Syntheses of fluorinated ligands to probe binding of antigenic determinants of Vibrio cholerae O:1, serotypes Inaba and Ogawa, to antibodies

Article abstract of DOI:10.1016/S0957-4166(99)00505-4

Derivatives of methyl α-glycosides of antigenic determinants of Vibrio cholerae O:1, serotypes Inaba and Ogawa, specifically fluorinated at position 2' or 4' have been synthesized by coupling the appropriately fluorinated derivatives of 3-deoxy-L-glycero-tetronic acid with the methyl α-glycosides of perosamine. The compound having the fluorine atom at position 2 was obtained by electrophilic addition of fluorine to the glycal derived from the parent antigenic determinant, serotypes Inaba, using Selectfluor((TM)) as a fluorination reagent. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0957-4166(99)00505-4

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 11 (2000) 595–606
Syntheses of fluorinated ligands to probe binding of antigenic
determinants of Vibrio cholerae O:1, serotypes Inaba and Ogawa,
to antibodies
Alex H. C. Chang,a Derek Horton a and Pavol Kovácˇ b,∗
aDepartment of Chemistry, American University, Washington, DC 20016, USA
bNational Institutes of Health, NIDDK, LMC, Bethesda, MD 20892-0815, USA
Received 20 October 1999; accepted 10 November 1999
Abstract
Derivatives of methyl α-glycosides of antigenic determinants of Vibrio cholerae O:1, serotypes Inaba and
Ogawa, specifically fluorinated at position 20 or 40 have been synthesized by coupling the appropriately fluorinated
derivatives of 3-deoxy-L-glycero-tetronic acid with the methyl α-glycosides of perosamine. The compound having
the fluorine atom at position 2 was obtained by electrophilic addition of fluorine to the glycal derived from the
parent antigenic determinant, serotypes Inaba, using Selectfluor™ as a fluorination reagent. © 2000 Elsevier Science
Ltd. All rights reserved.
1. Introduction
Reports in the literature1–4 suggest that carbohydrate antigens interact with antibodies, at least to
a significant extent, by hydrogen bonding. Information germane to understanding the details of these
interactions can be obtained by studies of binding, using as probes, deoxy and deoxyfluoro analogs
of components of the antigenic material. As part of our work towards a synthetic vaccine for cholera,
we have studied5 binding of monoclonal anti V. cholerae O:1 antibodies with a large series of ligands
related to the O-antigen, some of which were specifically deoxygenated. It was the purpose of this
work to synthesize specifically fluorinated derivatives of the methyl α-glycosides of the monosaccharide
determinants5,6 of the O-antigen of V. cholerae O:1, serotypes Inaba and Ogawa (1 and 2, respectively,
Fig. 1).
∗
Corresponding author. E-mail: kpn@helix.nih.gov
0957-4166/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00505-4
tetasy 3140

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Fig. 1. The methyl α-glycosides of the monosaccharide determinants of Vibrio cholerae O:1, serotypes Inaba (1, R=H) and
Ogawa (2, R=Me)
2. Results and discussion
Synthesis of the target compounds fluorinated at positions 20 involved preparation of appropriately
fluorinated derivatives of 3-deoxy-L-glycero-tetronic acid and their coupling with the known7–9 derivati-
ves of perosamines 3 and 4. Our previous study5 showed that replacement of the 3-deoxy-L-glycero-
tetronamido group for the D enantiomer did not result in abolition of binding. Therefore, with the
intention to examine binding of the corresponding fluoro analogs, 20-fluoro derivatives of 1 and 2 in
both D- and L-glycero series were prepared.
Syntheses of the 2-fluoro derivatives of 3-deoxy-D- and L-glycero-tetronolactone have been described.
Starting from enantiomerically pure D- and L-malic acid, Shiuey et al.10 described multi-step preparations
of both 2-fluorinated, enantiomerically pure enantiomers. Starting with the commercially available
α-hydroxy-γ-butyrolactone, Schwartz et al.11 prepared the racemic 3-deoxy-2-fluoro-tetronolactone.
Dimethylaminosulfur trifluoride (DAST) was the fluorinating reagent in both approaches. To prepare
2-fluoro derivatives 9–12 we followed, essentially, the latter protocol.11 Losses due to high volatility10 of
the fluoro lactones 6 and 8 could not be avoided by modifying the original protocol and conducting
evaporation of solutions in dichloromethane at atmospheric pressure. However, we have obtained
comparable yields of coupling products when we used crude vs chromatographically purified fluorinated
lactones 6 or 8 for introduction of the tetronamido side-chain. The fluorinated racemic lactone was
treated with each of the amines 3 and 4, and the resulting mixtures of diastereoisomers were separated
by chromatography. Compound 11 was also prepared from enantiomerically pure lactone 8, which was
obtained starting from the known12 2-O-acetyl derivative of lactone 7. The amorphous compound 11 was
characterized via the crystalline acetyl derivative 11a.
Our strategy to prepare analogs of 1 and 2 fluorinated at position 40 involved reactions of amines 3
and 4 with 4-deoxy-4-fluoro-3-deoxy-L-glycero-tetronic acid 24. Preparation of 24 required a derivative
of 3-deoxy-L-glycero-tetronic acid fully protected, except at HO-4. We have attempted opening of the O-
benzylidene ring in the known13 ester 18 using a variety of reagents,14–16 including that shown to effect
conversion of benzylidene acetals into secondary benzyl ethers.17 However, all attempts to prepare t-butyl
2-O-benzyl-3-deoxy-L-glycero-tetronate in such a way failed: the corresponding 4-benzyl ether13 19 was
formed instead. We have prepared compound 19 previously by opening the 2,4-O-benzylidene ring in
18 with borane–trimethylamine complex and AlCl3.13 That conversion, as we have found now, can be
conveniently performed with the same or better efficiency using the triethylsilane (TES)–trifluoroacetic
acid (TFA) reagent.18 Compound 19, obtained in this way in 78% yield, was acetylated and product 20
was subjected to catalytic hydrogenolysis to give 21. A small amount of 22, a product of acetyl group
migration, formed along with the desired compound 21 was also isolated and fully characterized. The
position of the acetyl group in 22 followed from the NMR spectral data. Similar acetyl group migration
has been observed previously12 in a related derivative of 3-deoxy-L-glycero-tetronic acid.

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597
Fluorination of ester 21 with diethylaminosulfur trifluoride (DAST) was uneventful. That acetyl group
migration did not occur under the condition of fluorination, and the presence of fluorine at the desired
1
position 4, was evident from the H NMR spectrum of 23 which showed a downfield shift of signals
for H-4a,b, compared to the chemical shift of these protons in 21. Also, as a result of fluorination
at C-4, the signal for H-4a,b appears in the spectrum of 23 as two 1-proton multiplets, showing a
3
typical JF,H coupling of 47.0 Hz. This is in contrast to the spectrum of 21, where the signal for H-
4a,b appears as one 2-proton multiplet. Because of high volatility of the fully protected, 4-fluorinated
t-butyl ester 23, considerable losses occurred when attempts were made to isolate this material using
protocols involving concentrations of large volumes of solutions, such as those resulting after aqueous
work-up and/or purification by column chromatography. Therefore, such operations were avoided, as
in the preparation of 6 and 8. The crude fluorinated t-butyl ester 23 was deprotected by treatment with
aqueous TFA. Removal of both of the acetyl and t-butyl protecting groups was evident from the 1H NMR
spectrum. Compared to the spectrum of 23, the high-field signals characteristic of the t-butyl group
were not present in the spectrum of 24, and the doublet of doublets for H-2 was shifted upfield, due
to the absence of the electron-withdrawing acetyl group at O-2. Reaction of 24 with each of amines 3
and 4, using [O-(7-azabezotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate] (HATU) as a
coupling reagent,19,20 then gave 13 and 14, respectively.

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A simple method for synthesizing compound 17 fluorinated at position 2 appeared to be the
recently described fluorination of glycals21 using Selectfluor™ (1-chloromethyl-4-fluoro-1,4-
diazoniabicyclo[2.2.2]octane). When applied to tri-O-acetyl-L-glucal in the presence of benzyl alcohol,21
the method was reported to afford benzyl 3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-α-D-mannopyranoside as
the major product. When glycal 25, obtained conventionally22 from 1,2,3-tri-O-acetyl-4,6-dideoxy-4-
(2,4-di-O-acetyl-3-deoxy-L-glycero-tetronamido)-α-D-mannopyranoside,23 was similarly treated, five
compounds were formed, as shown by TLC, and they were isolated by column chromatography. The
two fastest moving compounds were products of difluorination and were not further investigated. Next
eluted were three substances whose NMR spectra showed general features indicative that they belonged
to the desired class of compounds, namely that they contained the acetylated tetronamido side-chain, the
methyl aglycon, and were fluorinated at position 2. At this point it was important to establish which of
them, if any, was the desired compound 15.
Evidence that two of the compounds isolated (26 and 27) had the gluco configuration was a large
coupling observed for J2,3 (8–10 Hz). The anomeric configuration for these two substances was readily
assigned from the J1,2 coupling constants observed in their proton spectra, namely 3.8 and 7.7 Hz,
1
respectively. That the third compound isolated had the manno configuration also followed from the H
NMR spectrum (Tables 1 and 2), and the α-anomeric configuration, i.e. structure 15, was assigned from
the observed JH-1,C-1 carbon–proton coupling constant24 of 169.9 Hz (Table 3). Simple deacetylation
(Zemplén) of 15 then afforded the desired, target compound 17. The lower yield of the methyl α-D-manno
compound observed in the present case, compared to the derivative of benzyl α-D-mannopyranoside
reported previously,21 can be explained by the use of different synthons in these two independently
performed conversions.
Attempts to prepare the 3-deoxy-3-fluoro derivatives of 1 or 2 have failed thus far. It appears from our
preliminary work that positioning of fluorine at position 3 in derivatives of perosamine will be a more
formidable task than anticipated, and the chemistry involved in that synthesis will be reported at a later
date.
3. Experimental
Unless stated otherwise, optical rotations were measured at 25°C for solutions in CHCl3 (c 1) with
a Perkin–Elmer automatic polarimeter, model 241 MC. All reactions were monitored by thin-layer
chromatography (TLC). Detection of derivatives of perosamine was effected by charring with 5%
sulfuric acid in ethanol and, when applicable, by UV light. Lactones were detected by spraying with the
hydroxylamine–FeCl3 reagent.25 Compounds 21–24 which char poorly with 5% sulfuric acid in ethanol
were detected by spraying with 5% ethanolic phosphomolybdic acid. Preparative chromatography was
performed by gradient elution from columns of silica gel 60 (particle size 0.04–0.063 mm), using at
the onset of development a solvent mixture slightly less polar than that used for TLC. Assignments
1
of NMR (300 MHz for H and 75 MHz for 13C) signals, obtained at 25°C, were made by first-order
analysis of spectra supported by APT and/or DEPT experiments, homonuclear decoupling and/or homo-
and heteronuclear 2-dimensional correlation spectroscopy, using commercial software supplied with
the spectrometers (Varian Gemini or Varian Mercury). Chemical ionization mass spectra (CIMS) were
measured using ammonia as the reactive gas. Palladium-on-charcoal catalyst (5%, ESCAT 103) was
a product of Engelhard Industries. α-Hydroxy-γ-butyrolactone and Selectfluor™ (1-chloromethyl-4-
fluoro-1,4-diazoniabicyclo[2.2.2]octane) were purchased from Aldrich Chemical Company, and used
as supplied. HATU (O-7-azobenzotriazol-1-yl-1,1,3,3-tetramethyluronium hexafluorophosphate) was

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purchased from PerSeptive Biosystems. Unless stated otherwise, solutions in organic solvents were dried
with anhydrous sodium sulfate, and concentrated at 40°C/2 kPa.
3.1. Methyl 4-(2,3-dideoxy-2-fluoro-D- 10 and L-glycero-tetronamido)-4,6-dideoxy-α-D-mannopyrano-
side 9
DAST (7.55 mL, 56.8 mmol) was added at −70°C, by way of a syringe, to a solution of the
commercially available, racemic α-hydroxy-γ-butyrolactone (5 g) in dry CH2Cl2 (10 mL). The solution
was stirred for 12 h at room temperature, when TLC (4:1 EtOAc:hexane, lactone-specific detection,
see above) showed complete conversion of the starting material into one faster moving product. The
mixture was cooled to −20°C, methanol (∼1 mL) was added, and the mixture was allowed to reach
ambient temperature. Aqueous NaHCO3 was added portionwise until effervescence ceased, the mixture
was partitioned between CH2Cl2 and water, and the CH2Cl2 phase was concentrated. The residue was
chromatographed to give 2,3-dideoxy-2-fluoro-D,L-glycero-tetronolactone (6, 2.8 g, 55%). The 1H NMR
data agreed with those reported10 (for 13C NMR spectra, see Table 3). The spectra did not contain signals
suggesting the presence of either byproducts or residual solvents. CIMS: m/z 122 ([M+18]+).
One drop of pyridine was added to a mixture of amine 37,8 (1 g, 5.65 mmol) and lactone 6 (0.86 g,
8.26 mmol) and the mixture was stirred for 6 h at 100°C, when TLC (5:1 CH2Cl2:CH3OH) showed
that all starting amine had been consumed and that two products were formed. The mixture was
chromatographed to afford first the L diastereoisomer 9 (627 mg, 39.7%), mp 115.5–117.5 (from
acetone), [α]D +20.2 (c 1, H2O); CIMS: m/z 299 ([M+18]+). Anal. calcd for C11H20FNO6: C, 46.97;
H, 7.17; N, 4.98. Found: C, 46.91; H, 7.06; N, 4.93.
Eluted last was the D diastereoisomer 10 (516 mg, 32.6%), mp 133–135° (from EtOAc), [α]D + 69.3 (c
0.9, H2O); CIMS: m/z 299 ([M+18]+). Anal. calcd for C11H20FNO6: C, 46.97; H, 7.17; N, 4.98. Found:
C, 46.76; H, 7.03; N, 4.92. An intermediate, mixed fraction was also obtained.
3.2. Methyl 4-(2,3-dideoxy-2-fluoro-D- 12 and L-glycero-tetronamido)-4,6-dideoxy-2-O-methyl-α-D-
mannopyranoside 11
(a) Amine 49 (1.5 g, 7.8 mmol) was treated with the fluorinated racemic lactone 6 (1 g, 9.6 mmol)
as described for the preparation of 9 and 10 to give, after chromatography, first the amorphous L
diastereoisomer 11 (775 mg, 39.7%), [α]D +19.5 (c 1.3, CHCl3); CIMS: m/z 313 ([M+18]+).
Eluted last was the D diastereoisomer 12 (623 mg, 31.9%), mp 114.5–116°C (from EtOAc), [α]D
+71.7 (c 1, CHCl3); CIMS: m/z 313 ([M+18]+). Anal. calcd for C12H22FNO6: C, 48.81; H, 7.51; N, 4.74.
Found: C, 48.93; H, 7.48; N, 4.83. An intermediate, mixed fraction was also obtained.
(b) A solution of 2-O-acetyl-3-deoxy-D-glycero-tetronolactone26 (2.8 g) in 1 M TFA (60 mL) was
heated at 60°C for 3 h. TLC (1:3 hexane:EtOAc) showed disappearance of the starting material and
formation of a mixture of 3-deoxy-D-glycero-tetronolactone 7 and the corresponding free acid. The
solution was concentrated and toluene was evaporated from the residue (three times), to remove residual
TFA,9 resulting in virtually complete lactonization of the free acid, as shown by TLC and NMR
spectroscopy. Chromatography gave pure 3-deoxy-D-glycero-tetronolactone 7 (1.63 g, 82%).
The foregoing lactone 7 (0.318 g, 3.1 mmol) was fluorinated and the mixture was processed as already
described for the racemic lactone 6, except that all concentrations of solutions in CH2Cl2 were performed
at 45°C (bath) and atmospheric pressure. Without chromatography, the crude 2,3-dideoxy-2-fluoro-L-
glycero-tetronolactone 8 thus obtained was treated with amine 49 (475 mg, 2.5 mmol) as described in
(a), to give after chromatography 11 (270 mg, 36.7%), which was identical (NMR) with the compound

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603
just described. For characterization, compound 11 was acetylated conventionally, using acetic anhydride
and pyridine, to give methyl 3-O-acetyl-4-(4-O-acetyl-2,3-dideoxy-2-fluoro-L-glycero-tetronamido)-4,6-
dideoxy-2-O-methyl-α-D-mannopyranoside 11a, mp 65–67°C (from ether–isopropyl ether), [α]D +91.0
(c 0.8, CHCl3); CIMS: m/z 397 ([M+18]+). Anal. calcd for C16H26FNO8: C, 50.65; H, 6.91; N, 3.69.
Found: C, 50.47; H, 6.92; N, 3.72.
A sample of the fluorinated lactone 8 purified by chromatography gave NMR spectra identical to those
of 6, except for minor variations resulting from different conditions of measurements.
3.3. t-Butyl 4-O-benzyl-3-deoxy-L-tetronate 19
2,4-O-Benzylidene-(S)-1,2,4-butanetriol was prepared as described.13 Crystallization from ethyl ace-
tate gave material, mp 65.5–67°C, [α]D +12 (c 1.2). Previously,13 the specific optical rotation of [α]D
+106 was erroneously reported for the amorphous material. The foregoing alcohol was oxidized13 to give
the corresponding t-butyl ester 18.
Compound 18 (1.07, 4.03 mmol) was added to a mixture of triethylsilane (3.25 mL, 20.25 mmol)
in CH2Cl2 (15 mL), the mixture was cooled to −5°C, and TFA (1.6 mL, 20.25 mmol) was added
dropwise and with stirring. The cooling was removed, and the stirring was continued until TLC (4:1
hexane:EtOAc) showed that the reaction was complete (∼2 h). After neutralization with aq. NaHCO3,
the mixture was partitioned between water and CH2Cl2, the organic phase was dried, concentrated and
the residue was chromatographed to give 19 (0.81 g, 75.2%), which was identical with the previously
described material.13
3.4. t-Butyl 2-O-acetyl-4-O-benzyl-3-deoxy-L-tetronate 20
A solution of 19 (0.81 g) in a mixture of 1:1 pyridine:Ac2O (1 mL) was kept at room temperature
overnight. TLC (6:1 hexane:EtOAc) then showed that the conversion of the starting material to a single
faster moving product was complete. After concentration and elution from a small column of silica gel,
the title substance 20 was obtained in virtually theoretical yield as an oil, [α]D –34.4 (c 1.5, CHCl3);
CIMS: m/z 326 ([M+18]+). Anal. calcd for C17H24O5: C, 66.21; H, 7.84. Found: C, 66.40; H, 7.92.
3.5. t-Butyl 2-O- 21 and 4-O-acetyl-3-deoxy-L-tetronate 22
A mixture of 20 (11.66 g) and palladium-on-charcoal catalyst (2.5 g) in methanol was stirred at
room temperature and atmospheric pressure overnight. TLC (3:1 hexane:EtOAc) showed that the starting
material was consumed and that two products were formed. After filtration, the filtrate was concentrated
and the residue was chromatographed.
Eluted first was the 4-O-acetyl derivative 22 resulting from acetyl group migration (0.4 g, ∼5%).
The material solidified on standing, mp 32–33°C, [α]D −9.9 (c 0.7, CHCl3). Recrystallization from
common solvents was unsuccessful. The compound is highly volatile and sublimed when dried at room
temperature at 133 Pa. The analytical sample was kept in a desiccator over P2O5 and paraffin shavings
overnight at atmospheric pressure and room temperature. CIMS: m/z 236 ([M+18]+). Anal. calcd for
C10H18O5: C, 55.03; H, 8.31. Found: C, 55.02; H, 8.20.
Eluted next was the desired material 21 (7.1 g, 86%), in admixture with a small amount of 22, resulting
from the spontaneous conversion 21→22.

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3.6. Methyl 4,6-dideoxy-4-(3,4-dideoxy-4-fluoro-L-glycero-tetronamido)-α-D-mannopyranoside 13
DAST (15.45 mL, 0.116 M) was added at –60°C to a solution of 21 (6.3 g, 0.29 M) in THF (12.6
mL) containing Et3N (16.14 mL, 0.116 M). The cooling was removed and the mixture was stirred for
3 h. TLC (3:1 hexane:EtOAc) showed that starting material was virtually consumed and that one major
product was formed. After cooling (−20°C), methanol (15 mL) was added, the mixture was stirred for 2
h, and partitioned between aq. NaHCO3 and CH2Cl2. The organic phase was dried and concentrated at
atmospheric pressure, mainly to remove methanol. A solution of the residue in CH2Cl2 was filtered
through a layer of silica gel to remove bulk of the colored and base-line material, the eluate was
concentrated at atmospheric pressure, and the crude t-butyl 2-O-acetyl-4-fluoro-3,4-dideoxy-L-tetronate
(23, CIMS: m/z 238 ([M+18]+), thus obtained, was used for the next step.
A solution of the foregoing material in 1 M TFA (120 mL) was heated at 80°C for 4 h. TLC (1:1
EtOAc:MeOH) showed that the reaction was essentially complete and that one major product was
formed. The solution was concentrated, water was evaporated from the residue to remove TFA, and
the residue was chromatographed to obtain material (2.1 g) containing ∼70% (NMR) of 3,4-dideoxy-4-
fluoro-L-glycero-tetronic acid 24; CIMS: m/z 140 ([M+18]+).
A mixture of amine 3 (1 g, 6.28 mmol), acid 24 (1.09 g, 6.28 mmol, based on the purity of 24
already described), diisopropylethylamine (DIEA) (2.2 mL, 12.6 mmol), and HATU (4.8 g, 12.6 mmol)
in 1:1 MeOH:CH2Cl2 (4 mL) was stirred at room temperature for 2 h, when TLC (5:1 CH2Cl2:MeOH)
showed that most of the amine had been converted to a faster moving product. After concentration,
chromatography yielded 13 (0.98 g, 62%), mp 111.5–111.5°C (from acetone–EtOAc), [α]D +34 (c 0.9,
H2O); CIMS: m/z 299 ([M+18]+). Anal. calcd for C11H20FNO6: C, 46.97; H, 7.17; N, 4.98. Found: C,
46.99; H, 7.23; N, 4.96.
3.7. Methyl 4,6-dideoxy-4-(3,4-dideoxy-4-fluoro-L-glycero-tetronamido)-2-O-methyl-α-D-mannopyra-
noside 14
The amine 4 (1.4 g, 6.3 mmol) was treated with the fluorinated acid 24 (1.1 g, 6.3 mmol), as described
for the preparation of 13. After work-up, as already described, chromatography gave 14 (1.15 g, 53%),
mp 121–122°C (from EtOAc), [α]D +44.5 (c 1, CHCl3); CIMS: m/z 313 ([M+18]+). Anal. calcd for
C12H22FNO6: C, 48.81; H, 7.51; N, 4.74. Found: C, 48.99; H, 7.54; N, 4.68.
Compound 14 was further characterized as methyl 3-O-acetyl-4,6-dideoxy-4-(2-O-acetyl-3,4-dideoxy-
4-fluoro-L-glycero-tetronamido)-2-O-methyl-α-D-mannopyranoside 14a, which was obtained from 14
by conventional acetylation with acetic anhydride in pyridine, mp 106–107°C, [α]D +62.2 (c 1, CHCl3);
CIMS: m/z 397 ([M+18]+). Anal. calcd for C16H26FNO8: C, 50.65; H, 6.91; N, 3.69. Found: C, 50.54;
H, 6.97; N, 3.95.
3.8. 3-O-Acetyl-1,5-anhydro-2,4,6-trideoxy-4-(2,4-di-O-acetyl-3-deoxy-L-glycero-tetronamido)-α-D-
arabino-hex-1-enitol 25
1,2,3-Tri-O-acetyl-4,6-dideoxy-4-(2,4-di-O-acetyl-3-deoxy-L-glycero-tetronamido)-α-D-mannopyra-
noside23 16 (7.55 g) was treated with 40% HBr in AcOH for 1 h, when TLC (5:1 CH2Cl2:acetone)
showed that the reaction was complete. The mixture was concentrated with co-evaporation of toluene, to
remove excess reagents, to give the desired glycosyl bromide, 2,3-di-O-acetyl-4,6-dideoxy-4-(2,4-di-O-
acetyl-3-deoxy-L-glycero-tetronamido)-α-D-mannopyranosyl bromide, which was used for the next step
without further purification.

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605
Commercially available zinc dust was activated by successive washes with 10% HCl, water, acetone,
and ether. To a solution of the foregoing bromide in THF (50 mL) was added air-dried activated
zinc powder (12.4 g, 12 equiv.), the mixture was heated and 1-methylimidazole (1.2 mL, 1 equiv.)
was added when the inner temperature reached 45°C. The heating was continued for 45 min when
TLC (5:1 toluene:acetone) showed that the reaction was complete and two products had been formed.
Chromatography gave the faster moving, desired glycal 25 (3.6 g, 63%), mp 86.5–87.5°C, [α]D −54.8 (c
0.8, CHCl3); CIMS: m/z 357 ([M+18]+). Anal. calcd for C16H23NO8: C, 53.78; H, 6.49; N, 3.92. Found:
C, 53.93; H, 6.58; N, 3.92.
3.9. Methyl
3-O-acetyl-2,4,6-trideoxy-4-(2,4-di-O-acetyl-3-deoxy-L-glycero-tetronamido)-2-fluoro-
α-D-glucopyranoside 26, methyl 2,4,6-trideoxy-4-(3-deoxy-L-glycero-tetronamido)-2-fluoro-α-D-
mannopyranoside 15, and methyl 3-O-acetyl-2,4,6-trideoxy-4-(2,4-di-O-acetyl-3-deoxy-L-glycero-
tetronamido)-2-fluoro-β-D-glucopyranoside 27
To a mixture of glycal 25 (2.94 g, 8.2 mmol) and 4 Å molecular sieves (2 g) in acetonitrile (9 mL)
were added MeOH (3 mL), 2,6-di-t-butyl-4-methylpyridine (2.54 g, 12.4 mmol) and Selectfluor™(4.4 g,
12.4 mmol). The mixture was stirred in an argon atmosphere overnight, when TLC (3:2 toluene:EtOAc)
showed that all starting material was consumed and that, in addition to some base-line material, five
products had been formed. After filtration through a pad of Celite, the filtrate was concentrated and the
residue was chromatographed.
Eluted first were two compounds (∼30 mg each) whose CI mass spectra showed a peak at m/z 413,
indicating that each of these compounds contained two fluorine atoms. These were not investigated
further.
Eluted next was material enriched in a compound whose NMR data confirmed that it was the α-gluco
derivative 26. Crystallization (twice) from ethanol gave material (120 mg, 3.6%), melting at 154–155°C,
[α]D +121.5 (c 1.1, CHCl3); CIMS: m/z 425 ([M+18]+). Anal. calcd for C17H26FNO9: C, 50.12; H, 6.43;
N, 3.44. Found: C, 49.97; H, 6.43; N, 3.47.
Eluted next was material whose NMR data confirmed that it was the α-manno derivative 15 (0.234 g,
7%); [α]D +57.7 (c 0.9, CHCl3); CIMS: m/z 425 ([M+18]+).
Eluted last was material whose NMR data confirmed that it was the β-gluco derivative 27 (0.458 g,
14.7%), mp 96–98°C (from ether), [α]D +16.2 (c 1.4, CHCl3); CIMS: m/z 425 ([M+18]+). Anal. calcd
for C17H26FNO9: C, 50.12; H, 6.43; N, 3.44. Found: C, 50.26; H, 6.37; N, 3.36. Intermediate, mixed
fractions were also obtained.
3.10. Methyl 2,4,6-trideoxy-4-(3-deoxy-L-glycero-tetronamido)-2-fluoro-α-D-mannopyranoside
A solution of 15 in methanol was deacetylated (Zemplén) to give the desired 2-fluoro derivative 17
in virtually theoretical yield, mp 128–130°C (from EtOAc), [α]D +40.5C (c 0.8, H2O). Anal. calcd for
C11H26FNO6: C, 46.97; H, 7.17; N, 4.98. Found: C, 47.03; H, 7.26; N, 4.9.
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