Application of Appel reaction to the primary alcohol groups of fructooligosaccharides: Synthesis of 6,6′,6′′-trihalogenated 1-kestose derivatives

1-kestose ( O - β -D-fructofuranosyl-(2→1)- β -D-fructofuranosyl-(2→1)- α -D-glucopyranoside) is a potential short chain fructooligosaccharide with an inulin-type skeleton. Halogenation of 1-kestose was conducted via the Appel reaction with the use of carbon tetrahalide (CBr 4 or CCl 4 ) and triphenylphosphine, which was then followed by conventional acetylation. The per-O -acetylated form of 6,6’,6’’-trihalogenated derivatives of 1-kestose were conveniently isolated. Further deprotection of the per-O -acetylated form resulted in 6, 6’-,


Introduction
Inulin-type short-chain fructooligosaccharides (FOS) are fructose oligomers that consist of a terminal glucosyl unit and two to five fructosyl units.They are recognized as prebiotic indigestible organosaccharides 1 and due to this property, FOS demand has been increasing in the food industry. 21-Kestose (1, Figure 1) is one of FOS that has a β-D-fructofuranosyl group on O-1 of the D-fructosyl moiety of sucrose (2, Figure 1).4][5] The interest in 1-kestose 1, as a low-calorie food ingredient, continues to increase due to its sweetening power.FOS syrup enriched by 1-kestose 1 can be used as alternative sweetener for diabetics. 6To produce 1-kestose 1, the enzyme derived from the leaves of sugar beets have demonstrated transfructosylation activity in the present of sucrose 2; thus, the products of transfer were mainly 1-kestose 1 with smaller proportions of other oligosaccharides. 7ommercial cellulolytic enzymes have also been studied for preparation of FOS with high 1-kestose concentrations. 2gure 1.Structure of 1-kestose (1), sucrose (2), and undecaacetate of 1-kestose (3).
Since the specific substitution of sucrose primary hydroxyl groups by chloride enhances the sweetness activity, 8 halogenated carbohydrate derivatives can potentially be used as an alternative sweetener.The direct halogenation of the hydroxyl groups of carbohydrate is a convenient method to achieve the synthesis of halogenated carbohydrate on its primary hydroxyl groups.Thus, selective halogenation for carbohydrate has become an area of interest in organic chemistry.The Appel reaction is one of the most important reactions used to convert the primary hydroxyl group into a halo methylene group in the presence of triphenylphosphine and carbon tetrahalide. 9This method was used for selective halogenation of primary hydroxyl groups over secondary hydroxyl groups in carbohydrates.Halogenation (particularly for chlorination and bromination) of primary alcohols of sucrose 2 10 -the main precursor for enzyme-catalyst generation of 1kestose 1-has been studied since the 1970s 11 , but the reactivity of primary hydroxyl groups attained using the Appel reaction has not been completely identified due to limitation of structure analysis.Recently, we reported that sucrose 2 can be selectively brominated and chlorinated using the Appel reaction at only the 6and 6'-position with no halogenation at 1'-position supported by one-and two-dimensional (1D and 2D, respectively) NMR analysis. 12As for trisaccharide modification, halogenation of the primary hydroxyl groups of raffinose (O-α-D-galactopyranosyl-(1→6)-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside) has been reported previously. 13Raffinose reacted with sulfuryl chloride and the authors found that the chlorinated proportion at the primary position was followed by chlorination at the 4-position of the galactopyranosyl moiety secondary alcohols.To date, no study has been attempted to substitute primary alcohols of FOS using halogens.In midst of FOS, 1-kestose 1 has shown relatively high sweetness activity and is already commercially available.Therefore, its primary alcohol modification would potentially increase the synthesis of alternative sweeteners.
In this study, we aimed to synthesize halogenated 1-kestose 1 at the primary position by using the Appel reaction to comprehensively study the structure elucidation supported by 1D and 2D NMR analysis.

Results and Discussion
Direct substitution of 1-kestose 1 with 4.7 equiv.carbon tetrahalide (bromide or chloride) and 9.1 equiv.triphenylphosphine at 70 o C for two hours (Scheme 1) produced a complex mixture with complicated 1 H-NMR analysis due to observation of overlap signals, especially the modified primary centers.The mixture was separated after conventional acetylation.Further purification was conducted using an ether and hexane system as the representative mobile phase to isolate halogenated carbohydrate.The ethyl acetate or dichloromethane system cannot be used for the halogenated proportion. 12The mixture produced halogenated 1-kestose derivatives in the pre-O-acetylated form (bromination 4 and chlorination 5, Scheme 1).Scheme 1. Halogenation of 1-kestose (1) via Appel reaction with Ph 3 P and carbon tetrahalide to produce pre-O-acetylated form of halogenated 1-kestose derivatives (bromination 4 and chlorination 5).
The 1 H NMR analysis for peracetylated 4 and 5 showed two regions that contribute to the proton resonance of aliphatic sugar groups in the downfield region and halogenated methylene groups located in the upfield region.Based on 13 C NMR, three halogenated methylene in the upfield region were easily determined in the halogenated proportion in the 1-kestose derivatives, but the halogenated position remained unclear.Therefore, 2D NMR (COSY, HETCOR, HMQC, HMBC, NOESY, and TOCSY) was used to determine the halogenated position.For this purpose, CDCl 3 12,14 was used as the solvent to increase the visibility of the spin system during the analysis.Undecaacetate 1-kestose 3,15 -7 and SM-8), which was also, supported by HETCOR, HMQC, and HMBC analyses.

Conclusions
We studied the halogenation of 1-kestose using Appel reactions to synthesize trihalogenated 1-kestose at the 6-, 6′-, and 6′′-position.Isolation and structure elucidation were easily completed using the per-O-acetylated form which the 1D and 2D NMR supported the halogenation position.The synthesis and structure elucidation of novel compounds 4 and 5 contribute to the introduction of primary hydroxyl groups into FOS which can potentially be used as low-calorie sweeteners in the future.

Experimental Section
General.All reagents used were of analytical grade.NMR spectra were obtained in CDCl 3 or D 2 O by JEOL ECA500 (500 and 125 MHz) spectrometer (JEOL, Tokyo, Japan).Optical rotations were measured at 23 o C on a JASCO DIP370 polarimeter (JASCO, Tokyo, Japan).HRMS spectra were obtained with a Waters UPLC ESI-TOF mass spectrometer (Waters, Milford, CT, USA).