguader 80, E-08003 Barcelona (Spain)
c Instituto de Investigaciones Químicas, CSIC, Américo Vespucio s/n, Isla de la Cartuja,E-41092 Sevilla, Spain. Fax: +34 954460565; Tel: +34 954489559; E-mail:
General: Methyl 6-amino-6-deoxy-a-d-glucopyranoside 41 and 2,3,4,6-tetra-O-acetyl-b-d-glucopyranosyl isothiocyanate 62 were obtained according to literatutre procedures. N-acetyl 3,6-diamino-3,6-dideoxy-b-d-glucopyranosylamine 5 was prepared by conventional acetylation of ammonium 3,6-diazido-3,6-dideoxy-b-d-glucopyranosylcarbamate 39 (see Scheme 2) followed by azide reduction with 1,3-propanedithiol-triethylamine. Optical rotations were measured at room temperature in 1-cm or 1-dm tubes on a Perkin-Elmer 141 MC polarimeter. Infrared (IR) spectra were recorded on a Bomem Michelson MB-120 FTIR spectrophotometer. 1H (and 13C NMR) spectra were recorded at 300 (75.5), 500 (125.7) MHz with Bruker 300 AMX, 500 AMX and 500 DRX. Spectra recorded at 298 K showed broad signals due to slow rotation processes about the NH—C(S) bonds in the NMR time scale. Satisfactory resolutions were achieved after heating above 313 K. 1D TOCSY, 2D COSY, HMQC and HSQC experiments were used to assist on NMR assignments. Thin-layer chromatography (TLC) was carried out on aluminium sheets coated with Kieselgel 60 F254 (E. Merck), with visualization by UV light and by charring with 10% H2SO4. Column chromatography was carried out on Silica Gel 60 (E. Merck, 230-400 mesh). Gel permeation chromatography (GPC) of the fully unprotected pseudooligosaccharides was carried out on Sephadex G-10 (1:1 water/MeOH) or G-25 (water) columns (Pharmacia Amersham) attached to a Gradifrac system using a UV detector set at 248 nm. FAB mass spectra were obtained with a Kratos MS-80 RFA instrument. The operating conditions were the following: the primary beam consisted of Xe atoms with a maximum energy of 8 keV; the samples were dissolved in thioglycerol, and the positive ions were separated and accelerated over a potential of 7 keV; NaI was added as cationizing agent. MALDI-TOF mass spectra were acquired on a GSG System spectrometer operating in the positive-ion mode with an accelerating voltage of 28 keV. Samples were dissolved in water at millimolar concentration and mixed with a standard solution of 2,5- dihydroxybenzoic acid (DHB; 10 mg mL-1 in 10% aq EtOH, 2 mL) in 1:1 v/v relative proportions; 1 mL of the mixture was loaded onto the target plate, then allowed to air-dry at room temperature. ESI mass spectra were recorded in the positive mode on an Esquire 3000 ion-trap mass spectrometer (Bruker Daltonik GmbH). Typically, samples were dissolved in appropriate volumes of deionised water to give sample concentrations of 50 mg/l. Aliquots were mixed with 25:25:1 deionised water-methanol-trifluoro acetic acid, generally in a ratio of 1:10, to give a total volume of 200 ml. Samples were introduced by direct infusion, using a Cole-Parmer syringe at a flow rate of 2 ml/min. Ions were scanned between 300 and 3000 Da with a scan speed of 13000 Da/s at unit resolution using resonance ejection at the multipole resonance of one-third of the radio frequency (W = 781.25 kHz). Calibration of the mass spectrometer was performed using ES tuning mix (Hewlett Packard). Recorded data were processed using Bruker Daltonics Esquire 5.0 software (Bruker). Elemental analyses were performed at the Instituto de Investigaciones Químicas (Sevilla, Spain).
Synthesis of the AB building block 1
Scheme 1. Synthesis of sugar azido isothiocyanate 1: i) 90% TFA-water (quantitative); ii) NH4HCO3-16 m aq ammonia, 40 ºC, 36 h; iii) diethyl ethoxymethylenemalonate, MeOH (62% from 31); iv) 1:1 Ac2O-pyridine (96%); v) Cl2, CH2Cl2, 5 min, room temperature (97%); vi) CSCl2, CaCO3, CH2Cl2-water, 30 min, room temperature (64%).
2,3,4-Tri-O-acetyl-6-azido-6-deoxy-b-d-glucopyranosyl isothiocyanate 1: Compound 1 was prepared from 6-azido-6-deoxy-1,2-isopropylidene-a-d-glucofuranose 313 as depicted in Scheme 1. Deacetonation of 31 with 9:1 TFA-water at room temperature for 1 h afforded fully unprotected 6-azido-6-deoxy-d-glucose 324 in quantitative yield. To a solution of 32 (4.88 g, 24 mmol) in aq ammonia (16 m, 275 mL), NH4HCO3 (4.20 g, 53.2 mmol) was added and the mixture was stirred at 40 ºC for 36 h. The solvent was concentrated to half volume under reduced pressure, water (120 mL) was added and the solution was freeze-dried. The product thus obtained contained, almost exclusively, ammonium 6-azido-6-deoxy-b-d-glucopyranosylcarbamate 33, as seen by 13C NMR. Attemps to transform the carbamate salt into the corresponding glycosylamine by successive lyophilisation of aq solutions resulted in extensive formation of bis(glycosylamine) as well as in hydrolysis to give 32. Instead, crude 33 was dissolved in dry MeOH (50 mL) and diethyl ethoxymethylenemalonate (7.8 mL, 36 mmol) was added. The mixture was stirred at room temperature for 48 h, then concentrated and the residue purified by column chromatography (EtOAc®45:5:3 EtOAc-EtOH-H2O) to give N-(2,2-diethoxycarbonylvinyl)-6-azido-6-deoxy-b-d-glucopyranosylamine 34 (5.57 g, 62%). Conventional acetylation of 34 with 1:1 pyridine-acetic anhydride gave the corresponding triacetate 35 (96%).
Enamine 35 (700 mg, 1.4 mmol) was dissolved in a saturated solution of Cl2 in CH2Cl2 (15 mL) at 0 ºC. After 5 min, the solution was concentrated, Et2O (3 x 25 mL) was added and evaportae, the solid residue was washed with Et2O, filtered and dried to yield 2,3,4-tri-O-acetyl-6-azido-6-deoxy-b-d-glucopyranosylamine hydrochloride 36 (500 mg, 97%).
To a heterogeneous mixture of 36 (769 mg, 2.1 mmol) in H2O-CH2Cl2 (1:1, 20 mL) and CaCO3 (615 mg, 6.15 mmol, 3 eq), CSCl2 (239 mL, 1.5 eq) was added. The mixture was stirred 30 min in a round bottom flask provided with a system for evacuation of gases and diluted with CH2Cl2. The organic phase was separated, dried (MgSO4), concentrated, and the residue was purified by column chromatography (1:9®1:3 EtOAc-petroleum ether) to give 1 (0.5 g, 64%) as an amorphous solid. Overall yield from 31: 37% (six steps); Rf = 0.62 (1:1 EtOAc-petroleum ether); IR (KBr) nmax 2942, 2880, 2106, 2031, 1761, 1236, 909 cm-1; 1H NMR (500 MHz, CDCl3) Table 1 and d 2.09, 2.02, 2.00 (3 s, 9 H, 3 MeCO); 13C NMR (125.7 MHz, CDCl3) Table 2 and d 170.1, 169.3, 169.1 (MeCO), 145.0 (NCS), 20.6 (MeCO); FABMS: m/z = 395 ([M + Na]+); C13H16N4O7S: C, 41.93; H, 4.33; N, 15.05; found: C, 41.94; H, 4.21; N, 15.07.
Synthesis of the AB2 building block 2:
Scheme 2. Synthesis of sugar diazido isothiocyanate 2: i) 90% TFA-water (quantitative); ii) NH4-16 m aq ammonia, 40 ºC, 36 h; iii) diethyl ethoxymethylenemalonate, MeOH (52% from 37); iv) 1:1 Ac2O-pyridine (89%); v) Cl2, CH2Cl2, 5 min, room temperature (97%); vi) CSCl2, CaCO3, CH2Cl2-water, 30 min, room temperature (61%).
2,4-Di-O-acetyl-3,6-diazido-3,6-dideoxy-b-d-glucopyranosyl isothiocyanate 2: Compound 2 was prepared from 3,6-diazido-3,6-deoxy-1,2-isopropylidene-a-d-glucofuranose 375, via 38-42, following a reaction squence analogous to that described for the preparation of 1 from 31 (Scheme 2). Overall yield from 37: 27% (six steps); Rf = 0.69 (1:2 EtOAc-petroleum ether); [a]d = +9 (c 1.2, CH2Cl2); IR 2926, 2855, 2106, 2023, 1755, 1373, 1213, 1098, 1036 cm-1; 1H NMR (500 MHz, CDCl3) Table and d 2.19, 2.12 (2 s, 6 H, 2 MeCO); 13C NMR (75.5 MHz, CDCl3) Table 2 and d 169.1, 168.8 (2 MeCO); FABMS: m/z = 378 ([M+Na]+); C11H13N7O5S: C, 37.18; H, 3.69; N, 27.59; found: C, 37.15; H, 3.70; N, 27.54.
Synthesis of the ABC building block 3:
Scheme 3. Synthesis of sugar azidocarbamate isothiocyanate 3: i) 90% TFA-water (quantitative); ii) NH4HCO3-16 m aq ammonia, 40 ºC, 36 h; iii) diethyl ethoxymethylenemalonate, MeOH (62% from 43); iv) 1:1 1,3-propanedithiol-triethylamine, MeOH, room temperature, 16 h (90%); v) Boc2O, Na2CO3, dioxane, room temperature, overnight (85%); vi) TsCl, pyridine, room temperature, 16 h (90%); vii) NaN3, DMF, 50 ºC, 16 h (81%); viii) Ac2O-pyridine (83%); ix) Cl2, CH2Cl2, 5 min, room temperature (96%); x) CSCl2, CaCO3, CH2Cl2-water, 30 min, room temperature (73%).
2,4,6-Tri-O-acetyl-3-azido-3-deoxy-b-d-glucopyranosyl isothiocyanate 3: Compound 3 was prepared from 3-azido-3-deoxy-1,2:5,6-di-O-isopropylidene-a-d-glucofuranose 436 as depicted in Scheme 3. Hidrolysis of the acetonide groups of 37 with 90% TFA-water followed by incubation of the corresponding reducing derivative 44 with ammonium hydrogencarbonate-ammonium hydroxyde and further reaction of the carbamate salt intermediate 45 with diethyl ethoxymthylen malonate, as above described for the preparation of 34 and 40, afforded the glycosylenamine derivative 46 (62% from 43).
To a solution of 46 (3.21 g, 8.59 mmol) in freshly-distilled MeOH (50 mL) under Ar, Et3N (4.5 mL, 2 eq) and 1,3-propanedithiol (2.5 mL, 2 eq) were added. The reaction mixture was stirred at room temperature for 16 h monitoring by TLC (45:5:3 EtOAc-EtOH-H2O). The solvent was evaporated under reduced pressure and the residue was dissolved in water (25 mL) and extracted with CH2Cl2 (3 x 25 mL). Freeze-drying of the aqueous phase afforded 3-amino-3-deoxy-N-(2,2-diethoxycarbonylvinyl)-b-d-glucopyranosylamine 47 (2.7 g, 90%) as a white foam. Compound 47 was directly transformed into the corresponding tert-butyl carbamate derivative 48 by reaction with Boc2O (1.67 g, 1.1 eq) and Na2CO3 (1.63 g, 2.2 eq) in dioxane (11 mL). The reaction mixture was stirred at room temperature overnight, the solvents were evaporated and the residue was extracted with EtOAc-H2O (1:1, 50 mL), the organic phase was decanted, dried (MgSO4) and concentrated. Purification by column chromatography using 4:1 EtOAc-petroleum ether®EtOAc®45:5:3EtOAc-EtOH-H2O gave 48 (2.48 g, 80%).
Introduction of the azido group at the primary position C-6 of 48 was effected via the corresponding 6-O-tosyl derivative 49 by nucleophilic displacement with azide anion in DMF to give 6-azido-3-terc-butoxycarbonylamino-3,6-dideoxy-N-(2,2-diethoxycarbonylvinyl)-b-d-glucopyranosylamine 48 (73% overall for two steps). Transformation of 48 into the target azidocarbamate isothiocyanate 3 was carried out followin a reaction sequence analogous to that above described for the preparation of 1 from 34. Overall yield from 43: 24% (nine steps); [a]d = + 28.9 (c 1.05, CH2Cl2); Rf = 0.61 (1:1 EtOAc-petroleum ether); 1H NMR (500 MHz, CDCl3) Table 1 and d 2.10, 2.06 (2s, 6 H, 2 MeCO), 1.37 (s, 9 H, CMe3); 13C NMR (125.7 MHz, CDCl3) Table 2 and d 169.9, 169.8 (CO ester), 155.4 (CO carbamate), 144.7 (NCS), 80.3 (CMe3), 28.1 (CMe3), 20.6 (MeCO); FABMS: m/z = 452 [M+Na]+, 430 [M+H]+; C16H23N5O7S: C, 44.75; H, 5.40; N, 16.31; found: C, 44.93; H, 5.11; N, 16.18.
Table 1. 1H NMR data (500 MHz, CDCl3) of azido(carbamate) isothiocyanates 1- 3.
H-1 / H-2 / H-3 / H-4 / H-5 / H-6a / H-6b1 / 5.01d / 5.08t / 5.18t / 5.04t / 3.69dt / <------3.35d------>
2 / 4.93 d / 4.99 t / 3.61 t / 4.90 t / 3.63 ddd / 3.36 dd / 3.29 dd
3 / 5.01d / 4.85t / 3.95q / 4.83t / 3.70ddd / 3.38dd / 3.31dd
coupling constants (J, Hz)
J1,2 / J2,3 / J3,4 / J4,5 / J5,6a / J5,6b / J6a,6b
1 / 8.8 / 9.3 / 9.3 / 9.3 / 4.5 / 4.5 / ---
2 / 8.7 / 9.6 / 9.6 / 9.6 / 6.3 / 3.3 / 13.5
3 / 8.8 / 9.8 / 9.8 / 9.8 / 2.6 / 7.6 / 13.6
Table 2. 13C NMR data (125.7 MHz, CDCl3) of azido(carbamate) isothiocyanates 1- 3.
Chemical shifts (d, ppm)C-1 / C-2 / C-3 / C-4 / C-5 / C-6
1 / 83.4 / 71.7 / 72.3 / 68.8 / 75.3 / 50.8
2 / 83.6 / 71.6 / 64.0 / 69.0 / 76.1 / 50.8
3 / 84.1 / 72.2 / 55.1 / 68.9 / 76.3 / 50.8
Blockwise synthesis of thiourea-linked pseudooligosaccharides
Coupling reactions (®7, 10 and 13; 15 and 18; 20, 23, 26, and 29): A solution of the corresponding O-unprotected (poly)aminosugar (4, 9 and 12; 5 and 17; 4, 22, 25 and 28) and the per-O-acetylated sugar azido(carbamate) isothiocyanate counterpart (1; 2 and 6; 3, 1, 3 and 6, respectively; 1.1 equiv of isothiocyanate per amine group) in pyridine (7, 10, 13, 15, 20, 23 and 26) or in 1:1 water-acetone (18 and 29) at pH 8 (NaHCO3) was stirred for 5-24 h (TLC) at room temperature. The solvent was evaporated and the residue was chromatographed on silica gel (45:5:3 EtOAc-EtOH-water) to afford the thiourea adduct in 71-98% yield.
O-Deprotection reaction (8, 11 and 14; 16 and 19; 21, 24, 27 and 30): De-O-acetylation of the hemiacetylated compounds (7, 10 and 13; 15 and 18; 20, 23, 26, and 29) were effected in methanol at room temperature by addition of 0.1 equiv of NaOMe per mol of acetate and further neutralization with Amberlite IRA 120 (H+) ion-exchange resin. In the case of the dendritic and branched pseudoheptasaccharide mimetics 19 and 30, formation of a white precipitate was observed during the base treatment. The suspension was stirred for 30 min at room temperature and water was then added until a clear solution was obtained. The solution was further stirred for 30 min, neutralized with Amberlite IRA 120 (H+) ion-exchange resin and demineralized with Duolite MB 6113 (H+, OH-) ion-exchange resin. The unprotected derivatives were obtained in virtually quantitative yield (NMR). The final compounds were subjected to gel permeation chromatography (GPC) with either Sephadex G-10 (1:1 MeOH-water; 8, 11 and 14) or Sephadex G-25 (water; 19 and 30) to give analytically pure samples.
Reduction reaction (9, 12; 17; 22, 25 and 28): Azide reduction (8 and 11 and 14; 16; 21, 24 and 27) was effected in methanol under Ar by treatment with 1,3-propanedithiol (2 equiv) and triethylamine (2 equiv). The reaction was stirred at room temperature for 16 h, diluted with water and extracted with CH2Cl2. Freeze-drying of the aqueous solution afforded the corresponding (poly)aminosugar, in virtually quantitative yield (NMR, IR), as a white foam that was used directly in the next coupling reaction without further purification. In the case of compound 27, reduction of the terminal azido group was followed by acid hydrolysis of the carbamate groups by treatment with 1:1 TFA-water for 2 h at room temperature (®28). The acid was eliminated by coevaporation with water.