Aqueous semisynthesis of C-glycoside glycamines from agarose

Agarose was herein employed as starting material to produce primary, secondary and tertiary C-glycoside glycamines, including mono- and disaccharide structures. The semisynthetic approach utilized was generally based on polysaccharide-controlled hydrolysis followed by reductive amination. All reactions were conducted in aqueous media and without the need of hydroxyl group protection. We were able to identify optimal conditions for the reductive amination of agar hydrolysis products and to overcome the major difficulties related to this kind of reaction, also extending it to reducing anhydrosugars. The excess of ammonium acetate, methyl- or dimethylamine, and the use of a diluted basic (pH 11) reaction media were identified as important aspects to achieve improved yields, as well as to decrease the amount of byproducts commonly related to reductive amination of carbohydrates. This strategy allowed the transposition of the 3,6-anhydro-α-L-galactopyranose unit (naturally present in the agarose structure) to all glycamines synthesized, constituting an amino-substituted C-threofuranoside moiety, which is closely related to (+)-muscarine.


General methods
All reagents and solvents were of reagent grade. Agar, Type A, was purchased from Sigma. Reactions were monitored by thin layer chromatography (TLC) on commercially available pre-coated aluminium-backed plates (Merck Kieselgel 60 F 254 ).Visualization was carried by staining using a solution of ninhydrin (5% in ethanol) or resorcinol (2% in ethanol/H 2 SO 4 9:1) to visualize amines or sugars, respectively. Column chromatography was performed with the indicated eluents using Fluka silica gel 60 (particle size 220-440 mesh). Yields refer to chromatographically and spectroscopically pure compounds. For final amines the pH of the NMR samples was adjusted to 4.0 using 1 M HCl aqueous solution. All samples were lyophilized before analysis and were prepared with deuterium oxide (D 2 O). 1 H NMR, 13 C NMR and, HSQC 1 H-13 C) were obtained with a Bruker Avance DRX 400 or Bruker Avance 600 spectrometer (as indicated) operating at 400,1 MHz or 600,1 MHz, respectively for 1 H and 100,63 MHz for 13 C. 13 C NMR chemical shifts were determined by HSQC 1 H, 13 C correlation experiments. Chemical shifts (δ) were expressed in parts per million (ppm) and coupling constants (J) in Hertz (Hz) using the residual solvent peaks (H 2 O, δ 4,79) as internal standards. Multiplicities were described as singlet (s), doublet (d), triplet (t), doublet of doublets (dd), doublet of triplets (dt), broad (br), and multiplet (m). Mass spectra were acquired in positive mode using an ESI ion source Walters Micromass Quattro LC-MS/MS for low resolution mass spectra (LRMS) and Bruker Micro TOF-Q II XL for high resolution mass spectra (HRMS) using sodium formate solution as reference. Optical rotation values were obtained with a Jasco P-200 polarimeter equipped with a sodium light source. Specific rotation ([α] D ) of 1% (g/mL) compounds solutions in distillated water was calculated at 25 °C.

Synthesis
Commercial agar (5 g) was first dissolved in hot (90 °C) water (450 mL) and then 1 M TFA aqueous solution (50 mL) was added in one portion to make a final concentration of 0.1 M TFA. The resulting mixture was heated at 80 °C for 3 h, cooled to room temperature, diluted with water (500 mL), and then concentrated under vacuum. The resulting residue was dissolved in water (90 mL), diluted with iPrOH (90 mL), and then filtered through a glass-sintered filter. The filtrate was concentrated and coevaporated with methanol and lyophilized, resulting a light yellow powder as crude material (4.8 g, 96% yield w/w). Material assigned using literature reference.  β-D-Galactopyranosyl-(1'→4)-1-deoxy-1-amino-3,6-anhydro-α-L-galactitol (3)

Synthesis
Crude material containing agarobiose 2 (500 mg, 1.5 mmol) was dissolved in water (20 mL) then ammonium salts or ammonium hydroxide (40 equiv) was added and homogenized. pH 11 was adjusted with TEA and finally sodium cyanoborohydride (2 equiv, 3.0 mmol, 200 mg) was added in a single portion. The flask was tightly closed immediately. This mixture was placed in a 100 °C glycerin bath and stirred for 5 hours.
Hereafter the media was concentrated under reduced pressure, redissolved in water (100 mL) and stirred with strongly basic anion exchange resin (Amberlite IRA 410 -OH − form, 100 mL, 68 g) for 1 hour. The resin was filtered and washed with water (2 × 100 mL). The filtrate was dried under reduced pressure using coevaporation with EtOH to give a yellow crude product (≈550 mg). Then this material was dissolved in warm MeOH (50 mL), filtered through a borosilicate sintered funnel and dried under reduced pressure, resulting in a vibrant yellow-greenish crude product (≈330 mg). Finally the crude material was submitted to silica gel flash chromatography (eluent: MeOH/2M NH 4 OH 6:1) to give the pure aminoglycoside 3 as a white solid after beenig lyophilized (142 mg, 26% molar yield).
Physical and spectral assignment showed as a white solid after lyophilization (100 mg, 19% molar yield).

Method Bpure 8
Methylaminoglycoside 7 (40 mg, 0.12 mmol) was dissolved in water (2 mL) then formaldehyde (36% aqueous solution, 3.0 equiv, 0.36 mmol, 30 μL) was added and homogenized. pH 11 was adjusted with TEA and finally sodium cyanoborohydride (2 equiv, 0.24 mmol, 15 mg) was added in a single portion. The flask was tightly closed immediately. This mixture was placed in a 70 °C glycerin bath and stirred for 5 hours.
Reaction workup and purification were done as for the primary aminoglycoside 3 synthesis. After lyophilization, compound 8 was obtained as a white solid (23 mg, 55% molar yield, 13% from agarobiose).

S11
HSQC 1   MeOH (3 x 50 mL) to give a yellow syrup. This crude material was dissolved in 2M TFA solution (125 mL) and the resulting mixture was heated at 120 °C for 3 h, cooled to room temperature, diluted with H 2 O (100 mL), and then concentrated. The resulting residue was coevaporated with methanol three times to give a syrup. This material was dissolved in H 2 O (50 mL), cooled to 0 °C and then NaBH 4 (0.95 g, 25.0 mmol) was added in one portion. The resulting mixture was then stirred at room temperature for 1 h, diluted with AcOH (≈4.0 mL, pH ≈ 4.0), stirred for an additional 10 min, and concentrated to give a dark-brown syrup. This material was suspended in MeOH (15 mL) and acetone (50 mL), and then filtered through a glass-sintered filter. The filtrate was concentrated to afford a residue that was diluted with H 2 O (50 mL) and treated with Amberlite IRA 410 OHform (100 mL). The resulting mixture was stirred for 10 min, filtered through a glass-sintered filter, and washed thoroughly with H 2 O (150 mL). The combined filtrates were then treated with Amberlite IR120 (H + form 100 mL), stirred for 10 min, filtered through a glass-sintered filter, and washed thoroughly with H 2 O (200 mL). The combined filtrates were concentrated to give a dark yellow crude material (1.5 g) mainly constituted of the anhydro alditol.

Synthesis
Crude material containing aldehyde hydrate 12 (150 mg, 1.0 mmol) was dissolved in water (15 mL) then methylamine hydrochloride (20 equiv, 20 mmol, 1.33 g) was added and homogenized. pH 11 was adjusted with TEA and finally sodium cyanoborohydride (2 equiv, 2.0 mmol, 133 mg) was added in a single portion. The flask was tightly closed immediately. This mixture was placed in a 100 °C glycerin bath and stirred for 5 hours. Reaction workup and purification were done as for the primary aminoglycoside 3 synthesis. After chromatography the compound was obtaine as a white solid after lyophilization (32 mg, 22% molar yield from agarose).
Physical and spectral assignment