Synthesis and Characterization of Novel lacZ Gene Reporter Molecules: Detection of β-Galactosidase Activity Using ¹⁹F NMR of Polyglycosylated Fluorinated Vitamin B₆

Abstract

Gene therapy has emerged as a promising strategy for treatment of various diseases. However, widespread implementation is hampered by difficulties in assessing the success of transfection, in particular, the spatial extent of expression in the target tissue and the longevity of expression. Thus, the development of non-invasive reporter techniques based on appropriate molecules and imaging modalities may help to assay gene expression. We have previously demonstrated the ability to detect β-gal activity based on ¹⁹F NMR chemical shift associated with release of fluorophenyl aglycones from galactopyranoside conjugates. Use of fluoropyridoxol as the aglycone provides a potential less toxic alternative and we now report the design, synthesis and structural analysis of a series of novel polyglycosylated fluorinated vitamin B₆ derivatives as ¹⁹F NMR sensitive aglycones for detection of lacZ gene expression. In particular, we report the activity of 3, α⁴, α⁵-tri-O-(β-D-galactopyranosyl)-6-fluoropyridoxol 4, 3-O-(β-D-galactopyranosyl)-α⁴, α⁵-di-O-(β-D-glucopyranosyl)-6-fluoropyridoxol 12 and 3-O-(β-D-galactopyranosyl)-α⁴, α⁵-di-O-(α-D-mannopyranosyl)-6-fluoropyridoxol 13. 4, 12, and 13 all show promising characteristics including highly sensitive ¹⁹F NMR response to β-gal activity (Δδ = 9.0 ~ 9.4 ppm), minimal toxicity for substrate or aglycone, and good water solubility. However, the differential glycosylation of 12 and 13 appears more advantageous for assessing lacZ gene expression in vivo.

INTRODUCTION

Gene therapy holds great promise for the treatment of diverse diseases, but widespread implementation is hindered by difficulties in assessing the success of transfection. The development of non-invasive in vivo reporter techniques based on appropriate molecules and imaging modalities would be of considerable value for assessing the location, magnitude, and persistence of expression.

The lacZ gene encoding β-galactosidase (β-gal) is widely used in molecular biology as a reporter gene to assay clonal insertion, transcriptional activation, protein expression, and protein interaction. Many colorimetric reporter molecules have been described to detect β-gal activity and these for the basis of highly effective spectrophotometric assays in vitro.^1–3 However, optical methods are less practical for applications in animals in vivo or ultimately in man in the clinic due to extensive light scattering and absorption by tissues. Towards such applications new reporter molecules are being developed. Recently, Tung et al.⁴ presented a near infrared approach in vivo based on 9H-(1, 3-dichloro-9, 9-dimethylacridin-2-one-7-yl) β-D-galactopyranoside to detect β-gal activity in transfected tumors in live mice. Lee et al.⁵ described use of a radiolabeled competitive inhibitor 2-(4-[¹²⁵I/¹²³I]iodophenyl)ethyl-1-thio-β-D-galactopyranoside to detect β-gal activity in mice. Louie et al.⁶ introduced an NMR approach using 1-[2-(β-D-galactopyranosyloxy)propyl]-4, 7, 10-tris(carboxymethyl)-1, 4, 7, 10-tetraazacyclododecane) gadolinium (III) based on proton MRI contrast to detect β-gal activity in developing frog embryos following direct injection of substrate into eggs. We have been developing in vivo reporter molecules based on ¹⁹F NMR with structures exploiting fluorophenol, trifluorophenol, and fluoropyridoxol aglycones.^7–11 To date our published investigations have focused on development of reporter molecules and we have demonstrated detection of β-gal activity in cultured tumor cells with preliminary examples of detectability in tumors in living mice ¹². In a continuing effort to develop enhanced approaches for in vivo detection of β-gal, we now report the synthesis, and evaluation of polyglycosylated fluorinated vitamin B₆ reporter molecules, designed to enhance water solubility, cellular penetration and enzyme response.

DESIGN

The diversity of substrates and reporter molecules for β-gal activity is indicative of broad substrate specificity. Agents have been tailored for specific imaging modalities or with particular characteristics, such as thermal stability suitable for autoclaving. However, some substrates suffer from poor aqueous solubility and inability to reach targets in vivo and some aglycone products are toxic. Our initial investigations used fluorophenyl β-D-galactopyranosides.^{7, 9} This approach was particularly facile, being a simple analogy of the classic “yellow” agent o-nitrophenyl β-D-galactopyranoside (ONPG). However, the product aglycone appears somewhat toxic, being closely similar to the uncoupler dinitrophenol. We were able to reduce the requisite concentration of reporter molecule by introducing a trifluoromethyl reporter moiety in place of a single fluorine atom, but this is characterized by a much smaller chemical shift response.¹¹ Toxicity could be largely avoided by using 6-fluoropyridoxol (1, FPOL) as the aglycone and we recently demonstrated proof of principle. Introduction of a D-galactose at the 3 phenolic group of FPOL, 3-O-(β-D-galactopyranosyl)-6-fluoropyridoxol (GFPOL) yielded a ¹⁹F NMR gene expression reporter exhibiting a large chemical shift response to β-gal cleavage, but having only moderate kinetic sensitivity to β-gal.⁸ GFPOL was also modestly water soluble.

We considered that introduction of additional sugar moieties could enhance water solubility and potentially improve enzyme sensitivity. Pertinent to this approach were reports that modification the α⁴- and α⁵-position hydroxymethyl moieties of FPOL produces modification of its pK_a with relatively minor changes in chemical shift and chemical shift range.¹³ Further, Escherichia coli (lacZ) β-gal catalyses the hydrolysis of galactopyranosides by cleavage of the C-O bond between D-galactose and the aglycone with a double-displacement mechanism involving the formation (‘glycosylation’ step) and breakdown (‘deglycosylation’ step) of a glycosyl-enzyme intermediate via oxocarbonium-ion-like transition states. It has been observed that hydrogen bonding interaction between the enzyme and the glycosidic substrate is important in the formation of the enzyme-substrate complex and to the hydrolysis rate.^{14, 15} The involvement of fluorine atoms in hydrogen bonding is well documented and exemplified by some of the strongest known hydrogen bonds.¹⁶ Considerable evidence suggests that a C-F moiety can act as a weak proton acceptor and may form hydrogen bonds between the enzyme and the substrate.^17–21

We have found evidence of intramolecular hydrogen bonding between α⁵-OH and 6-F in ¹H-NMR spectra of FPOL and its derivatives. For example, the signal of α⁵-OH in α⁴-OH and α⁵-OH unprotected analogues, such as FPOL or 3-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-galactopyranosyl)-6-fluoropyridoxol always appears downfield and is coupled with 5-CH₂ as triplet due to the α⁵-OH exchange-limitation by the α⁵-OH and 6-F hydrogen bonding. Meanwhile, α⁴-OH occurs as an upfield singlet. Introduction of two additional carbohydrate residues at α⁴ and α⁵-hydroxymethyl positions of GFPOL would inhibit α⁵-OH and 6-F hydrogen bonding and facilitate hydrogen bonding between the enzyme and the new substrates. Thus, substrate affinity should increase and both water solubility and enzyme sensitivity could be improved, while retaining the virtues of GFPOL. ⁸

RESULTS AND DISCUSSION

Syntheses

Our initial approach used a one-pot technique to introduce three D-galactose moieties at the 3 phenolic and α⁴-, α⁵-hydroxymethylic sites, simultaneously. Reaction of 1 with 3.3 equivalents of 2, 3, 4, 6-tetra-O-acetyl-α-D-galactopyranosyl bromide 2 in anhydrous dichloromethane catalyzed by Hg(CN)₂ afforded the fully galactopyranosylated 6-fluoropyridoxol (3) in 89% yield, which was deacetylated with NH₃/MeOH giving the free galactopyranoside 3, α⁴, α⁵-tri-O-(β-D-galactopyranosyl)-6-fluoropyridoxol 4 in quantitative yield (Figure 1). The ESI-MS of 3 showed the expected molecular ion at m/z 1178 and quasimolecular ion at m/z 1179 [M+H], corresponding to the fully adorned derivative with three fully acetylated galactosides. The identity of 3 was established using ¹H and ¹³C NMR. The anomeric protons H-1′, H-1″ and H-1‴ of D-galactoses linked to 3, α⁴- and α⁵-positions of FPOL at 5.24, 4.66 and 4.52 ppm, respectively, with three well resolved doublets (J_1,2 = 8.0 Hz), as well as J_2,3 ~ 10 Hz confirming that all D-galactoses are in the β-configuration with the ⁴C₁ chair conformation, whereas in the ¹³C NMR spectrum, the anomeric carbons C-1′, C-1″ and C-1‴ occurred at 103.34 and 100.22 ppm.

Figure 1. Reagents and conditions.

(a) 2, 3, 4, 6-tetra-O-acetyl-α-D-galactopyranosyl bromide 2, Hg(CN)₂, 4Å M.S., CH₂Cl₂, r.t., 12 h, 89%; (b) NH₃-MeOH, 0 °C→r.t., 24 h, quantitative yields.

4 was stable in buffer and gave a single sharp ¹⁹F NMR signal. Exposure of 4 to β-gal indicated that all three β-D-galactopyranosyl C_1(gal)-O linkages are sensitive resulting in multiple ¹⁹F signals around 3 ppm and 12 ~ 20 ppm (Figure 2). These results demonstrate the principle of polyglycosylation to enhance water solubility, while retaining sensitivity to β-gal, but the complex spectra suggested a need for a more sophisticated approach. We therefore designed two further molecules 3-O-(β-D-galactopyranosyl)-α⁴, α⁵-di-O-(β-D-glucopyranosyl)-6-fluoropyridoxol 12 and 3-O-(β-D-galactopyranosyl)-α⁴, α⁵-di-O-(α-D-mannopyranosyl)-6-fluoropyridoxol 13, featuring differential glycosylation: galactosylation at the 3 phenolic group being sensitive to β-gal, and glucopyranosylation or mannopyranosylation at the α⁴, α⁵-hydroxymethyl groups to aid water solubility, but resist β-gal activity. Retro-synthetic analysis suggested two approaches through differentially protected intermediates as key synthons.

Figure 2.

¹⁹F-NMR spectra of 3, α⁴, α⁵-tri-O-(β-D-galactopyranosyl)-6-fluoropyridoxol 4 (10.1 mg, 15 mmol, lower) and its products resulting from addition of β-gal (E801A, 15 units) in PBS (pH=7.4) at 37 °C (upper). Spectra acquired in 51 s and enhanced with an exponential line broadening 40 Hz; β-D-Galp = β-D-galactopyranosyl.

6-Fluoro-α⁴, α⁵-isopropylidenepyridoxol 5 was previously prepared as part of the synthesis of 6-fluoro-3, α⁴-isopropylidenepyridoxol.^{8, 13} Testing various acids as catalysts showed 2% H₂SO₄ acetone solution to provide the best yield of 5 (26%). The regioselectivity of the acetonation reaction was confirmed by comparing ¹H-NMR of 5 and 6-fluoro-3, α⁴-isopropylidenepyridoxol, in which the 5-CH₂ signal of 5 appeared at 5.03 ppm as a singlet, while in 6-fluoro-3, α⁴-isopropylidenepyridoxol it appeared at 4.97 ppm as a doublet (J_H-5,HO-5 = 1.2 Hz) due to the coupling of 5-OH. 13 Treatment of 5 with 2 using the Koenigs-Knorr glycosylation gave 3-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-galactopyranosyl)-α⁴, α⁵-isopropylidene-6-fluoropyrodoxol 6 in 85% yield. The δ_H-1′ at 4.64 ppm is a well-resolved doublet (J_1,2 = 8.0 Hz) and δ_C-1′ at 100.03 ppm demonstrated that the D-galactose was in the β-configuration. The correlation between 2-CH₃ and H-1′ of sugar ring from the NOSEY spectrum of 6 verified that 2, 3, 4, 6-tetra-O-acetyl-β-D-galactopyranosyl residue connected at 3 phenolic site providing further evidence that the acetonation had occurred regioselectively on 4, 5 hydroxymethyl groups.

3-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-galactopyranosyl)-6-fluoropyridoxol 7 was obtained by cleavage of acetonide 6, but the yields were quite low (≤15%), based on several hydrolysis conditions, such as 80% AcOH, 1% HCl or 90% CF₃CO₂H in MeOH, CH₂Cl₂ or 1,4-dioxane at various temperatures (60 ~ 100 °C). A moderate amount of 1 was recoverable indicating that the β-D-galactopyranosyl C_1′(gal)-O₃ bond became weak and sensitive to acid hydrolysis, presumably due to the presence of the 6-fluorine atom. Condensation of 7 with 2, 3, 4, 6-tetra-O-acetyl-α-D-glucopyranosyl bromide 8 or 2, 3, 4, 6-tetra-O-acetyl-α-D-mannopyranosyl bromide 9 in dry CH₂Cl₂ with Hg(CN)₂ as a promoter gave 3-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-galactopyranosyl)-α⁴, α⁵-di-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-glucopyranosyl)-6-fluoropyridoxol 10 or 3-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-galactopyranosyl)-α⁴, α⁵-di-O-(2, 3, 4, 6-tetra-O-acetyl-α-D-mannopyranosyl)-6-fluoropyridoxol 11 in yields of 80% or 78%, respectively. Deacetylation of 10 or 11 in NH₃/MeOH from 0°C to room temperature gave the target molecules 12 and 13 in quantitative yields (Figure 3). However, the overall yields for 12 and 13 through the five-step reactions were only of 3% with limiting steps in the α⁴, α⁵-isopropylidene group formation and hydrolysis procedures.

Figure 3. Reagents and conditions.

(a) 2% H₂SO₄, acetone, r.t. 4~5 h, 26%; (b) 2, 3, 4, 6-tetra-O-acetyl-α-D-galactopyranosyl bromide 2, Hg(CN)₂, 4Å M.S., CH₂Cl₂, r.t., 12 h, 85%; (c) 80% AcOH, 80°C, 4~5 h, 15%; (d) 2, 3, 4, 6-tetra-O-acetyl-α-D-glucopyranosyl bromide 8 or 2, 3, 4, 6-tetra-O-acetyl-α-D-mannopyranosyl bromide 9, Hg(CN)₂, 4Å M.S., CH₂Cl₂, r.t., 12 h, 80%(→10) or 78%(→11), respectively; (e) NH₃-MeOH, 0°C→r.t., 24 h, quantitative yields.

The acidic 3 phenolic group para to 6-fluorine atom in FPOL should be easily converted into the monoanion under mild base conditions, ^{8, 13, 22} suggesting an alternate approach to selectively benzylate the 3-OH under carefully controlled conditions. Benzyl bromide (1.1 equiv.) was added dropwise over a period of 4 ~ 5 h to the well-stirred reaction mixture of 1 in a dichloromethane-aqueous biphasic system (pH 10~ 11) using tetrabutylammonium bromide (TBAB) as the phase-transfer catalyst yielding 3-O-benzyl-6-fluoropyridoxol 14 in 76% yield. The structure was established on the basis of the coupling characteristics of α⁴, α⁵-CH₂ as doublets (J_H-4,HO-4 = 6.0 Hz, J_H-5,HO-5 = 5.4 Hz) and α⁴, α⁵-OH as triplets in the ¹H-NMR spectrum. Condensation of 14 with 8 or 9 gave 3-O-benzyl-α⁴, α⁵-di-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-glucopyranosyl)-6-fluoropyridoxol 15 or 3-O-benzyl-α⁴, α⁵-di-O-(2, 3, 4, 6-tetra-O-acetyl-α-D-mannopyranosyl)-6-fluoropyridoxol 16 in satisfactory yields. Removal of the benzyl-protecting group afforded acceptors α⁴, α⁵-di-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-glucopyranosyl)-6-fluoropyridoxol 17 or α⁴, α⁵-di-O-(2, 3, 4, 6-tetra-O-acetyl-α-D-mannopyranosyl)-6-fluoropyridoxol 18 in quantitative yields, which were subjected to a procedure similar to that described above for the preparation of galactosides giving 10 or 11 in high yields (88% or 85%, respectively). After work up and deacetylation, the target compounds 12 and 13 were obtained in 57% and 52% overall yields over five-step reactions (Figure 4).

Figure 4. Reagents and conditions.

(a) benzyl bromide (1.1 equiv.), CH₂Cl₂-H₂O, pH 10~11, 50°C, TBAB, 4~5 h, 76%; (b) 2, 3, 4, 6-tetra-O-acetyl-α-D-glucopyranosyl bromide 8 or 2, 3, 4, 6-tetra-O-acetyl-α-D-mannopyranosyl bromide 9, Hg(CN)₂, 4Å M.S., CH₂Cl₂, r.t., 12 h, 90%(→15) or 85%(→16), respectively; (c) 25psi H₂, Pd/C, r.t., 12 h, quantitative yields; (d) 2, 3, 4, 6-tetra-O-acetyl-α-D-galactopyranosyl bromide 2, Hg(CN)₂, 4Å M.S., CH₂Cl₂, r.t., 12 h, 88%(→10) or 85%(→11), respectively; (e) NH₃-MeOH, 0°C→r.t., 24 h, 95%(→12) or 94%(→13), respectively.

Recognizing the differential reactivity of the 3 phenolic group over the hydroxymethyl groups, most recently, we have successfully specifically galactopyranosylated 1 on the 3 phenolic group directly with 2 using the above phase-transfer catalysis technique yielding 7.⁸ Figure 5 depicts a very efficient route to synthesize the target compounds 12 and 13 using just three-steps with higher overall yields (67% and 65%, respectively).

Figure 5. Reagents and conditions.

(a) 2, 3, 4, 6-tetra-O-acetyl-α-D-galactopyranosyl bromide 2, CH₂Cl₂-H₂O, pH 10~11, r.t., TBAB, 4~5 h, 88%; (b) 2, 3, 4, 6-tetra-O-acetyl-α-D-glucopyranosyl bromide 8 or 2, 3, 4, 6-tetra-O-acetyl-α-D-mannopyranosyl bromide 9, Hg(CN)₂, 4Å M.S., CH₂Cl₂, r.t., 12 h, 80%(→10) or 78%(→11), respectively; (c) NH₃-MeOH, 0°C→r.t., 24 h, 95%(→12) or 94%(→13), respectively.

Characteristics

4, 12 and 13 each gave a single narrow ¹⁹F NMR signal between δ −2.0 ~ −3.3 ppm essentially invariant (Δδ ≤ 0.06 ppm) with pH in the range 3 to 12 and temperatures from 25 to 37 °C in whole rabbit blood, 0.9% saline or PBS. Addition of β-gal (E801A) in PBS at 37 °C to 4, 12 and 13 caused rapid hydrolysis releasing the aglycones α⁴, α⁵-di-O-(β-D-galactopyranosyl)-6-fluoropyridoxol, α⁴, α⁵-di-O-(β-D-glucopyranosyl)-6-fluoropyridoxol and α⁴, α⁵-di-O-(α-D-mannopyranosyl)-6-fluoropyridoxol, which also appeared as single narrow ¹⁹F signals between δ −11.20 ~ −12.40 ppm (Δδ = 9.0 ~ 9.4 ppm) (Table 1). Action of β-gal on 4 was complicated by action on each of the galactose residue apparently randomly to generate multiple signals representing 1 together with partially galactosylated products (Figure 2). The β-gal hydrolysis of 4, 12, and 13 proceeded in a smooth manner indicating that the liberated aglycones have no inhibitory effects on β-gal (Figure 6). The kinetic curves suggest straightforward first-order kinetics, which were much more rapid for all substrates than for GFPOL. 12 and 13 gave single products upon exposure to β-gal (Figure 7). Addition of 12 and 13 to stably transfected human breast MCF-7-lacZ tumor cells showed cleavage of 12 or 13 (Figure 8) and this proceeded in an initially smooth monotonic manner at rates of 18.6 or 19.6 μmol/min per million MCF7-lacZ cells, respectively. 12 and 13 have much higher aqueous solubility than GFPOL (75 mM, vs. 12, 196 mM and 13, 173 mM in PBS).

Table 1.

¹⁹F chemical shifts ⁺ and hydrolytic rates ^*

Reporters	4	12	13	GFPOL
δF (substrate)	−3.02	−2.85	−2.14	−3.22
δF (product)	−12.37	−12.16	−11.22	−11.21
ΔδF	9.35	9.31	9.08	7.99
ν(μmol/min/unit)	34.0	35.0	38.0	4.3

⁺ppm with respect to sodium trifluoroacetate.

^*β-gal (E801A) added at 37°C in PBS (0.1 M, pH=7.4).

Figure 6.

The kinetic hydrolysis time courses of 4 (◆), 12 ( ), 13 (□) (15.0 mmol each) and GFPOL (○) (10.0 mmol) by β-gal (E801A, 15 units) hydrolysis in PBS (0.1 M, pH=7.4, 600 μL) at 37 °C.

Figure 7.

¹⁹F-NMR spectra of 3-O-(β-D-galactopyranosyl)-α⁴, α⁵-di-O-(β-Dglucopyranosyl)-6-fluoropyridoxol 12 (10.1 mg, 15 mmol, lower) and its hydrolysis by β-gal (E801A, 15 units) in PBS (0.1 M, pH=7.4, 600 μL) at 37 °C (upper). Spectra acquired in 205 s and enhanced with exponential line broadening = 40 Hz.

Figure 8.

¹⁹F-NMR spectra of 3-O-(β-D-galactopyranosyl)-α⁴, α⁵-di-O-(β-D-glucopyranosyl)-6-fluoropyridoxol 12 (5.1 mg, 7.5 mmol) with stably transfected MCF7-lacZ cells (5×10⁶) in PBS (0.1M, pH=7.4, 600 μL) at 37 °C. Spectra acquired in 51 s and enhanced with an exponential line broadening = 100 Hz. (DUFPOL: α⁴, α⁵-di-O-(β-D-glucopyranosyl)-6-fluoropyridoxol).

The products α⁴, α⁵-di-O-(β-D-galactopyranosyl)-6-fluoropyridoxol (DGFPOL), α⁴, α⁵-di-O-(β-D-glucopyranosyl)-6-fluoropyridoxol (DUFPOL) and α⁴, α⁵-di-O-(α-D-mannopyranosyl)-6-fluoropyridoxol (DMFPOL) of the action of β-gal on 4, 12 and 13 also exhibit large ¹⁹F NMR chemical shift response to pH (Δδ = ~ 11.0 ppm) in the range of pH 1 ~ 12 (Figure 9, Table 2), but there is no spectral overlap with the substrates.

Figure 9.

¹⁹F NMR chemical shifts pH titration curve of DGFPOL, DUFPOL and DMFPOL in 0.9% saline at 37°C. (DGFPOL: α⁴, α⁵-di-O-(β-D-galactopyranosyl)-6-fluoropyridoxol; DUFPOL: α⁴, α⁵-di-O-(β-D-glucopyranosyl)-6-fluoropyridoxol; DMFPOL: α⁴, α⁵-di-O-(α-D-mannopyranosyl)-6-fluoropyridoxol).

Table 2.

Acidities and ¹⁹F NMR/pH properties of DGFPOL, DUFPOL, DMFPOL and FPOL in saline at 25 °C

pH Indicators	DGFPOL	DUFPOL	DMFPOL	FPOL24
pKa	7.95	8.08	8.18	8.20
δF acid	−8.34	−8.15	−7.44	−9.85
δF base	−19.05	−18.85	−18.15	−19.61

⁺ppm with respect to sodium trifluoroacetate.

Conclusion

These results provide further evidence for the broad specificity of β-gal and the feasibility of modifying substrate structures to enhance enzyme sensitivity and water solubility. The additional sugar residues in 4, 12, and 13 compared with GFPOL all lead to faster cleavage kinetics with β-gal. Significantly, the differential glycosylation provides structures that respond to β-gal with generation of single products. The results with stably transfected breast cancer cells indicate the potential for future studies in vivo.

EXPERIMENTAL

General methods

NMR spectra were recorded on a Varian Inova 400 spectrometer (400 MHz for ¹H, 100 MHz for ¹³C, 376 MHz for ¹⁹F) with CDCl₃, or DMSO-d₆ as solvents. ¹H and ¹³C chemical shifts are referenced to TMS as internal standard, and ¹⁹F to a dilute solution of NaTFA in a capillary as external standard (37 °C). Compounds were characterized by acquisition of ¹H, ¹³C, DEPT, ¹H-¹H COSY or NOESY experiments at 25 °C. Microanalyses were performed on a Perkin-Elmer 2400CHN microanalyser. Mass spectra were obtained by positive and negative ESI-MS using a Micromass Q-TOF hybrid quadrupole/time-of-flight instrument (Micromass UK Ltd.). Reactions requiring anhydrous conditions were performed under nitrogen or argon. Hg(CN)₂ was dried before use at 50 °C for 1h, CH₂Cl₂ was dried over Drierite, and acetonitrile was dried on CaH₂ and kept over molecular sieves under N₂. Solutions in organic solvents were dried with anhydrous sodium sulfate, and concentrated in vacuo below 45 °C. 2, 3, 4, 6-tetra-O-acetyl-α-D-galactopyranosyl bromide 2 and 2, 3, 4, 6-tetra-O-acetyl-α-D-glucopyranosyl bromide 8 were purchased from the Sigma Chemical Company. 2, 3, 4, 6-tetra-O-acetyl-α-D-mannopyranosyl bromide 9 was prepared according to the literature method.²³ Column chromatography was performed on silica gel (200 ~ 300 mesh) by elution with cyclohexane-EtOAc and silica gel GF₂₅₄ (Aldrich) used for analytical TLC. Detection was effected by spraying the plates with 5% ethanolic H₂SO₄ (followed by heating at 110 °C for ~10 min.) or by direct UV illumination of the plate.

For enzyme kinetic experiments, 4, 12 and 13 (10.1 mg, 15 mmol) were dissolved in PBS (0.1 M, pH=7.4, 600 μL), and a PBS solution of β-gal (0.1 M, pH=7.4, 15 μL, 1 unit/μL, E801A, Promega, Madison, WI, USA) was added and NMR data were acquired immediately at 37 °C.

MCF7-lacZ human breast cancer cells stably transfected to express β-gal were grown in culture under standard conditions and harvested. 12 or 13 were added to suspension of cells (5×10⁶) in PBS and observed by NMR for 1 h.

Syntheses

3, α⁴, α⁵-tri-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-galactopyranosyl)-6-fluoro-pyridoxol 3

A solution of 2, 3, 4, 6-tetra-O-acetyl-α-D-galactopyranosyl bromide 2 (1.35 g, 3.3 mmol, 1.1 equiv.) in anhydrous CH₂Cl₂ (8 mL) was added dropwise to the solution of 6-fluoropyridoxol 1 (0.18 g, 1.0 mmol) and Hg(CN)₂ (1.01 g, 4.0 mmol) in dry MeCN (10 mL) containing powdered molecular sieves (4 Å, 2.0 g) with vigorous stirring at r.t. under argon in the dark for 12h. The mixture was diluted with CH₂Cl₂ (30 mL), filtered through Celite, washed, dried (Na₂SO₄) and concentrated in vacuo. The residue was purified on a silica gel column (1:3 cyclohexane-EtOAc) to yield 3 (1.05 g, 89%) as syrup, R_f 0.30 (1:3 cyclohexane-EtOAc), NMR (CDCl₃), δ_H: 5.24 (1 H, d, J_1′,2′ = 8.0 Hz, H-1′), 5.04 (1 H, dd, J_2′,3′ = 9.8 Hz, H-2′), 4.73 (1 H, dd, J_3′,4′ = 3.4 Hz, H-3_′), 3.98 (1 H, dd, J_4′,5′ = 2.4 Hz, H-4′), 4.02~4.10 (3 H, m, H-5′, H-6′), 4.66 (1 H, d, J_1″,2″ = 8.0 Hz, H-1″), 4.52 (1 H, d, J_1‴,2‴ = 8.0 Hz, H-1‴), 5.15 (2 H, dd, J_2″,3″ = J_2‴,3‴ = 10.0 Hz, H-2″, H-2‴), 5.07 (2 H, dd, J_3″,4″ = J_3‴,4‴ = 3.6 Hz, H-3″, H-3‴), 5.52 (2 H, dd, J_4″,5″ = J_4‴,5‴ = 3.2 Hz, H-4″, H-4‴), 3.88 (2 H, m, H-5″, H-5‴), 4.18 (2 H, dd, J_5″,6a″ = J_5‴,6a‴ = 3.6 Hz, J_6a″,6b″ = J_6a‴,6b‴ = 9.2 Hz, H-6a″, H-6a‴), 4.11 (2 H, dd, J_5″,6b″ = J_5‴,6b‴ = 6.8 Hz, H-6b″, H-6b‴), 4.48 (2 H, d, J_{CH2-4a,CH2-4b} = J_{CH2-5a,CH2-5b} = 13.2 Hz, CH₂-4a, CH₂-5a), 4.12 (2 H, d, J_{CH2-4a,CH2-4b} = J_{CH2-5a,CH2-5b} = 13.2 Hz, CH₂-4b, CH₂-5b), 2.43 (3 H, s, CH₃-2), 2.18, 2.17, 2.16, 2.15, 2.12, 2.11, 2.10, 2.09, 2.08, 2.07, 2.06, 2.05 (36 H, 12s, 12×CH₃CO) ppm; δ_C: 170.84, 170.79, 170.77, 170.73, 170.68, 170.54, 170.53, 170.49, 170.45, 170.35, 170.31, 170.28 (12×CH₃CO), 146.47 (d, ³J_F-C = 14.5 Hz, Py-C₂), 148.16 (d, ⁴J_F-C = 3.8 Hz, Py-C₃), 133.10 (s, Py-C₄), 112.51 (d, ²J_F-C = 31.3 Hz, Py-C₅), 155.04 (d, ¹J_F-C = 231.2 Hz, Py-C₆), 103.34 (s, C-1′), 100.22 (s, C-1″, C-1‴), 70.75 (s, C-2′), 71.14 (s, C-3′), 70.61 (s, C-4′), 71.56 (s, C-5′), 67.03 (s, C-6′), 67.45 (s, C-2″, C-2‴), 68.39 (s, C-3″, C-3‴), 66.31 (s, C-4″, C-4‴), 68.55 (s, C-5″, C-5‴), 61.99 (s, C-6″, C-6‴), 61.54 (s, CH₂-4), 61.67 (s, CH₂-5), 21.03, 20.94, 20.90, 20.89, 20.87, 20.85, 20.83, 20.79, 20.77, 20.76, 20.74, 20.72 (12s, 12×CH₃CO), 18.77 (s, CH₃-3) ppm. ESIMS: m/z 1178 [M⁺] (26%), 1179 [M+1] (14%). Anal. Calcd. for C₅₀H₆₄NO₃₀F(%): C, 50.96, H, 5.48, N, 1.19; Found: C, 50.93, H, 5.46, N, 1.15.

3, α⁴, α⁵-tri-O-(β-D-galactopyranosyl)-6-fluoropyridoxol 4

A solution of 3 (0.9 g) in anhydrous MeOH (20 mL) containing 0.5 M NH₃ was vigorously stirred from 0 °C to r.t. overnight until TLC showed complete reaction and evaporated to dryness in vacuo. Chromatography of the crude syrup on silica gel with EtOAc/MeOH (4:1) afforded 4 (0.52 g) as a syrup in quantitative yield, R_f 0.10 (1:4 MeOH-EtOAc), NMR (DMSO-d₆), δ_H: 4.95 (1 H, d, J_1′,2′ = 8.2 Hz, H-1′), 4.76 (1 H, dd, J_2′,3′ = 10.0 Hz, H-2′), 4.91 (1 H, dd, J_3′,4′ = 2.8 Hz, H-3′), 5.11 (1 H, dd, J_4′,5′ = 2.3 Hz, H-4′), 3.77 (1 H, m, H-5′), 3.90 (1 H, dd, J_5′,6a′ = 6.4 Hz, J_6a′,6b′ = 12.4 Hz, H-6a′), 3.68 (1 H, dd, J_5′,6b′ = 3.6 Hz, H-6b′), 4.22 (2 H, d, J_1″,2″ = J_1‴,2‴ = 8.0 Hz, H-1″, H-1‴), 3.29 (2 H, dd, J_2″,3″ = J_2‴,3‴ = 10.6 Hz, H-2″, H-2‴), 3.51 (2 H, dd, J_3″,4″ = J_3‴,4‴ = 3.2 Hz, H-3″, H-3‴), 3.62 (2 H, dd, J_4″,5″ = J_4‴,5‴ = 2.4 Hz, H-4″, H-4‴), 3.46 (2 H, m, H-5″, H-5‴), 3.66 (2 H, dd, J_5″,6a″ = J_5‴,6a‴ = 3.6 Hz, J_6a″,6b″ = J_6a‴,6b‴ = 10.4 Hz, H-6a″, H-6a‴), 3.39 (2 H, dd, J_5″,6b″ = J_5‴,6b‴ = 6.6 Hz, H-6b″, H-6b‴), 4.48 (2 H, d, J_{CH2-4a,CH2-4b} = J_{CH2-5a,CH2-5b} = 13.0 Hz, CH₂-4a, CH₂-5a), 4.44 (2 H, d, J_{CH2-4a,CH2-4b} = J_{CH2-5a,CH2-5b} = 13.0 Hz, CH₂-4b, CH₂-5b), 2.32 (3H, s, CH₃-2) ppm; δ_C: 144.65 (d, ³J_F-C = 14.5 Hz, Py-C₂), 147.87 (d, ⁴J_F-C = 3.9 Hz, Py-C₃), 137.36 (d, ⁴J_F-C = 3.8 Hz, Py-C₄), 115.15 (d, ²J_F-C = 32.3 Hz, Py-C₅), 154.26 (d, ¹J_F-C = 226.6 Hz, Py-C₆), 103.19 (s, C-1′), 101.67 (s, C-1″, C-1‴), 70.36 (s, C-2′), 73.94 (s, C-3′), 69.44 (s, C-4′), 76.08 (s, C-5′), 62.88 (s, C-6′), 72.10 (s, C-2″, C-2‴), 73.50 (s, C-3″, C-3‴), 68.26 (s, C-4″, C-4‴), 75.02 (s, C-5″, C-5‴), 60.60 (s, C-6″, C-6‴), 68.77 (s, CH₂-4), 68.92 (s, CH₂-5), 19.19 (s, CH₃-3) ppm. ESIMS: m/z 673 [M⁺] (6%), 674 [M+1] (10%). Anal. Calcd. for C₂₆H₄₀NO₁₈F(%): C, 46.34, H, 5.99, N, 2.08; Found: C, 46.30, H, 5.96, N, 2.05.

α⁴, α⁵-O-isopropylidene-6-fluoropyridoxol 5

A suspension of 1 (0.50 g, 2.67 mmol) in anhydrous acetone (40 mL) containing 2% c. H₂SO₄ was stirred for 4 ~ 5h, at the end of which time TLC (4:1 cyclohexane-EtOAc) indicated complete reaction, then cold saturated Na₂CO₃ solution was added with vigorous stirring up to pH between 8 ~ 9. The precipitate was filtered off and concentration of the reaction mixture under reduced pressure followed by purification on flash silica gel column (4:1 cyclohexane-EtOAc) gave 5 (0.64 g, 26%) as a syrup, R_f 0.34 (4:1 cyclohexane-EtOAc), NMR (CDCl₃), δ_H: 7.45 (1 H, s, HO-3), 5.03 (2 H, s, CH₂-5), 4.57 (2 H, s, CH₂-4), 2.33 (3 H, s, CH₃-2), 1.55 (6 H, s, 2×CH₃) ppm; δ_C: 146.40 (d, ³J_F-C = 14.5 Hz, Py-C₂), 144.32 (d, ⁴J_F-C = 3.8 Hz, Py-C₃), 130.68 (s, Py-C₄), 111.21 (d, ²J_F-C = 32.8 Hz, Py-C₅), 152.20 (d, ¹J_F-C = 231.2 Hz, Py-C₆), 100.15 (s, CMe₂), 58.70 (d, ³J_F-C = 3.0 Hz, CH₂-5), 54.51 (s, CH₂-4), 31.62 (s, C(CH₃)₂), 17.58 (s, CH₃-2) ppm. Anal. Calcd. for C₁₁H₁₄NO₃F(%): C, 58.13, H, 6.21, N, 6.17; Found: C, 58.08, H, 6.16, N, 6.11.

3-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-galactopyranosyl)-α⁴, α⁵-O-isopropylidene-6-fluoropyridoxol 6

To a solution of 5 (0.62 g, 2.72 mmol) and Hg(CN)₂ (0.88 g, 3.50 mmol) in dry CH₂Cl₂ (10 mL) containing freshly activated 4Å molecular sieves (2.0 g) was added dropwise 2 (1.23 g, 3.0 mmol, 1.1 equiv.). The mixture was stirred overnight in the dark at r.t. under N₂ until TLC indicated complete reaction. Work up as for 3 gave 6 (1.29 g, 85%), R_f 0.40 (2:3 cyclohexane-EtOAc), NMR (CDCl₃), δ_H: 4.64 (1 H, d, J_1′,2′ = 8.0 Hz, H-1′), 5.25 (1 H, dd, J_2′,3′ = 10.0 Hz, H-2′), 5.02 (1 H, dd, J_3′,4′ = 3.6 Hz, H-3′), 5.41 (1 H, dd, J_4′,5′ = 3.2 Hz, H-4′), 3.97 (1 H, m, H-5′), 4.21 (1 H, dd, J_5′,6a′ = 4.4 Hz, J_6a′,6b′ = 11.2 Hz, H-6a′), 4.13 (1 H, dd, J_5′,6b′ = 7.2 Hz, H-6b′), 5.10 (1 H, d, J_{CH2-4a,CH2-4b} = 8.0 Hz, CH₂-4a), 4.67 (1 H, d, J_{CH2-4a,CH2-4b} = 8.0 Hz, CH₂-4b), 5.14 (1 H, d, J_{CH2-5a,CH2-5b} = 9.6 Hz, CH₂-5a), 5.12 (1 H, d, J_{CH2-5a,CH2-5b} = 9.6 Hz, CH₂-5b), 2.42 (3 H, s, CH₃-2), 2.17, 2.09, 2.08, 1.99 (12 H, 4s, 4×CH₃CO), 1.61, 1.59 (6 H, 2s, 2×CH₃) ppm; δ_C: 170.78, 170.39, 170.26, 170.11 (4s, 4×CH₃CO), 145.48 (d, ³J_F-C = 15.2 Hz, Py-C₂), 133.16 (d, ⁴J_F-C = 4.0 Hz, Py-C₃), 126.26 (s, Py-C₄), 116.95 (d, ²J_F-C = 32.1 Hz, Py-C₅), 154.30 (d, ¹J_F-C = 229.0 Hz, Py-C₆), 101.41 (s, CMe₂), 100.03 (s, C-1′), 68.70 (s, C-2′), 70.82 (s, C-3′), 67.12 (s, C-4′), 71.53 (s, C-5′), 64.28 (s, C-6′), 55.38 (s, CH₂-4), 61.58 (s, CH₂-5), 31.88 (s, C(CH₃)₂), 20.90, 20.89, 20.82, 20.77 (4s, 4×CH₃CO), 18.77 (s, CH₃-2) ppm. Anal. Calcd. for C₂₅H₃₂NO₁₂F(%): C, 53.84, H, 5.79, N, 2.51; Found: C, 53.79, H, 5.74, N, 2.49.

3-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-galactopyranosyl)-6-fluoropyridoxol 7

A mixture of 6 (1.25 g, 2.50 mmol) in 80% AcOH (40 mL) was stirred at 80 °C for 4 ~ 5 h, till TLC (1:3 cyclohexane-EtOAc) showed reaction complete. The cooled mixture was neutralized with cold saturated Na₂CO₃ solution, extracted with EtOAc (4×30 mL), concentrated and purified by flash silica gel column with 1:4 cyclohexane-EtOAc giving 7 (0.17 g, 15%) as a syrup, R_f 0.18 (1:4 cyclohexane-EtOAc), NMR (CDCl₃), δ_H: 4.79 (1 H, d, J_1′,2′ = 8.0 Hz, H-1′), 5.55 (1 H, dd, J_2′,3′ = 10.6 Hz, H-2′), 5.10 (1 H, dd, J_3′,4′ = 3.6 Hz, H-3′), 5.41 (1 H, dd, J_4′,5′ = 3.6 Hz, H-4′), 3.88 (1 H, m, H-5′), 4.24 (1 H, dd, J_5′,6a′ = 4.4 Hz, J_6a′,6b′ = 12.0 Hz, H-6a′), 4.09 (1 H, dd, J_5′,6b′ = 6.0 Hz, H-6b′), 5.01 (2 H, d, J_{CH2-4a,CH2-4b} = J_{CH2-5a,CH2-5b} = 12.4 Hz, CH₂-4a, CH₂-5a), 4.62 (1 H, d, J_{CH2-4a,CH2-4b} = 12.4 Hz, CH₂-4b), 4.66 (1 H, d, J_{CH2-5a,CH2-5b} = 12.4 Hz, CH₂-5b), 3.50 (1 H, m, α⁴-HO, exchangeable with D₂O), 3.56 (1 H, m, α⁵-HO, exchangeable with D₂O), 2.47 (3 H, s, CH₃-2), 2.23, 2.17, 2.02, 2.00 (12 H, 4s, 4×CH₃CO)ppm; δ_C: 170.32, 170.28, 170.18, 169.48 (4×CH₃CO), 150.33 (d, ³J_F-C = 15.2 Hz, Py-C₂), 147.62 (d, ⁴J_F-C = 4.6 Hz, Py-C₃), 146.32 (d, ³J_F-C = 4.5 Hz, Py-C₄), 120.17 (d, ²J_F-C = 32.0 Hz, Py-C₅), 157.60 (d, ¹J_F-C = 235.8 Hz, Py-C₆), 102.39 (s, C-1′), 68.91 (s, C-2′), 70.74 (s, C-3′), 67.19 (s, C-4′), 71.93 (s, C-5′), 61.98 (s, C-6′), 55.91 (s, CH₂-4), 59.60 (s, CH₂-5), 20.99, 20.85, 20.70, 20.67 (4s, 4×CH₃CO), 19.46 (s, CH₃-2) ppm. Anal. Calcd. for C₂₂H₂₈NO₁₂F(%): C, 51.05, H, 5.46, N, 2.71; Found: C, 51.00, H, 5.39, N, 2.68.

Alternately 7 was synthesized from 1 directly by phase transfer catalysis: to a well stirred CH₂Cl₂ (10 mL)-H₂O (10 mL) biphasic mixture (pH 10 ~ 11) of 1 (0.5 g, 2.67 mmol) and TBAB (0.1 g, 0.31 mmol), a solution of 2 (1.21 g, 2.94 mmol, 1.1 equiv.) in CH₂Cl₂ (10 mL) was added dropwise over a period of 4 ~ 5h at r.t., and the stirring continued for an additional hour. The products were extracted (EtOAc; 4×20 mL), washed free of alkali, dried (Na₂SO₄), and concentrated. The residue was purified by column chromatography on silica gel (1:4 cyclohexane-EtOAc) to afford 7 (1.08 g, 88%) as syrup, which is identical in all respects to the product obtained above.

3-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-galactopyranosyl)-α⁴, α⁵-di-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-glucopyranosyl)-6-fluoropyridoxol 10 and 3-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-galactopyranosyl)-α⁴, α⁵-di-O-(2, 3, 4, 6-tetra-O-acetyl-α-D-manno-pyranosyl)-6-fluoropyridoxol 11

Condensation of 7 (0.5 g, 1.1 mmol) with 2, 3, 4, 6-tetra-O-acetyl-α-D-glucopyranosyl bromide 8 or 2, 3, 4, 6-tetra-O-acetyl-α-D-mannopyranosyl bromide 9 (1.0 g, 2.40 mmol, 1.1 equiv.) in dry CH₂Cl₂ (10 mL) with Hg(CN)₂ (0.63 g, 2.50 mmol) as a promoter, according to the procedures described for the preparation of 3 and 6, furnished 3-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-galactopyranosyl)-α⁴, α⁵-di-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-glucopyranosyl)-6-fluoropyridoxol 10 and 3-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-galactopyranosyl)-α⁴, α⁵-di-O-(2, 3, 4, 6-tetra-O-acetyl-α-D-mannopyranosyl)-6-fluoropyridoxol 11, respectively.

3-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-galactopyranosyl)-α⁴, α⁵-di-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-glucopyranosyl)-6-fluoropyridoxol 10 (1.04 g, 80%), syrup, R_f 0.30 (1:3 cyclohexane-EtOAc), NMR (CDCl₃), δ_H: 5.06 (1 H, d, J_1′,2′ = 7.8 Hz, H-1′), 5.28 (1 H, dd, J_2′,3′ = 8.8 Hz, H-2′), 4.98 (1 H, dd, J_3′,4′ = 4.8 Hz, H-3′), 4.73 (1 H, dd, J_4′,5′ = 2.8 Hz, H-4′), 3.95 (1 H, m, H-5′), 4.19 (1 H, dd, J_5′,6a′ = 3.6 Hz, J_6a′,6b′ = 10.8 Hz, H-6a′), 4.02 (1 H, dd, J_5′,6b′ = 5.2 Hz, H-6b′), 5.36 (1 H, d, J_1″,2″ = 8.0 Hz, H-1″), 5.39 (1 H, d, J_1‴,2‴ = 8.0 Hz, H-1‴), 5.12 (1 H, dd, J_2″,3″ = 7.2 Hz, H-2″), 5.15 (1 H, dd, J_2‴,3‴ = 6.8 Hz, H-2‴), 5.04 (1 H, dd, J_3″,4″ = 3.2 Hz, H-3″), 5.07 (1 H, dd, J_3‴,4‴ = 3.6 Hz, H-3‴), 4.76 (1 H, dd, J_4″,5″ = 2.8 Hz, H-4″), 4.78 (1 H, dd, J_4‴,5‴ = 2.8 Hz, H-4‴), 3.91 (1 H, m, H-5″), 3.93 (1 H, m, H-5‴), 4.08 (1 H, dd, J_5″,6a″ = 3.2 Hz, J_6a″,6b″ = 9.4 Hz, H-6a″), 4.10 (1 H, dd, J_5‴,6a‴ = 3.0 Hz, J_6a″,6b″ = 10.0 Hz, H-6a‴), 4.04 (1 H, dd, J_5″,6b″ = 7.6 Hz, H-6b″), 4.07 (1 H, dd, J_5‴,6b‴ = 6.8 Hz, H-6b‴), 4.55 (2 H, d, J_{CH2-4a,CH2-4b} = J_{CH2-5a,CH2-5b} = 11.2 Hz, CH₂-4a, CH₂-5b), 4.49 (1 H, d, J_{CH2-4a,CH2-4b} = 11.2 Hz, CH₂-4b), 4.91 (1 H, d, J_{CH2-5a,CH2-5b} = 11.2 Hz, CH₂-5a), 2.34 (3 H, s, CH₃-2), 2.05, 1.98, 1.97, 1.96, 1.95, 1.94, 1.93, 1.92, 1.91, 1.90, 1.89, 1.88 (36 H, 12s, 12×CH₃CO) ppm; δ_C: 170.83, 170.80, 170.76, 170.72, 170.70, 170.56, 170.28, 170.20, 170.17, 170.00, 169.82, 169.75 (12×CH₃CO), 152.14 (d, ³J_F-C = 16.0 Hz, Py-C₂), 149.81 (s, Py-C₃), 138.42 (d, ³J_F-C = 11.4 Hz, Py-C₄), 117.48 (d, ²J_F-C = 32.0 Hz, Py-C₅), 157.56 (d, ¹J_F-C = 233.5 Hz, Py-C₆), 102.66 (s, C-1′), 98.00 (s, C-1″), 98.06 (s, C-1‴), 71.09 (s, C-2′), 68.65 (s, C-2″), 68.95 (s, C-2‴), 74.46 (s, C-3′), 70.84 (s, C-3″), 71.51 (s, C-3‴), 70.05 (s, C-4′), 68.13 (s, C-4″), 68.22 (s, C-4‴), 75.10 (s, C-5′), 72.20 (s, C-5″), 74.24 (s, C-5‴), 63.75 (s, C-6′), 61.91 (s, C-6″), 63.86 (s, C-6‴), 56.77 (s, CH₂-4), 57.16 (s, CH₂-5), 21.20, 20.95, 20.93, 20.91, 20.89, 20.87, 20.85, 20.75, 20.67, 20.62, 20.58, 20.54 (12s, 12×CH₃CO), 19.76 (s, CH₃-3) ppm. ESIMS: m/z 1178 [M⁺] (28%), 1179 [M+1] (12%). Anal. Calcd. for C₅₀H₆₄NO₃₀F(%): C, 50.96, H, 5.48, N, 1.19; Found: C, 50.92, H, 5.44, N, 1.16.

3-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-galactopyranosyl)-α⁴, α⁵-di-O-(2, 3, 4, 6-tetra-O-acetyl-α-D-mannopyranosyl)-6-fluoropyridoxol 11 (1.01 g, 78%), syrup, R_f 0.35 (1:3 cyclohexane-EtOAc), NMR (CDCl₃), δ_H: 4.80 (1 H, d, J_1′,2′ = 8.2 Hz, H-1′), 5.13 (1 H, dd, J_2′,3′ = 9.8 Hz, H-2′), 5.36 (1 H, dd, J_3′,4′ = 4.2 Hz, H-3′), 5.30 (1 H, dd, J_3″,4″ = 3.6 Hz, H-4′), 4.01 (1 H, m, H-5′), 4.33 (1 H, dd, J_5′,6a′ = 3.2 Hz, J_6a′,6b′ = 10.0 Hz, H-6a′), 4.11 (1 H, dd, J_5′,6b′ = 4.6 Hz, H-6b′), 4.71 (2 H, d, J_1″,2″ = J_1‴,2‴ = 2.4 Hz, H-1″, H-1‴), 4.74 (2 H, dd, J_2″,3″ = J_2‴,3‴ = 6.2 Hz, H-2″, H-2‴), 5.22 (2 H, dd, J_3″,4″ = J_3‴,4‴ = 3.8 Hz, H-3″, H-3‴), 3.95 (2 H, dd, J_4″,5″ = J_4‴,5‴ = 2.0 Hz, H-4″, H-4‴), 4.02 (2 H, m, H-5″, H-5‴), 4.11 (2 H, dd, J_5″,6a″ = J_5‴,6a‴ = 2.0 Hz, J_6a″,6b″ = J_6a″,6b″ = 7.4 Hz, H-6a″, H-6a‴), 4.07 (2 H, dd, J_5″,6b″ = J_5‴,6b‴ = 5.6 Hz, H-6b″, H-6b‴), 4.87 (2 H, d, J_{CH2-4a,CH2-4b} = J_{CH2-5a,CH2-5b} = 13.6 Hz, CH₂-4a, CH₂-5b), 4.67 (1 H, d, J_{CH2-4a,CH2-4b} = J_{CH2-5a,CH2-5b} = 13.6 Hz, CH₂-4b, CH₂-5a), 2.33 (3 H, s, CH₃-2), 2.07, 2.04, 2.03, 2.00, 1.99, 1.98, 1.97, 1.96, 1.95, 1.94, 1.93, 1.92 (36 H, 12s, 12×CH₃CO); δ_C: 171.27, 171.23, 171.15, 171.06, 170.87, 170.83, 170.76, 170.63, 170.58, 170.44, 170.29, 170.25 (12×CH₃CO), 153.06 (d, ³J_F-C = 16.0 Hz, Py-C₂), 149.41 (d, ⁴J_F-C = 4.6 Hz, Py-C₃), 145.38 (d, ³J_F-C = 4.6 Hz, Py-C₄), 117.21 (d, ²J_F-C = 31.3 Hz, Py-C₅), 159.55 (d, ¹J_F-C = 235.0 Hz, Py-C₆), 103.62 (s, C-1′), 98.32 (s, C-1″), 98.61 (s, C-1‴), 70.75 (s, C-2′), 70.22 (s, C-2″), 70.26 (s, C-2‴), 71.83 (s, C-3′), 70.36 (s, C-3″), 70.39 (s, C-3‴), 69.38 (s, C-4′), 67.04 (s, C-4″), 68.60 (s, C-4‴), 72.54 (s, C-5′), 71.90 (s, C-5″), 72.45 (s, C-5‴), 61.47 (s, C-6′), 62.46 (s, C-6″), 63.53 (s, C-6‴), 56.44 (s, CH₂-4), 56.46 (s, CH₂-5), 21.29, 21.21, 21.19, 21.17, 21.13, 21.10, 21.08, 21.05, 21.00, 20.95, 20.90, 20.88 (12s, 12×CH₃CO), 20.35 (s, CH₃-3) ppm. ESIMS: m/z 1178 [M⁺] (20%), 1179 [M+1] (17%). Anal. Calcd. for C₅₀H₆₄NO₃₀F(%): C, 50.96, H, 5.48, N, 1.19; Found: C, 50.94, H, 5.45, N, 1.16.

3-O-(β-D-galactopyranosyl)-α⁴, α⁵-di-O-(β-D-glucopyranosyl)-6-fluoro-pyridoxol 12 and 3-O-(β-D-galactopyranosyl)-α⁴, α⁵-di-O-(α-D-mannopyranosyl)-6-fluoropyridoxol 13 Compounds 10, 11 (1.00 g, 0.85 mmol) were deacetylated as described above for 4, to yield 12 and 13 in quantitative yields.

3-O-(β-D-galactopyranosyl)-α⁴, α⁵-di-O-(β-D-glucopyranosyl)-6-fluoropyridoxol 12 (0.57 g), foam solid, R_f 0.20 (1:4 MeOH-EtOAc), NMR (DMSO-d₆), δ_H: 5.01 (1 H, d, J_1′,2′ = 8.2 Hz, H-1′), 5.22 (1 H, dd, J_2′,3′ = 9.0 Hz, H-2′), 4.92 (1 H, dd, J_3′,4′ = 4.6 Hz, H-3′), 4.70 (1 H, dd, J_4′,5′ = 2.6 Hz, H-4′), 3.91 (1 H, m, H-5′), 4.12 (1 H, dd, J_5′,6a′ = 3.2 Hz, J_6a′,6b′ = 10.2 Hz, H-6a′), 4.00 (1 H, dd, J_5′,6b′ = 5.6 Hz, H-6b′), 5.14 (2 H, d, J_1″,2″ = 10.0 Hz, H-1″, H-1‴), 4.82 (2 H, dd, J_2″,3″ = J_2‴,3‴ = 8.2 Hz, H-2″, H-2‴), 4.69 (2 H, dd, J_3″,4″ = J_3‴,4‴ = 3.4 Hz, H-3″, H-3‴), 4.93 (2 H, dd, J_4″,5″ = J_4‴,5‴ = 3.2 Hz, H-4″, H-4‴), 3.65 (2 H, m, H-5″, H-5‴), 3.55 (2 H, dd, J_5″,6a″ = J_5‴,6a‴ = 4.8 Hz, J_6a″,6b″ = J_6a‴,6b‴ = 12.0 Hz, H-6a″, H-6a‴), 3.31 (2 H, dd, J_5″,6b″ = J_5‴,6b‴ = 5.6 Hz, H-6b″, H-6b‴), 4.29 (1 H, d, J_{CH2-4a,CH2-4b} = 7.6 Hz, CH₂-4a), 4.36 (1 H, d, J_{CH2-4a,CH2-4b} = 7.6 Hz, CH₂-5a), 4.20 (2 H, d, J_{CH2-4a,CH2-4b} = J_{CH2-5a,CH2-5b} = 7.6 Hz, CH₂-4b, CH₂-5b), 4.18 ~ 3.65 (12 H, br, HO-2′, 3′, 4′, 6′, 2″, 3″, 4″, 6″, 2‴, 3‴, 4‴, 6‴, exchangeable with D₂O), 2.42 (3 H, s, CH₃-2); δ_C: 144.43 (d, ³J_F-C = 15.0 Hz, Py-C₂), 136.26 (d, ⁴J_F-C = 3.8 Hz, Py-C₃), 124.40 (d, ³J_F-C = 3.8 Hz, Py-C₄), 120.39 (d, ²J_F-C = 32.8 Hz, Py-C₅), 148.98 (d, ¹J_F-C = 259.7 Hz, Py-C₆), 103.65 (s, C-1′), 101.76 (s, C-1″, C-1‴), 72.34 (s, C-2′), 71.28 (s, C-2″, C-2‴), 74.55 (s, C-3′), 73.88 (s, C-3″, C-3‴), 69.82 (s, C-4′), 68.89 (s, C-4″, C-4‴), 76.62 (s, C-5′), 77.29 (s, C-5″, C-5‴), 61.36 (s, C-6′), 60.95 (s, C-6″, C-6‴), 60.54 (s, CH₂-4), 60.78 (s, CH₂-5), 19.88 (s, CH₃-3) ppm. ESIMS: m/z 673 [M⁺] (8%), 674 [M+1] (14%). Anal. Calcd. for C₂₆H₄₀NO₁₈F(%): C, 46.34, H, 5.99, N, 2.08; Found: C, 46.32, H, 5.97, N, 2.07.

3-O-(β-D-galactopyranosyl)-α⁴, α⁵-di-O-(α-D-mannopyranosyl)-6-fluoropyridoxol 13 (0.57 g), foam solid, R_f 0.26 (1:4 MeOH-EtOAc), NMR (DMSO-d₆), δ_H: 5.00 (1 H, d, J_1′,2′ = 8.0 Hz, H-1′), 5.23 (1 H, dd, J_2′,3′ = 10.0 Hz, H-2′), 5.16 (1 H, dd, J_3′,4′ = 3.8 Hz, H-3′), 5.08 (1 H, dd, J_3″,4″ = 3.2 Hz, H-4′), 4.21 (1 H, m, H-5′), 4.51 (1 H, dd, J_5′,6a′ = 3.6 Hz, J_6a′,6b′ = 10.2 Hz, H-6a′), 4.31 (1 H, dd, J_5′,6b′ = 4.8 Hz, H-6b′), 4.84 (2 H, d, J_1″,2″ = J_1‴,2‴ = 2.6 Hz, H-1″, H-1‴), 4.68 (2 H, dd, J_2″,3″ = J_2‴,3‴ = 6.0 Hz, H-2″, H-2‴), 5.02 (2 H, dd, J_3″,4″ = J_3‴,4‴ = 3.6 Hz, H-3″, H-3‴), 4.05 (2 H, dd, J_4″,5″ = J_4‴,5‴ = 2.2 Hz, H-4″, H-4‴), 3.94 (2 H, m, H-5″, H-5‴), 4.21 (2 H, dd, J_5″,6a″ = J_5‴,6a‴ = 2.4 Hz, J_6a″,6b″ = J_6a″,6b″ = 8.4 Hz, H-6a″, H-6a‴), 4.17 (2 H, dd, J_5″,6b″ = J_5‴,6b‴ = 6.5 Hz, H-6b″, H-6b‴), 4.77 (2 H, d, J_{CH2-4a,CH2-4b} = J_{CH2-5a,CH2-5b} = 11.6 Hz, CH₂-4a, CH₂-5b), 4.57 (1 H, d, J_{CH2-4a,CH2-4b} = J_{CH2-5a,CH2-5b} = 11.6 Hz, CH₂-4b, CH₂-5a), 2.45 (3 H, s, CH₃-2), 4.30 ~ 3.70 (12 H, br, HO-2′, 3′, 4′, 6′, 2″, 3″, 4″, 6″, 2‴, 3‴, 4‴, 6‴, exchangeable with D₂O); δ_C: 151.07 (d, ³J_F-C = 15.3 Hz, Py-C₂), 148.61 (d, ⁴J_F-C = 4.8 Hz, Py-C₃), 144.27 (d, ³J_F-C = 3.6 Hz, Py-C₄), 116.29 (d, ²J_F-C = 32.0 Hz, Py-C₅), 157.25 (d, ¹J_F-C = 233.5 Hz, Py-C₆), 103.68 (s, C-1′), 98.56 (s, C-1″), 98.68 (s, C-1‴), 71.76 (s, C-2′), 70.66 (s, C-2″), 70.86 (s, C-2‴), 72.38 (s, C-3′), 71.46 (s, C-3″), 71.32 (s, C-3‴), 70.48 (s, C-4′), 66.87 (s, C-4″), 67.90 (s, C-4‴), 73.64 (s, C-5′), 72.20 (s, C-5″), 72.65 (s, C-5‴), 62.77 (s, C-6′), 63.56 (s, C-6″), 64.83 (s, C-6‴), 57.54 (s, CH₂-4), 58.41 (s, CH₂-5), 20.12 (s, CH₃-3) ppm. ESIMS: m/z 673 [M⁺] (5%), 674 [M+1] (9%). Anal. Calcd. for C₂₆H₄₀NO₁₈F(%): C, 46.34, H, 5.99, N, 2.08; Found: C, 46.31, H, 5.97, N, 2.05.

3-O-Benzyl-6-fluoropyridoxol 14

To a well stirred CH₂Cl₂ (10 mL)-H₂O (10 mL) biphasic mixture (pH 10 ~ 11) of 1 (0.50 g, 2.67 mmol) and TBAB (0.10 g, 0.31 mmol), a solution of benzyl bromide (0.51 g, 2.94 mmol, 1.1 equiv.) in CH₂Cl₂ (10 mL) was added dropwise over a period of 4 ~ 5h, while the reaction temperature was maintained at 50 °C, and the stirring continued for an additional hour. Products were extracted (CH₂Cl₂, 4×20 mL), washed free of alkali, dried (Na₂SO₄), and concentrated, the residue was purified by column chromatography on silica gel with 1:2 cyclohexane-EtOAc to afford major product 14 (0.56 g, 76%), white crystalline, R_f 0.38 (1:2 cyclohexane-EtOAc), NMR (CDCl₃), δ_H: 7.39 (5 H, m, Ar-H), 4.90 (2 H, s, PhCH₂), 4.75 (2 H, d, J_H-5,HO-5 = 5.4 Hz, CH₂-5), 4.72 (2 H, d, J_H-4,HO-4 = 6.0 Hz, CH₂-4), 3.57 (1 H, t, J_H-5,HO-5 = 5.4 Hz, α⁵-OH, exchangeable with D₂O), 3.49 (1 H, t, J_H-4,HO-4 = 6.0 Hz, α⁴-OH, exchangeable with D₂O), 2.44 (3 H, s, CH₃-2); δ_C: 151.34 (d, ³J_F-C = 9.6 Hz, Py-C₂), 146.97 (d, ⁴J_F-C = 2.9 Hz, Py-C₃), 149.55 (d, ³J_F-C = 3.1 Hz, Py-C₄), 119.09 (d, ²J_F-C = 20.8 Hz, Py-C₅), 156.30 (d, ¹J_F-C = 216.2 Hz, Py-C₆), 136.33, 128.96, 128.88, 128.57 (Ph-C), 55.99 (s, PhCH₂, CH₂-4), 56.76 (s, CH₂-5), 19.31 (s, CH₃-2) ppm. Anal. Calcd. for C₁₅H₁₆NO₃F(%): C, 64.96, H, 5.82, N, 5.05; Found: C, 64.95, H, 5.79, N, 5.04.

3-O-Benzyl-α⁴, α⁵-di-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-glucopyranosyl)-6-fluoro-pyridoxol 15 and 3-O-Benzyl-α⁴, α⁵-di-O-(2, 3, 4, 6-tetra-O-acetyl-α-D-manno-pyranosyl)-6-fluoropyridoxol 16

Glycosylation of 14 (0.46 g, 2.0 mmol) with 8 or 9 (1.83 g, 4.45 mmol, 1.1 equiv.) was carried out as for 3, 10 and 11 to give 15 and 16, respectively.

3-O-Benzyl-α⁴, α⁵-di-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-glucopyranosyl)-6-fluoropyridoxol 15 (0.32 g, 95%), syrup, R_f 0.35 (3:2 cyclohexane-EtOAc), NMR (CDCl₃), δ_H: 7.41 (5 H, m, Ar-H), 5.36 (1 H, d, J_1′,2′ = 8.2 Hz, H-1′), 5.41 (1 H, d, J_1″,2″ = 8.2 Hz, H-1″), 5.14 (2 H, dd, J_2′,3′ = J_2″,3″ = 7.4 Hz, H-2′, H-2″), 4.45 (2 H, dd, J_3′,4′ = J_3″,4″ = 3.3 Hz, H-3′, H-3″), 4.84 (2 H, dd, J_4′,5′ = J_4″,5″ = 3.8 Hz, H-4′, H-4″), 3.96 (2 H, m, H-5′, H-5″), 4.80 (2 H, dd, J_5′,6a′ = J_5″,6a″ = 2.6 Hz, J_6a′,6b′ = J_6a″,6b″ = 10.1 Hz, H-6a′, H-6a″), 4.10 (2 H, dd, J_5′,6b′ = J_5″,6b″ = 3.0 Hz, H-6b′, H-6b″), 4.94 (2 H, s, PhCH₂), 4.55 (1 H, d, J_{CH2-4a,CH2-4b} = 10.4 Hz, CH₂-4a), 4.48 (1 H, d, J_{CH2-4a,CH2-4b} = 10.4 Hz, CH₂-4b), 4.60 (1 H, d, J_{CH2-5a,CH2-5b} = 11.0 Hz, CH₂-5a), 4.52 (1 H, d, J_{CH2-5a,CH2-5b} = 11.0 Hz, CH₂-5b), 2.37 (3 H, s, CH₃-2), 2.00, 1.99, 1.98, 1.97, 1.96, 1.95, 1.94, 1.93 (24 H, 8s, 8×CH₃CO); δ_C: 170.84, 170.76, 170.31, 170.29, 170.26, 169.95, 169.92, 169.84 (8×CH₃CO), 152.18 (d, ³J_F-C = 14.5 Hz, Py-C₂), 142.64 (d, ⁴J_F-C = 4.6 Hz, Py-C₃), 150.12 (d, ³J_F-C = 3.8 Hz, Py-C₄), 116.20 (d, ²J_F-C = 32.0 Hz, Py-C₅), 157.40 (d, ¹J_F-C = 234.3 Hz, Py-C₆), 136.37, 129.00, 128.94, 128.87, 128.16, 127.77 (Ph-C), 100.23 (s, C-1′), 100.41 (s, C-1″), 71.41 (s, C-2′, C-2″), 72.08 (s, C-3′), 72.19 (s, C-3″), 68.34 (s, C-4′), 68.51 (s, C-4″), 72.86 (s, C-5′), 72.93 (s, C-5″), 61.86 (s, C-6′), 61.98 (s, C-6″), 60.98 (s, CH₂-4), 61.28 (s, CH₂-5), 20.88, 20.85, 20.82, 20.75, 20.73, 20.60, 20.59, 20.58 (8s, 8×CH₃CO), 19.43 (s, CH₃-3) ppm. ESIMS: m/z 937 [M⁺] (35%), 938 [M+1] (25%). Anal. Calcd. for C₄₃H₅₂NO₂₁F(%): C, 55.05, H, 5.59, N, 1.49; Found: C, 55.03, H, 5.57, N, 1.48.

3-O-Benzyl-α⁴, α⁵-di-O-(2, 3, 4, 6-tetra-O-acetyl-α-D-mannopyranosyl)-6-fluoropyridoxol 16 (0.30 g, 90%), syrup, R_f 0.40 (3:2 cyclohexane-EtOAc), NMR (CDCl₃), δ_H: 7.38 (5 H, m, Ar-H), 5.38 (1 H, d, J_1′,2′ = 2.6 Hz, H-1′), 5.41 (1 H, d, J_1″,2″ = 2.6 Hz, H-1″), 5.36 ~ 3.95 (18 H, m, H-2′, 3′, 4′, 5′, 6′, 2″, 3″, 4″, 5″, 6″, PhCH₂, CH₂-4, CH₂-5), 2.38 (3 H, s, CH₃-2), 2.02, 2.00, 1.99, 1.98, 1.97, 1.96, 1.95, 1.94 (24 H, 8s, 8×CH₃CO); δ_C: 171.25, 171.18, 170.89, 170.85, 170.78, 170.66, 170.60, 170.48 (8×CH₃CO), 153.28 (d, ³J_F-C = 15.8 Hz, Py-C₂), 145.48 (d, ⁴J_F-C = 4.8 Hz, Py-C₃), 150.16 (d, ³J_F-C = 3.8 Hz, Py-C₄), 116.30 (d, ²J_F-C = 31.0 Hz, Py-C₅), 157.77 (d, ¹J_F-C = 206.8 Hz, Py-C₆), 98.42 (s, C-1′), 100.03 (s, C-1″), 72.60 ~ 56.54 (13C, C-2′, 3′, 4′, 5′, 6′, 2″, 3″, 4″, 5″, 6″, PhCH₂, CH₂-4, CH₂-5), 21.23, 20.94, 20.92, 20.90, 20.88, 20.86, 20.84, 20.80, 20.78 (8s, 8×CH₃CO), 18.37 (s, CH₃-3) ppm. ESIMS: m/z 937 [M⁺] (32%), 938 [M+1] (20%). Anal. Calcd. for C₄₃H₅₂NO₂₁F(%): C, 55.05, H, 5.59, N, 1.49; Found: C, 55.01, H, 5.55, N, 1.45.

α⁴, α⁵-Di-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-glucopyranosyl)-6-fluoropyridoxol 17 and α⁴, α⁵-di-O-(2, 3, 4, 6-tetra-O-acetyl-α-D-mannopyranosyl)-6-fluoropyridoxol 18 A mixture of 15 or 16 (0.29 g, 0.30 mmol) and Pd-C (5%, 50 mg) in MeOH (40 mL) was stirred for 24h at r.t. under H₂ (25 psi). Evaporated filtrate gave 17, 18 in quantitative yields.

α⁴, α⁵-Di-O-(2, 3, 4, 6-tetra-O-acetyl-β-D-glucopyranosyl)-6-fluoropyridoxol 17 (0.26 g), syrup, R_f 0.28 (1:3 cyclohexane-EtOAc), NMR (CDCl₃), δ_H: 7.33 (1 H, s, HO-3, exchangeable with D₂O), 5.30 (1 H, d, J_1′,2′ = 8.4 Hz, H-1′), 5.35 (1 H, d, J_1″,2″ = 8.4 Hz, H-1″), 5.09 (2 H, dd, J_2′,3′ = J_2″,3″ = 7.6 Hz, H-2′, H-2″), 4.35 (2 H, dd, J_3′,4′ = J_3″,4″ = 3.4 Hz, H-3′, H-3″), 4.80 (2 H, dd, J_4′,5′ = J_4″,5″ = 3.6 Hz, H-4′, H-4″), 3.89 (2 H, m, H-5′, H-5″), 4.77 (2 H, dd, J_5′,6a′ = J_5″,6a″ = 2.4 Hz, J_6a′,6b′ = J_6a″,6b″ = 10.6 Hz, H-6a′, H-6a″), 4.05 (2 H, dd, J_5′,6b′ = J_5″,6b″ = 3.2 Hz, H-6b′, H-6b″), 4.51 (1 H, d, J_{CH2-4a,CH2-4b} = 10.3 Hz, CH₂-4a), 4.45 (1 H, d, J_{CH2-4a,CH2-4b} = 10.3 Hz, CH₂-4b), 4.57 (1 H, d, J_{CH2-5a,CH2-5b} = 11.1 Hz, CH₂-5a), 4.49 (1 H, d, J_{CH2-5a,CH2-5b} = 11.1 Hz, CH₂-5b), 2.35 (3 H, s, CH₃-2), 1.99, 1.98, 1.97, 1.96, 1.95, 1.94, 1.93, 1.91 (24 H, 8s, 8×CH₃CO); δ_C: 170.82, 170.78, 170.65, 170.58, 170.46, 169.85, 169.82, 169.80 (8×CH₃CO), 152.28 (d, ³J_F-C = 14.2 Hz, Py-C₂), 148.28 (d, ⁴J_F-C = 3.2 Hz, Py-C₃), 142.69 (d, ³J_F-C = 4.8 Hz, Py-C₄), 116.26 (d, ²J_F-C = 32.2 Hz, Py-C₅), 157.56 (d, ¹J_F-C = 231.4 Hz, Py-C₆), 100.35 (s, C-1′), 100.54 (s, C-1″), 71.37 (s, C-2′, C-2″), 72.18 (s, C-3′), 72.29 (s, C-3″), 68.38 (s, C-4′), 68.56 (s, C-4″), 72.83 (s, C-5′), 72.88 (s, C-5″), 61.82 (s, C-6′), 61.89 (s, C-6″), 60.90 (s, CH₂-4), 61.19 (s, CH₂-5), 20.85, 20.83, 20.82, 20.80, 20.78, 20.76, 20.73, 20.65 (8s, 8×CH₃CO), 19.32 (s, CH₃-3) ppm. ESIMS: m/z 847 [M⁺] (30%), 848 [M+1] (21%). Anal. Calcd. for C₃₆H₄₆NO₂₁F(%): C, 50.99, H, 5.47, N, 1.65; Found: C, 50.96, H, 5.45, N, 1.62.

α⁴, α⁵-Di-O-(2, 3, 4, 6-tetra-O-acetyl-α-D-mannopyranosyl)-6-fluoropyridoxol 18 (0.26 g), syrup, R_f 0.27 (1:3 cyclohexane-EtOAc), NMR (CDCl₃), δ_H: 7.33 (1 H, s, HO-3, exchangeable with D₂O), 5.33 (1 H, d, J_1′,2′ = 2.7 Hz, H-1′), 5.37 (1 H, d, J_1″,2″ = 2.7 Hz, H-1″), 5.45 ~ 4.07 (16 H, m, H-2′, 3′, 4′, 5′, 6′, 2″, 3″, 4″, 5″, 6″, CH₂-4, CH₂-5), 2.35 (3 H, s, CH₃-2), 2.01, 2.00, 1.99, 1.98, 1.97, 1.96, 1.95, 1.94 (24 H, 8s, 8×CH₃CO); δ_C: 171.33, 171.21, 170.85, 170.83, 170.76, 170.61, 170.56, 170.53 (8×CH₃CO), 153.67 (d, ³J_F-C = 15.8 Hz, Py-C₂), 149.08 (d, ⁴J_F-C = 3.0 Hz, Py-C₃), 145.68 (d, ³J_F-C = 4.6 Hz, Py-C₄), 118.23 (d, ²J_F-C = 31.2 Hz, Py-C₅), 157.59 (d, ¹J_F-C = 223.1 Hz, Py-C₆), 98.67 (s, C-1′), 100.33 (s, C-1″), 72.8 ~ 56.56 (12C, C-2′, 3′, 4′, 5′, 6′, 2″, 3″, 4″, 5″, 6″, CH₂-4, CH₂-5), 20.99, 20.97, 20.93, 20.90, 20.88, 20.86, 20.84, 20.80 (8s, 8×CH₃CO), 18.45 (s, CH₃-3) ppm. ESIMS: m/z 847 [M⁺] (25%), 848 [M+1] (18%). Anal. Calcd. for C₃₆H₄₆NO₂₁F(%): C, 50.99, H, 5.47, N, 1.65; Found: C, 50.97, H, 5.44, N, 1.63.