C -Glycoside D-galacturonates suitable as glycosyl acceptors for the synthesis of allyl C -homo- and rhamno-galacturonan modules 1

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Results and Discussion
In our previous paper 2 methyl (4) and benzyl (5) glycosyl acceptors were obtained by different routes starting from D-galactose and D-galacturonic acid respectively.Exploring several protecting group manipulations, an effective route was found for the preparation of the key intermediate 3 (Scheme 1) through the isopropylidene compound 1 and its benzylation under basic conditions, where no β-elimination was observed. 2,3Selective benzylation via 3,4-Obenzylstannyl intermediates resulted in methyl (4) and benzyl (5) glycosyl acceptors, both suitable as acceptors in glycosylation reactions.

Scheme 2
In general, benzyl esters are less stable than the corresponding methyl esters.On the other hand, benzyl esters can be removed easily by hydrogenation.In order to get a defined pattern of protected and unprotected carboxylic esters in pectin fragments, both methyl and benzyl esters were prepared. 6,7Thus, it was of interest to incorporate galacturonate 8, protected as the benzyl ester, into our program for the synthesis of the glycosyl donors 14α/β.Therefore, the 4-Oposition was acetylated with acetic anhydride in dry pyridine to provide 10 in 98% yield.
Regioselective benzylation of 6 via 3,4-O-butylstannyl intermediates gave galacturonates 7 and 8 in 80% overall yield (ratio 3:1).The galacturonate 7 was used as a glycosyl acceptor for the synthesis of homo-and rhamnogalacturonan oligosaccharides. 3,9  the 1 H NMR (500 MHz) spectrum, the acetylation of 6 caused the expected downfield shift to the H-4 ring proton signal from  4.32 ppm (8) to 5.82 ppm (10).In the 13 C NMR spectrum, the signal at  20.53 ppm and 169.68 ppm confirmed the presence of the acetyl group at the 4-position.The deallylation was evidenced by the expected upfield shift of the H-1 ring proton signal from  4.48 ppm (10) to 5.40 ppm (12).The small coupling constant J1,2 = 3.5 Hz of H-1 and the C-1 signal at δ 92.18 ppm are caused by the α-configuration of 8.
The introduction of the trichloroacetimidate group at the anomeric center of 11 in the presence of DBU produced the α-D-galactopyranosyl trichloroacetimidate 13α (Rf 0.76) and the β-trichloroacetimidate 12β (Rf 0.71).After HPLC chromatography compounds 13α and 13β were obtained in pure in 76% total yield (ratio 2:1).In the 1 H NMR (500 MHz) spectrum the small coupling constant J1,2 = 3.5 Hz for compound 13α, and the considerably larger coupling constant J1,2 = 8.2 Hz for compound 13βconfirmed the proposed structure.Moreover, 13 C NMR signals at 94.54 ppm for compound 13α, and at 97.78 ppm for compound 13β assured the stereochemistry at the anomeric center of the glycosyl donor 13α and 13β respectively.
The coupling of galacturonate acceptor 4 with a slight excess of the α-configured donor 13α was promoted by trimethylsilyl trifluoromethanesulfonate (TMSOTf). 3,11,12Standard work-up of the reaction mixture provided the (1→4)-linked disaccharides 16αand 16βin 53% total yield in a ratio 3:1 (Scheme 3).The same result was observed when 13βwas used as glycosyl donor.Although the Rf.values of the resulting diaccharides were so close together, the disaccharides 15α and 15βwere successfully separated by using gradient HPLC.Still, both of the separated fractions 15α and 15β contained traces of the corresponding anomer.The 1 H NMR spectra of 15α showed a doublet signal at  5.16 ppm with vicinal coupling constant J1′,2′ = 3.5 Hz for H-1′, and a triple doublet signal at  4.24 with vicinal coupling constants J1,2 = 2.5 Hz, J1,Ha = 6.0 Hz, J1,Hb = 8.5 Hz for H-1.In addition, the 13 C NMR signal for C-1 ′ was found to fall within the expected range of 98.80 Hz with JC-1,H-1′ = 171.0Hz coupling constant for the -coupled disaccharide 15α.In the case of 15βthe value of the C-1′ signal, which was determined at Hz, matches the -linked disaccharide.Subsequent experiments have shown that the -or β-configured trichloroacetimidate group at the anomeric center of 13α/β exerted no influence on the outcome of stereoselectivity of the glycosylations investigated here. 3,14For that reason, in the subsequent experiments we used the α/β mixture of 13α/β.

Scheme 3
The coupling of glycosyl acceptor 5 with a slight excess of the 14α/β was initiated by TMSOTf as a glycosylation promoter. 3,11,12Standard work-up of the reaction mixture provided the (1→4)-linked disaccharides 16 and 16in 44% total yield in a ratio of 2:1 (Scheme 3).
Unfortunately, the αand β-coupled disaccharides 16α and 16β were difficult to separate this time.The  C NMR showed the signal for C-1′ has the expected range of 99.0 ppm for the coupled disaccharide 16α, whereas the -coupled disaccharide 16βshowed a signal at 102.3 ppm.The other 1 H and 13 C NMR data were also fully consistent with the assigned structures.
In connection with the synthesis of rhamnogalacturonan fragments type I, we required a simple approach to benzylated rhamnopyranosides with an O-acetyl group at 2-postion.To achieve this, acetobromorhamnose 17 was prepared according to the literature. 14Methyl orthoester 18 was prepared in 81% yield by the reaction of bromide 17 with dry EtOH in the presence of 2,4,6-collidine and tetrabutylammonium bromide (Scheme 4). 15,16The structure of the obtained orthoester 18 was supported by the analytical data. 17Compound 18 was then deacetylated by the Zemplén procedure to give 18, but the subsequent neutralization decreased the yield of 20 dramatically.Therefore, deacetylation of orthoester 18 and the benzylation of the resulting compound 21 under basic conditions were carried out without further purification of 20.Instead of using Zemplèn conditions, deacetylation was achieved by refluxing 18 with KOH in dry toluene. 18heme 4 Figure 1.An ORTEP diagram of compound 20, the aromatic ring of the benzyl group at O4 is disordered over two positions around the C11-C12 axis.Only one set of atoms is shown in Figure 1, the other is omitted for clarity.
After addition of benzyl chloride and classical work up orthoester 20 was obtained in 82% yield as colorless crystals suitable for X-ray investigation (Figure 1).NOESY studies (Figure 2) were employed to address the conformation and the absolute configuration of the orthoester 20.
In the 1 H-NMR spectrum of 20 the exchange of the acetyl groups at O-3 and O-4 positions by benzyl groups caused a significant upfield shift (ca.1.5 ppm) of the geminal ring proton, with respect to the precursor 17. 17 Furthermore, the H-1 signal of 20 appears at relatively high field  5.26 ppm with a coupling constant J1,2 = 2.5 Hz.The NOESY spectrum (Figure 2) shows correlation between H1H2,H1H3, H1H5, CCH3 (orthoester) OCH2CH3 (orthoester), and H2  OCH2CH3 (orthoester).From these results, it could be concluded that the rhamnopyranose ring of 20 is in the 1 C4 chair conformation, and in the 1,2-O-(1-ethoxyethylidene) derivative 20 the ethoxy group exists in an exo-orientation (S-configuration).The distortion of the chair towards a half-chair conformation, as reported by Perlin, 19,20 which is to be expected from the fusion of the five-membered orthoester group at positions 1 and 2 of the sugar molecule, was not observed for 20.As shown in Scheme 5, the rhamnopyranose donors 21αβ were coupled with the C-glycoside galacturonate acceptors 4 in a ratio of 1.1:1 in the presence of trimethylsilyl trifluoromethanesulfonate. 10-12 This coupling produced the desired α-(1→4)-coupled disaccharide 23 in 18% yield, 21α/β (donors) in 26% yield, and the accompanying transesterfication 21,22 product of the acceptor 25 in 23% yield.To improve the yield of the disaccharide 23, the bromide donor 22 was synthesized from compounds 21 by the reaction with oxalyl bromide in dry dichloromethane to give the bromosugar 22 (Scheme 4) in 95% yield. 3,11Compound 22 was unstable on storage and was used directly without further purification.

Conclusions
Introduction of the trichloroacetimidate group at the anomeric center of 9 23,24 was reexamined, and the resulting trichloroacetimidate derivatives 13 were obtained in 98% total yield (α/β Trichloroacetimidate derivatives 14 were synthesized in 76% total (α/β yield and incorporated in our program as glycosyl donors.The (1→4)-linked disaccharide derivatives 15 were obtained in 53% total yield with low stereoselectivity 3,23,24 (α,β 3:1).The α-linked disaccharide 15 and its β-linked anomer were successfully separated, and characterized by the aid of NMR spectroscopy.The change of the methyl ester to a benzyl ester in both glycosyl acceptor and donor had no strong influence on the outcome of the glycosylation.Disaccharide derivatives 16 were obtained in 44% total yield (α/β 2:1).Separation of 16α and 16β was cumbersome and NMR investigation could be done only with enriched fractions.Glycosyl derivatives 21α/β and the corresponding bromide 25,26 22 were prepared as glycosyl donors in high yield from the orthoacetate 18. Glycosylation reaction of C-allylated galacturonate 5 with rhamnose acetates 21α,β gave only a moderate yield (18%) compared with results for the glycosylation of O-allylated galacturonates 3 .In order to improve the yield, the more reactive bromide 22 was used.Glycosylation of acceptors 4 and 5 with 22 gave the αlinked disaccharides 23 and 24 in 54% and 56% yields, respectively.

Introduction of the trichloroacetamidate group at the anomeric center of 11.
To a solution of compound 11 (330 mg, 0.77 mmol) in dry dichloromethane (5 mL), trichloroacetonitrile (2.84 mL, 28.4 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 23 L, 0.15 mmol) were added under argon at -20 ºC.The reaction mixture was stirred at that temperature for one hour, and then for an additional hour at room temperature (TLC eluent B; Rf. 0.39, Rf. 0.27).Finally, the mixture was concentrated.The residue was suspended in ethyl acetate (30 mL) and filtered over a layer of silica gel.The remaining solids were washed with ethyl acetate (3 × 8 mL); the combined filtrate and washings were dried and concentrated.The residue was purified by HPLC (eluent ethyl acetate gradient 0% → 50% in petrol ether v/v) to provide the product.

Introduction of the trichloroacetimidate group at the anomeric center of 12.
To a solution of compound 12 (510 mg, 1.0 mmol) in dry dichloromethane (7 ml), trichloroacetonitrile (3.7 mL, 37 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU 30 l, 0.20 mmol) were added under argon at -20 ºC.The reaction mixture was stirred at that temperature for one hour, and then for an additional hour at room temperature (TLC eluent F; Rf. 0.77, Rf. 0.73).Finally, the mixture was concentrated; the residue was suspended in ethyl acetate (30 mL) and filtered over a layer of silica gel.The remaining solids were washed with ethyl acetate (3 × 8 mL), the combined filtrate and washings were dried and concentrated.The residue was purified by HPLC (eluent ethyl acetate gradient 0% → 50% in petrol ether v/v) to provide 14α (Rf 0.77, 210 mg, 40%), 14β (Rf 0.73, 100 mg, 19%), and αβ mixture (90 mg, 17%).(20).Compound 19 (4.40 g, 13.8 mmol) was dissolved in dry toluene (20 mL) and potassium hydroxide (9.60 g, powder) was added.The mixture was heated to reflux, and benzyl chloride (14.5 mL) was added dropwise.The reaction mixture was heated under reflux for 2.5 h and cooled to room temperature, and ice-water was added.The organic phase was diluted with toluene and washed with ice-water until neutral.The toluene solution was concentrated to an oil, which was then shifted to flash chromatography (eluent G, Rf 0.61) to give a syrupy product, which crystallized from petrol ether-ethyl acetate to give 20 (4.7 g, 82%) as colorless crystals: mp 76-78 ºC; [α]D 22 + 2.9 (c 1.0 chloroform); 1 H NMR (500 MHz, CDCl3) t J 6.9 Hz, C(OCH2CH3)CH3], 1.31 (d, 3H, J 6. 3   61 mmol) was hydrolysed by sequential treatment with 70% aqueous acetic acid (28 mL) at room temperature for 7 min (TLC eluent K, Rf 0.52).The solution was then concentrated and acetic acid in the syrupy residue was removed by co-evaporation with toluene.The 1 H NMR spectrum indicated the product to be essentially pure.Without further purification, the crude syrupy residue was dissolved in dry pyridine (106.5 mL).Acetic anhydride (53 mL) was added at 0 ºC and the reaction mixture stirred at room temperature for 1h (TLC eluent I, Rf 0.71).The mixture was poured into ice-water and the aqueous layer extracted with chloroform.The combined organic extracts was washed with cold 1% HCl, ice-water, cold sat aq NaHCO3, then again with ice-water, dried and concentrated.The syrupy residue was purified by flash chromatography (eluent A).   (22).To a solution of 21α/β (500 mg, 1.17 mmol) in dry dichloromethane (7 mL), was added oxalyl bromide 70,78 (162 l) was added under argon at -40 ºC.After an additional one hour at that temperature, the reaction mixture was kept at room temperature for one hour (TLC eluent F, Rf 0.75).The mixture was concentrated and repeatedly co-evaporated with toluene to give crude product of 20 (500 mg, 96 %).The compound 20 was unstable for storage and was used directly without further purification.(23).(a) Via 21.Glycosyl acceptor 5 (0.4 mmol), glycosyl donor 19 (α/β) (0.5 mmol), and powdered activated molecular sieves (4Å, 4.0 g) were dried azeotropically with toluene, and then subjected to high vacuum for 2h.The mixture was dissolved in dry dichloromethane (8 mL), and the reaction mixture was stirred for 2 h under argon at room temperature in the dark.After cooling to -70 ºC, TMSOTf (83 l, 0.5 mmol) was added, and stirring was continued for 3 h at that temperature.The reaction mixture was then allowed to warmup to room temperature and stirring was continued for an additional 18 h (TLC eluent F).The reaction mixture was passed through a layer of alkaline alumina by elution with chloroform.The eluate was concentrated to 30 mL and then washed with cold aq sat NaHCO3 (2 × 15 mL), ice-water (2 × 15 mL), dried and concentrated.The crude residue was purified by HPLC (eluent ethyl acetate gradient 0% → 25% in petrol ether v/v) to give disaccharide 23 (Rf