Simple and Efficient Method for the Synthesis of Galactofuranosides

An efficient and improved one-pot method for the synthesis of galactofuranosides via iodine-promoted cyclization of galactose diethyl dithioacetal in the presence of alcohol, acting both as solvent and nucleophile, is described. The reaction is carried out at room temperature. Alcohols, such as methanol, cyclohexanol and tert-butanol, were used as nucleophiles for the reaction using 2%, 3% and 5% iodine promoter, respectively. A key finding in this study was that the iodine-promoted cyclization of galactose diethyl dithioacetal with alcohol led to selective formation of β-galactofuranoside allowing the efficient preparation of derivatives of this monosaccharide.


INTRODUCTION
Monosaccharides found in mammalian cells are in the six-membered pyranose conformation.In lower organisms, such as bacteria, parasites, and fungi, several monosaccharides are observed to be in their furanose form, wherein the linear six carbon sugar is cyclized into a five membered ring.The most common furanoses are the arabinofuranose, galactofuranose, and fructo-furanose (Lowary, 2003).Hexopyranosides are thermodynamically more stable than their furanoside counterparts due to steric, and electronic factors (Plavec et al., 1996).Among the common furanosides, D-galactofuranose (D-Galf) is the only one that occurs naturally.Both the alpha () and beta () anomers of galactofuranose are produced by various lower organisms, with the beta anomer being more prevalent (Lowary, 2003).Several pathogenic https://doi.org/10.26534/kimika.v27i2.38-49organisms are thought to produce this thermodynamically less stable and more sterically-hindered conformation of sugar as an important element for their survival.These include Mycobacterium, Trypanosoma, Leishmania and Aspergillus which are responsible for certain species-associated diseases that are difficult to treat (Tefsen et al., 2012).
When hexoses are treated with alcohols under Fischer glycosylation conditions (acid in an alcoholic solvent), furanosides are formed initially, which are then converted to their pyranoside forms (Capon, 1969).Furanosides are considered the kinetic products of this reaction and are often formed in excellent yields as a mixture of anomers, if proper conditions are chosen.D-Galactose was converted to a mixture of anomeric methyl furanosides via a ferric chloride-catalyzed Fischer glycosylation (Lubineau and Fischer, 1991).Fraser-Reid and coworkers also reported a Fischer glycosidation of D-galactose under kinetic conditions using npentenyl alcohol and camphorsulfonic acid (CSA) at 90-100 °C for six hours to afford npentenyl galactofuranoside (5) as a mixture of anomers (Figure 1, Arasappan and Fraser-Reid, 1995).The reaction also gave the corresponding anomeric mixture of pyranosides (6), albeit in poor yield.(Arasappan and Fraser-Reid, 1995).

Figure 1. Fischer glycosidation of D-galactose
A modified method for glycosylation of 4penten-1-ol with D-galactose was done in tetrahydrofuran using ferric chloride as the promoter and calcium chloride as an additive.The reaction afforded, after 54 hours at room temperature and in situ acetylation, a mixture of furanosides and the corresponding pyranosides with a net preference for β-furanoside and pyranoside derivatives (Velty et al., 1997).The favorable formation of the furanoside products over the thermodynamically more stable pyranosides was attributed to the complexation of the furanoside forms by Ca +2 ion (Angyal, 1980).
An unconventional approach to the synthesis of galactofuranosides from galactopyranose involves the reaction of protected 1,4anhydrogalactopyranose derivatives with alcohols in the presence of camphorsulfonic acid for 3-4 days via regioselective ring opening of the bicyclic system to yield the desired galactofuranosides (Kovensky and Sinay, 2000).Another method used acyclic glycosyl dithioacetals as precursors in the synthesis of glycosyl furanosides (Fischer, 1894;Green, 1966;Pacsu and Green, 1936).One example involved the preparation of 1-chloro-1-(ethylthio) derivatives (10) of D-glucose and Dgalactose from the corresponding dithioacetal pentaacetates (Figure 3, Wolfrom et al., 1944).The S,O acetals (11) formed were found to undergo regio-and stereoselective cyclization following deacetylation and treatment with a mixture of HgO and HgCl2 in either methanol or ethanol to give the corresponding β-Dfuranosides (12).(Wolfrom et al., 1944).
An elegant extension of this method was demonstrated by McAuliffe and Hindsgaul (1997) by treatment of a peracetylated diethyl dithioacetal of D-galactose with acetyl chloride and boron trifluoride diethyl etherate to give the acyclic 1-chloro-(ethylthio) derivative 10.Glycosidation of acceptor 13 with donor 10 in the presence of AgOTf and Ag2CO3, provided S,O-acetal 14. Deacetylation of 14 under Zémplen conditions (Zemplén et al., 1953) followed by addition of HgO and HgCl2 to the methanolic solution and subsequent acetylation gave β-galactofuranoside 16 (Figure 4, McAuliffe and Hindsgaul, 1997).
Relevant to these methods is a report involving the 1,3-dibromo-5,5-dimethylhydantoin (DBDH)-mediated activation of glycosyl dithioacetals leading to the formation of glycofuranosides (Madhusudan and Misra, 2005).However, a major disadvantage of these methods is the use of toxic heavy metal salts and expensive reagents.A related methodology that circumvents this limitation is the treatment of glucosyl, mannosyl and arabinosyl dithioacetals with a dilute solution of iodine (I2) in methanol to give the corresponding glycosyl furanosides (Szarek et al., 1986).
This reaction was only applied to arabinose, glucose and mannose-derived dithioacetals, but not to those derived from galactose.We previously reported adapting this method for the iodine-induced dithioacetal cyclization in octanol towards the synthesis of octyl galactofuranosides (Completo and Lowary, 2008).This was the first reported synthesis of ßoctyl galactofuranoside using galactose-derived dithioacetal.We now report here an extension of the utility of our method by using other simple and sterically-hindered alcohols, acting as both solvent and nucleophile, in the synthesis of corresponding galactofuranosides (Figure 6).

EXPERIMENTAL METHODS
General Methods.Reactions were carried out in oven-dried glassware.All reagents were purchased from commercial sources and were used without further purification unless noted.Unless stated otherwise, all reactions were carried out at room temperature under a positive pressure of nitrogen and were monitored by TLC on Silica Gel 60 F254 (0.25 mm, E. Merck).TLC spots were detected under UV light or by charring with acidified panisaldehyde solution in ethanol.All organic solutions were dried with anhydrous MgSO4.Unless otherwise indicated, all column chromatography was performed on Silica Gel (40-60 M).The ratio between silica gel and crude product ranged from 100 to 50:1 (w/w).Optical rotations were measured at 22 ± 2 °C using Perkin Elmer 241 Polarimeter at the Department of Chemistry, University of Alberta, Edmonton, Canada. 1 H NMR spectra were recorded using 500 MHz Agilent or 400 MHz JEOL Lambda NMR spectrometers and chemical shifts were referenced to either trimethylsilane (TMS) (0.0 ppm, CDCl3), CD3OD (3.30 ppm, CD3OD) or HOD (4.78 ppm, D2O). 1 H data were reported as though they were first order. 13C NMR spectra were recorded at 125 MHz or 100 MHz using Agilent or JEOL Lambda NMR spectrometers, respectively. 13C NMR chemical shifts were referenced to internal CDCl3 (77.23 ppm, CDCl3), or CD3OD (48.9 ppm, CD3OD) or external acetone (31.07,D2O).Assignments of resonances in NMR spectra were made using 1 H-1 H COSY and HMQC experiments.Organic solutions were concentrated under vacuum at <40 °C.All electrospray ionization (ESI-MS) mass spectral data were obtained using Bruker micrOTOF-Q II mass spectrometer.Mass spectra were recorded on samples suspended in mixtures of THF with MeOH and added NaCl.

RESULTS AND DISCUSSION
Several methods for the conversion of acyclic sugar dithioacetals to β-D-furanosides have been reported but the use of toxic heavy metals and expensive reagents preclude its general utility (McAuliffe and Hindsgaul, 1997;Madhusudan and Misra, 2005).
A modified method circumventing this limitation is the iodine-promoted cyclization of glucosyl, mannosyl and arabinosyl-derived dithioacetals in methanol to give the corresponding glycosyl furanosides (Szarek et al, 1986).Likewise, we were the first to report the use of galactose-derived dithioacetal in a similar reaction (Completo and Lowary, 2008).The major advantage of this dithioacetal cyclization reaction in preparing galactofuranosides is its simplicity.Not only does this technique avoid the use of toxic heavy metals, it is also inexpensive, simple and, most importantly, provides high yield of galactofuranosides without or minimal formation of galactopyranosides.We applied this method in the synthesis of methyl and octyl galactofuranosides as intermediates for the preparation of molecular probes of a mycobacterial cell wall enzyme galactofuranosyltransferase (Rose et al., 2006;Belánová et al., 2008).
The proposed mechanism for the iodinepromoted cyclization reaction is shown in Figure 7.The initial step involves the nucleophilic attack of one of the sulfur atoms of 2 on the electrophilic iodine to form sulfonium ion 19.Iodine is a soft acid (Pearson, 1987) and it easily complexes with the sulfur atom of one of the thiol groups to form a sulfonium ion.The sulfonium ion, being a good leaving group, can then be easily cleaved to form thiocarbenium 20.Subsequent cyclization of 20 gives thioglycoside 21, which then reacts with another molecule of iodine to generate intermediate 22. Loss of ethyl sulfenium iodide leads to oxacarbenium intermediate 23, which can be attacked by an alcohol such as methanol, acting as both solvent and nucleophile, to give methyl glycoside 3.

Synthesis of Methyl galactofuranoside 3
and Perbenzoylated methyl galactofuranoside 24.Further extension of this method was applied in the synthesis of a perbenzoylated methyl galactofuranoside 24.
To implement this approach, D-galactose 1 was treated with ethanethiol and HCl to give diethyl dithioacetal 2 in 47% yield using an established method (Wolfrom, 1940).This was followed by the iodine-promoted cyclization of intermediate 2 to methyl galactofuranoside 3 (Figure 8).Table 1 shows the different iodine concentrations used to obtain the optimal conditions in the synthesis of methyl galactofuranoside 3. We observed that the reaction rate was dependent on iodine concentration.Although the use of higher concentrations of iodine (3-5%) resulted in shorter reaction times, this, however, resulted in the formation of significant amount of salt that hindered purification of the product.It was only during reactions beyond 24 h that we start to observe (via thin layer chromatography) the increase in the formation of the  anomer product.Thus, it was necessary to use lower iodine concentration (2%) without significant effect in the ratio (average :β ratio of 8-9:1) and yield (74-78%) obtained for the product.Thus, cyclization of intermediate 2 was effected by treatment with 2% I2 (by weight, 0.08 M) in methanol to give intermediate 3 (Figure 8).The reaction was quenched by adding solid Na2S2O3 until the solution turned colorless.The solution was then neutralized with solid NaHCO3 and the solvent was evaporated under reduced pressure.However, the formation of solid salts during work-up and the polar nature of the product (intermediate 3) made purification difficult.This prompted us to perform the benzoylation reaction without purification of crude intermediate 3. Therefore, pyridine was added to the crude methyl galactofuranoside 3, followed by dropwise addition of benzoyl chloride.
The resulting perbenzoylated product, 24, was obtained in 71 % yield over two steps after purification by column chromatography.Using 1 H and 13 C NMR spectroscopy, we were able to discriminate between the formation of the  and -configuration of the product.The singlet signal of the anomeric hydrogen (H = 5.80 ppm) established the trans relationship between H-1 and H-2.A cis relationship between H-1 and H-2 would have shown a larger coupling constant (J1,2 = 3-5 Hz) (Cyr and Perlin, 1979).Also, the chemical shift of the anomeric carbon of 24 was shifted downfield (c = 106.9ppm) compared to the more upfield shift (c ~ 103 ppm) of the anomeric carbon of an α-product (Cyr and Perlin, 1979).In addition to this, the C-2 and C-4 signals resonated at 82.2 and 81.2 ppm, respectively.These signals are significantly more downfield than the carbons in pyranosides and, thus, conclusively establish the furanose ring form of 24.The observed 1 H NMR and 13 C NMR data were consistent with the previously reported characteristic signal pattern for β-D-galactofuranosides (Cyr and Perlin, 1979) and with our results (Completo and Lowary, 2008).
Synthesis of Cyclohexyl-/-galactofuranoside 4. Encouraged by these observations, we explored the general applicability of this method using more sterically hindered alcohols such as a secondary alcohol cyclohexanol.We anticipated that the hindered nature of cyclohexanol will require higher iodine concentration, thus, 3% iodine was used for the reaction.Indeed, it took 24 hours of continuous stirring to guarantee completion of the reaction.The cyclohexyl galactofuranoside 4 was obtained in 55% yield with 1:5.4 : product ratio.The 1 H NMR spectral data showed the alpha anomeric proton as a doublet at 5.17 ppm, while the beta anomeric proton gave a doublet at 5.12 ppm.This was confirmed by their 3 J1,2 values of 4.6 and 2.8 Hz, respectively, that is consistent with the characteristics of alkyl furanosides (Cyr and Perlin, 1979).The 13 C NMR spectra showed the  and  anomeric carbons resonating at 100.16 and 106.24 ppm, respectively.These data were consistent with the expected chemical shifts for hexofuranosides (Bock and Pedersen, 1983).
Synthesis of tert-Butyl-/-galactofuranoside 5.The use of tertiary alcohol, such as tert-butyl alcohol, was also explored.Due to the more hindered nature of tert-butyl alcohol and its weaker nucleophilicity, higher iodine concentration (5% w/v) was used to form the tert-butyl-/-galactofuranoside.The 1 H NMR signal for the  and -protons were observed at 5.5 ppm and 5.15 ppm having 3 J1,2 values of 0 and 2.5 Hz, respectively, in a 1:1 ratio.A cis relationship between H-1 and H-2 of furanosides is expected to give a 3 J1,2 coupling constant of greater than 3, while a trans relationship should have a coupling constant between 0 to 3 (Cyr and Perlin, 1979).The experimental 3 J1,2 values obtained were consistent with the expected literature values indicative of  and  Galf anomeric hydrogens.From the 13 C NMR spectrum, the chemical shift of the anomeric carbon was observed at 104.30 ppm, consistent with the signal of the expected anomeric carbon position for galactofuranosides (Bock and Pedersen, 1983).The 13 C chemical shifts for C-2 and C-4 were observed at 83.56 and 84.02 ppm, respectively.These are consistent with characteristic 13 C signals for furanose ring carbons resonating more downfield than pyranose ring carbons, thus further establishing that tert-butyl--galactofuranoside was formed.
The use of varying concentration of iodine is dependent on the steric bulkiness of the alkyl component of the alcohol used and which greatly affects the reaction time.The use of more sterically-hindered alcohol required higher iodine concentration as the promoter for the reaction to proceed to completion, else more than 24 h is needed to complete the reaction.Further method optimization is ongoing in our laboratory to improve the yield of the reaction using more hindered alcohols.To date, this is the first time that an iodinepromoted dithioacetal cyclization was used in preparing more sterically hindered alkyl furanosides.The application of this method in the synthesis of disaccharides is also being pursued in our laboratory.

CONCLUSION
In summary, we have demonstrated that a simple and efficient iodine-promoted cyclization of galactose-derived dithioacetal towards the synthesis of galactofuranoside derivatives can be effected using, not just primary alcohols, but also more stericallyhindered alcohols acting as both solvent and nucleophile.The results showed that alcohols with varying steric bulk such as methanol, cyclohexanol and tert-butanol acting as nucleophiles using 2%, 3% and 5% iodine as promoter, respectively, led to the selective formation of β-galactofuranoside derivatives in moderate to good yields.Further method optimization is underway in our laboratory to improve the yield of the reaction.