5-azido-5-deoxy-2 :3-isopropylidene-D-arabinose compounds; their method of manufacture and their use for the synthesis of ARA-N3, KDO-N3 and 4EKDO-N3

Abstract

Disclosed are new compounds of formulae: ##STR00001##
Wherein: R.sup.1 and R.sup.2 can be independently H; a C.sub.1 to C.sub.6 alkyl including methyl, ethyl, propyl, butyl; aryl including phenyl, para-methoxyphenyl; or R.sup.1,R.sup.2 together with the carbon C-6 can be a cyclopentylidene or cyclohexylidene; each of these groups being substituted or not; and R.sup.3 can be a C.sub.1 to C.sub.6 alkyl including methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl; or aryl including phenyl, methylphenyl, ethylphenyl, each of these groups being substituted or not. The invention also relates to their method of manufacture and their use for the synthesis of Ara-N.sub.3, Kdo-N.sub.3 and 4eKdo-N.sub.3.

Claims

1. A compound of formula V or VI, selected from: ##STR00051## wherein: R.sup.1 and R.sup.2 are each independently H; a C.sub.1 to C.sub.6 alkyl; aryl; or R.sup.1, R.sup.2 together with the carbon C-6 are a cyclopentylidene or cyclohexylidene; and R.sup.3 is a C.sub.1 to C.sub.6 alkyl; or aryl.

2. A method of preparation of compound of formula VI according to claim 1 or 5-Azido-5-deoxy-2:3-isopropylidene-D-arabinose, comprising the chemical reaction of: either a compound of formula V: ##STR00052## wherein: R.sup.1 and R.sup.2 are each independently H; a C.sub.1 to C.sub.6 alkyl; aryl; or R.sup.1, R.sup.2 together with the carbon C-6 are a cyclopentylidene or cyclohexylidene; and R.sup.3 is a C.sub.1 to C.sub.6 alkyl; or aryl; or the specific compound (5), named 5-azido-5-deoxy-2:3-isopropylidene-D-arabinose O-methyloxime: ##STR00053## with an aqueous solution of an organic or inorganic acid in the presence of an aldehyde.

3. A method of preparation of compound of formula V according to claim 1 or 5-azido-5-deoxy-2:3-isopropylidene-D-arabinose O-methyloxime, comprising the chemical reaction of: either a compound of formula IV: ##STR00054## wherein: R.sup.1 and R.sup.2 are each independently H; a C.sub.1 to C.sub.6 alkyl; aryl; or R.sup.1, R.sup.2 together with the carbon C-6 are a cyclopentylidene or cyclohexylidene; and R.sup.3 is a C.sub.1 to C.sub.6 alkyl; or aryl; and R.sup.4 is a C.sub.1 to C.sub.6 alkyl; a C.sub.1 to C.sub.6 perfluoroalkyl; or aryl; or the specific compound (4), named 2:3-isopropylidene-5-O-methanesulfonyl-D-arabinose O-methyloxime: ##STR00055## with an organic or inorganic azide salt in a non-polar solvent or in polar aprotic solvent.

4. A method of preparation of compound of formula IV or of specific compound (4) according to claim 3, comprising the chemical reaction of: either a compound of formula III: ##STR00056## wherein: R.sup.1 and R.sup.2 can be are each independently H; a C.sub.1 to C.sub.6 alkyl; aryl; or R.sup.1, R.sup.2 together with the carbon C-6 are a cyclopentylidene or cyclohexylidene; and R.sup.3 is a C.sub.1 to C.sub.6 alkyl; or aryl; or the specific compound (3), named 2:3-isopropylidene-D-arabinose O-methyloxime: ##STR00057## with a sulfonyl chloride or sulfonic anhydride in the presence of an organic base and in the presence or not of a polar aprotic solvent.

5. A method for the synthesis of 5-azido-5-deoxy-D-arabinofuranose or Ara-N.sub.3 (7): ##STR00058## comprising reacting synthesis intermediate of formula VI according to claim 1 or 5-Azido-5-deoxy-2:3-isopropylidene-D-arabinose, under reaction conditions providing removal of protecting group of compound of formula VI with an aqueous solution of an organic or inorganic acid in a polar aprotic solvent or non-polar solvent.

6. A method for the synthesis of: either a compound of formula VIIIa: ##STR00059## wherein: R.sup.1 and R.sup.2 are each independently H; a C.sub.1 to C.sub.6 alkyl; aryl; or R.sup.1,R.sup.2 together with the carbon C-6 are a cyclopentylidene or cyclohexylidene; and R.sup.5 is a C.sub.1 to C.sub.6 alkyl; or benzyl; or the specific compound (8a), named ethyl (4R)-4-{(4R,5R)-5-[(R)-2-azido-1-hydroxyethyl]-2,2-dimethyl-1,3-dioxolan-4-yl)-4-hydroxy-2-methylenebutanoate: ##STR00060## by reaction of compound of formula VI of claim 1 or 5 Azido-5-deoxy-2:3-isopropylidene-D-arabinose, with alkyl 2-(halomethyl)acrylate, in the presence of a metal, and the presence or not of an aqueous solution of an organic or inorganic acid, in a protic solvent or in a mixture of water and a polar aprotic solvent.

7. A method for the preparation of: either a compound of formula IXa: ##STR00061## wherein: R.sup.1 and R.sup.2 are each independently H; a C.sub.1 to C.sub.6 alkyl; aryl; or R.sup.1,R.sup.2 together with the carbon C-6 are a cyclopentylidene or cyclohexylidene; and R.sup.5 is a C.sub.1 to C.sub.6 alkyl; or benzyl; or the specific compound (9a), named (4R)-4-{(4R,5R)-5-[(R)-2-azido-1-hydroxyethyl]-2,2-dimethyl-1,3-dioxolan-4-yl)-4-hydroxy-2-oxobutanoate: ##STR00062## comprising performing an ozonolysis of compound of formula VIIIa or of specific compound (8a) according to claim 6, with ozone in a protic solvent , at a temperature between 100 and 0 C., and then the unstable intermediate compound being reacted with a reducing agent, in a protic solvent.

8. A method for the preparation of ammonium 8-azido-3,8-dideoxy-D-manno-oct-2-ulosonate or Kdo-N.sub.3 (10a): ##STR00063## comprising deprotecting compound of formula IXa or specific compound (9a) according to claim 7, with an aqueous solution of an organic or inorganic acid.

9. A method for the synthesis of: either a compound of formula VIIIb: ##STR00064## wherein: R.sup.1 and R.sup.2 can be are each independently H; a C.sub.1 to C.sub.6 alkyl; aryl; or R.sup.1,R.sup.2 together with the carbon C-6 are a cyclopentylidene or cyclohexylidene; and R.sup.5 is a C.sub.1 to C.sub.6 alkyl; or benzyl; or the specific compound (8b), named ethyl (4S)-4-{(4R,5R)-5-[(R)-2-azido-1-hydroxyethyl]-2,2-dimethyl-1,3-dioxolan-4-yl)-4-hydroxy-2-methylenebutanoate: ##STR00065## by reaction of compound of formula VI or 5-Azido-5-deoxy-2:3-isopropylidene-D-arabinose according to claim 1 with alkyl 2-(halomethyl)acrylate, in the presence of a metal, and the presence or not of an aqueous solution of an organic or inorganic acid, in a protic solvent or in a mixture of water and a polar aprotic solvent.

10. A method for the preparation of: either a compound of formula IXb: ##STR00066## wherein: R.sup.1 and R.sup.2 are each independently H; a C.sub.1 to C.sub.6 alkyl; aryl; or R.sup.1,R.sup.2 together with the carbon C-6 are a cyclopentylidene or cyclohexylidene; and R.sup.5 is a C.sub.1 to C.sub.6 alkyl; or benzyl; or the specific compound (9b), named (4S)-4-{(4R,5R)-5-[(R)-2-azido-l-hydroxyethyl]-2,2-dimethyl-1,3-dioxolan-4-yl)-4-hydroxy-2-oxobutanoate: ##STR00067## comprising performing an ozonolysis of compound of formula VIIIb or of specific compound (8b) of claim 9, with ozone in a protic solvent , at a temperature between 100 and 0 C., and then the unstable intermediate compound being reacted with a reducing agent, in a protic solvent.

11. A method for the preparation of ammonium 8-azido-3,8-dideoxy -D-gluco-oct-2-ulosonate or 4eKdo-N3 (10b): ##STR00068## comprising deprotecting compound of formula IXb or specific compound (9b) of claim 10, with an aqueous solution of an organic or inorganic acid.

12. A compound selected from the group consisting of: a) ##STR00069## wherein: R.sup.1 and R.sup.2 are each independently H; a C.sub.1 to C.sub.6 alkyl; aryl; or R.sup.1, R.sup.2 together with the carbon C-6 are a cyclopentylidene or cyclohexylidene; and R.sup.5 is a Cto C.sub.6 alkyl; or benzyl; and b) ##STR00070## wherein: R.sup.1 and R.sup.2 are each independently H; a C.sub.1 to C.sub.6 alkyl; aryl; or R.sup.1, R.sup.2 together with the carbon C-6 are a cyclopentylidene or cyclohexylidene; and R.sup.5 is a C.sub.1 to C.sub.6 alkyl; or benzyl; and c) ##STR00071## wherein: R.sup.1 and R.sup.2 are each independently H; a C.sub.1 to C.sub.6 alkyl; aryl; or R.sup.1, R.sup.2 together with the carbon C-6 are a cyclopentylidene or cyclohexylidene; and R.sup.5 is a Cto C.sub.6 alkyl; or benzyl; and d) ##STR00072## wherein: R.sup.1 and R.sup.2 are each independently H; a C.sub.1 to C.sub.6 alkyl; aryl; or R.sup.1, R.sup.2 together with the carbon C-6 are a cyclopentylidene or cyclohexylidene; and R.sup.5 is a C.sub.1 to C.sub.6 alkyl; or benzyl; and e) ##STR00073## wherein: R.sup.1 and R.sup.2 are each independently H; a C.sub.1 to C.sub.6 alkyl; aryl; or R.sup.1, R.sup.2 together with the carbon C-6 are a cyclopentylidene or cyclohexylidene; and R.sup.3 is a C.sub.1 to C.sub.6 alkyl; or aryl; and R.sup.4 is a C.sub.1 to C.sub.6 alkyl; a C.sub.1 to C.sub.6 perfluoroalkyl; or aryl.

13. Method for the detection of bacteria, comprising contacting a compound of formula VIIIa or VIIIb, according to claim 12, with a culture medium comprising the bacteria.

14. A method of preparation of compound of formula V or of specific compound (5) according to claim 1, comprising the chemical reaction of: either a compound of formula IV: ##STR00074## wherein: R.sup.1 and R.sup.2 are each independently H; a C.sub.1 to C.sub.6 alkyl; aryl; or R.sup.1, R.sup.2 together with the carbon C-6 are a cyclopentylidene or cyclohexylidene; and R.sup.3 is a C.sub.1 to C.sub.6 alkyl; or aryl; and R.sup.4 is a C.sub.1 to C.sub.6 alkyl; a C.sub.1 to C.sub.6 perfluoroalkyl; or aryl; or the specific compound (4), named 2:3-isopropylidene-5-O-methanesulfonyl-D-arabinose O-methyloxime: ##STR00075## with an organic or inorganic azide salt in a non-polar solvent or in polar aprotic solvent.

15. A method of preparation of compound of formula IV or of specific compound (4) according to claim 14, comprising the chemical reaction of: either a compound of formula III: ##STR00076## wherein: R.sup.1 and R.sup.2 are each independently H; a C.sub.1 to C.sub.6 alkyl; aryl; or R.sup.1, R.sup.2 together with the carbon C-6 are a cyclopentylidene or cyclohexylidene; and R.sup.3 is a C.sub.1 to C.sub.6 alkyl; or aryl; or the specific compound (3), named 2:3-isopropylidene-D-arabinose O-methyloxime: ##STR00077## with a sulfonyl chloride or sulfonic anhydride in the presence of an organic base and in the presence or not of a polar aprotic solvent.

Description

DETAILED DESCRIPTION OF THE BEST EMBODIMENT

(1) According to the invention, and in reference to Scheme 1 here-below, the target compounds (10a) (8-azido-8-deoxy-Kdo) and (10b) (8-azido-8-deoxy-4eKdo) were synthesized in 11 steps from D-arabinose with an overall yield of 17 mol % and a purity of more than 95% for compound (10a) and 6 mol % overall yield and a purity of more than 95% for (10b). The key step in both syntheses is an indium-mediated allylation [1] of a common aldehyde intermediate (6) which in turn is obtained from an oxime ether intermediate [2]. And the target compound (7) (5-azido-Ara) was synthetized in 7 steps from D-arabinose with an overall yield of 26 mol % and a purity of more than 95%.

(2) According to specific embodiments, the reaction of D-arabinose with O-methylhydroxylamine in dry pyridine afforded the corresponding O-methyloxime. The resulting intermediate was then protected into its bis-isopropylidene form compound (2) by reaction with 2,2-dimethoxypropane and a catalytic amount of p-toluenesulfonic acid. This compound was obtained as a mixture of E and Z-oxime ethers but no preparative purification was attempted since these oxime ethers were planned to be hydrolysed later.

(3) Selective deprotection of the terminal acetonide in 80% aqueous acetic acid under reduced pressure (200 mbar) yielded compound (3) in 58% yield from D-arabinose. The azido derivative (5) was obtained by selective mesylation of the primary alcohol in pyridine followed by a nucleophilic substitution using sodium azide in dimethylformamide (78% over 2 steps).

(4) To obtain the common aldehyde intermediate (6), a first attempted ozonolysis of oxime ethers (5) followed by reduction of unstable intermediates with dimethyl sulfide, yielded only 23% of compound (6) after chromatographic purification. Oxidative methods to regenerate the aldehyde from oximes using cerium ammonium nitrate [3] or a mixture of trimethylsilyl chloride and sodium nitrite [4] were unsuccessful.

(5) Finally, target aldehyde (6) was obtained by hydrolysis of oxime ethers (5) in aqueous acetic acid by adding a stoichiometric amount of formaldehyde to trap the released O-methylhydroxylamine. This common intermediate (6) was first transformed into 5-azido-5-deoxy-D-arabinose (7) by removal of the isopropylidene group with a mixture of trifluoroacetic acid and water in dichloromethane. Hence, 5-azido-5-deoxy-D-arabinofuranose (7) was obtained in 58% from compound (5).

(6) Barbier type indium-mediated allylation of aldehyde (6) with ethyl 2-(bromomethyl)acrylate gave the two erythro (8a) and threo (8b) diastereomers, the erythro isomer being the major product in a 2:1 ratio as predicted by Felkin-Anh model [5]. Confirmation of these absolute configurations was obtained by conversion of each diastereomer into compounds (10a) and (10b), respectively, and comparing their NMR spectra with that of previously synthesized 8-azido-8-deoxy-Kdo [6].

(7) Hence, the two diastereomers were separated by flash chromatography over silica gel and each product was separately submitted to ozonolysis in methanol to provide after dimethylsufide addition, the alpha-keto esters (9a) and (9b), respectively. Final hydrolysis of the esters and the acetonides were achieved with 10% aqueous trifluoroacetic acid at 40 C. The instability of ulosonic acids to acidic conditions [7] made this step critical and evaporation of solvents had to be done on the rotary evaporator without warming followed by co-evaporation with toluene to allow complete elimination of the acid. Finally, the residue was neutralised with aqueous ammonia giving compounds (10a) and (10b) in 71% (from (8a)) and 46% (from (8b)) respectively. Compound (10a) proved to be identical to the previously reported 8-azido-3,8-dideoxy-D-manno-octulosonate. This approach also afforded its 4-epimer, 8-azido-3,8-dideoxy-D-gluco-octulosonate (10b).

(8) It is apparent that chemical reaction scheme I or scheme 1, here-below, starting from the commercially available D-arabinose to reach at will either known Kdo-N.sub.3, or known 4eKdo-N.sub.3 or even known Ara-N.sub.3 forms an integral part of the invention and is claimed in all its aspects, in part or in combination.

(9) ##STR00036##
Wherein:
R.sup.1 and R.sup.2 can be independently H; a C.sub.1 to C.sub.6 alkyl including methyl, ethyl, propyl, butyl; aryl including phenyl, para-methoxyphenyl; or R.sup.1,R.sup.2 together with the carbon C-6 can be a cyclopentylidene or cyclohexylidene; each of these groups being substituted or not;
and R.sup.3 can be a C.sub.1 to C.sub.6 alkyl including methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl; or aryl including phenyl, methylphenyl, ethylphenyl, each of these groups being substituted or not;
and R.sup.4 can be a C.sub.1 to C.sub.6 alkyl including methyl, ethyl, propyl; a C.sub.1 to C.sub.6 perfluoroalkyl including trifluoromethyl, pentafluoroethyl; or aryl including para-methylphenyl, para-nitrophenyl; each of these groups being substituted or not;
and R.sup.5 can be a C.sub.1 to C.sub.6 alkyl including methyl, ethyl, propyl, butyl, pentyl, hexyl; each of these groups being substituted or not.

INVENTION EXAMPLES

(10) Materials and Methods:

(11) Thin layer chromatography was performed over Merck 60 F254 with detection by UV, and/or by charring with sulphuric acid or KMnO.sub.4 or phosphomolybdic acid solutions. Silica gel 60 40-63 m was used for flash column chromatography.

(12) NMR spectra were taken on Bruker Avance 300 or 500 MHz spectrometers, using the residual protonated solvent as internal standard. Chemical shifts are given in parts per million (ppm) and coupling constants are reported as Hertz (Hz). Splitting patterns are designated as singlet (s), doublet (d), triplet (t), doublet of doublet (dd), doublet of doublet of doublet (ddd). Splitting patterns that could not be interpreted or easily visualized are designated as multiplet (m).

(13) Mass spectra were taken on a Waters LCT Premier XE (ToF), with electrospray ionization in the positive (ESI+) or in the negative (ESI) mode of detection.

(14) IR-FT spectra were recorded on a Perkin Elmer Spectrum 100 spectrometer. Characteristic absorptions are reported in cm.sup.1.

(15) Specific optical rotations were measured at 20 C. with an Anton Paar MCP 300 polarimeter in a 10-cm cell at 20 C. and 589 nm.

(16) All biological and chemical reagents were of analytical or cell culture grade, obtained from commercial sources, and used without further purifications.

(17) Escherichia coli and Legionella strains were purchased from ATCC-LGC or Leibniz Institute DSMZ (E. coli K12 ATCC 700926, E. coli O13 ATCC 15223, Legionella pneumophila Sg1 Philadelphia DSM 25020 and Legionella (Fluoribacter) gormanii ATCC 33342).

(18) Fluorescence microscopy experiments were performed on a confocal straight Leica SP8 (DM 6000), using a 63 PLAN APO oil immersion lens (Leica), an argon laser (488 nm) and a GaAsP Hybrid detector (Hamamatsu). The microscope was operated with LAS-X program. ImageJ program was used for image treatment.

Invention Example 1

Synthesis of 5-azido-5-deoxy-2:3-isopropylidene-D-arabinose O-methyloxime (5)

(19) ##STR00037##

(20) To a solution of (4E/4Z) (810 mg, 2.72 mmol, 1.0 eq.) in N,N-dimethylformamide (30.0 mL, 0.10 M), sodium azide (531 mg, 8.17 mmol, 3.0 eq.) was added and the reaction mixture was heated at 80 C. for 15 hours. Solvent was then removed under reduced pressure and the residue was purified by flash column chromatography (cyclohexane/ethyl acetate 9:1) to yield a mixture of 5-azido-5-deoxy-2:3-isopropyl idene-D-arabinose O-methyloxime (5E/5Z) (NMR ratio 7:3, 637 mg, 96%) as yellowish oil. A fraction of (5E) was isolated by flash column chromatography (dichloromethane/MTBE 97:3) for its characterizations. Purity of more than 95% by NMR.

(21) Rf (cyclohexane/ethyl acetate 8:2): 0.30.

(22) IR (cm.sup.1): 3458, 2989, 2939, 2823, 2100, 1630, 1443, 1373, 1213, 1164, 1066, 1036, 885, 865.

(23) HRMS (ESI.sup.+): [M+H].sup.+ (C.sub.9H.sub.17N.sub.4O.sub.4.sup.+) Calc. m/z: 245.1245, found: 245.1250.

(24) Isomer (5E):

(25) Rf (dichloromethane/MTBE 97:3): 0.23.

(26) .sup.1H-NMR (500 MHz, CDCl.sub.3) : 7.42 (d, 1H, J.sub.1,2 5.8 Hz, H-1); 4.54 (dd, 1H, J.sub.2,3 7.2, J.sub.1,2 5.8 Hz, H-2); 3.98 (dd, 1H, J.sub.2,3 7.2, J.sub.3,4 6.4 Hz, H-3); 3.92 (dddd, 1H, J.sub.3,4 6.4, J.sub.4,5b 6.2, J.sub.4,OH 3.9, J.sub.4,5a 3.8 Hz, H-4); 3.85 (s, 3H, CH.sub.3O); 3.46 (dd, 1H, J.sub.5a,5b 12.5, J.sub.4,5a 3.8 Hz, H-5a); 3.42 (dd, 1H, J.sub.5a,5b 12.5, J.sub.4,5b 6.2 Hz, H-5b); 2.61 (d, 1H, J.sub.4,OH 3.9 Hz, OH); 1.41 (s, 3H, CH.sub.3C); 1.39 (s, 3H, CH.sub.3C).

(27) .sup.13C-NMR (125 MHz, CDCl.sub.3) : 148.4 (C-1); 110.6 (C-6); 78.8 (C-3); 75.8 (C-2); 71.5 (C-4); 62.3 (CH.sub.3O); 53.7 (C-5); 27.0, 26.8 (2 CH.sub.3C).

(28) Isomer (5Z):

(29) .sup.1H-NMR (500 MHz, CDCl.sub.3) : 6.86 (d, 1H, J.sub.1,2 6.1 Hz, H-1); 4.94 (dd, 1H, J.sub.2,3 7.2, J.sub.1,2 6.1 Hz, H-2); 3.93 (s, 3H, CH.sub.3O); 3.87 (ddd, 1H, J.sub.3,4 7.5, J.sub.4,5b 6.4, J.sub.4,5a 2.8 Hz, H-4); 3.82 (dd, 1H, J.sub.3,4 7.5, J.sub.2,3 7.2 Hz, H-3); 3.47 (dd, 1H, J.sub.5a,5b 12.8, J.sub.4,5a 2.8 Hz, H-5a); 3.39 (dd, 1H, J.sub.5a,5b 12.8, J.sub.4,5b 6.4 Hz, H-5b); 1.40 (s, 3H, CH.sub.3C); 1.38 (s, 3H, CH.sub.3C).

(30) .sup.13C-NMR (75 MHz, CDCl.sub.3) : 150.8 (C-1); 111.0 (C-6); 80.0 (C-3); 72.9 (C-2); 72.5 (C-4); 62.8 (CH.sub.3O); 53.5 (C-5); 26.9, 26.5 (2 CH.sub.3C).

Invention Example 2

Synthesis of 5-azido-5-deoxy-2:3-isopropylidene-D-arabinose (6)

(31) ##STR00038##

(32) To a solution of (5E/5Z) (820 mg, 3.36 mmol, 1.0 eq.), as prepared from Example 1, in 80% (v/v) aqueous acetic acid (120 mL), formaldehyde (0.8 mL) was added and the reaction mixture was stirred for 1 hour at room temperature. Solvents were removed under reduced pressure and co-evaporation with toluene was done to assure complete elimination of acetic acid. The crude compound 5-azido-5-deoxy-2:3-isopropyl idene-D-arabinose (6) (682 mg) was used directly in the next step without further purification.

(33) Colorless Oil

(34) Rf (cyclohexane/ethyl acetate 7:3): 0.56.

(35) IR (cm.sup.1): 3408, 2988, 2936, 2100, 1733, 1440, 1373, 1238, 1213, 1164, 1063, 863.

(36) .sup.1H-NMR (500 MHz, CDCl.sub.3) : 9.79 (d, 1H, J.sub.1,2 1.2 Hz, H-1); 4.41 (dd, 1H, J.sub.2,3 6.4, J.sub.1,2 1.2 Hz, H-2); 4.04 (dd, 1H, J.sub.2,3 6.4, J.sub.3,4 6.1 Hz, H-3); 3.90 (ddd, 1H, J.sub.4,5b 6.4, J.sub.3,4 6.1, J.sub.4,5a 3.4 Hz, H-4); 3.51 (dd, 1H, J.sub.5a,5b 12.8, J.sub.4,5a 3.4 Hz, H-5a); 3.43 (dd, 1H, J.sub.5A,5B 12.8, J.sub.4,5B 6.4 Hz, H-5B); 1.47 (s, 3H, CH.sub.3C); 1.37 (s, 3H, CH.sub.3C).

(37) HRMS (ESI.sup.+): [2M+Na].sup.+ (C.sub.16H.sub.26N.sub.6NaO.sub.8.sup.+) Calc. m/z: 453.1704, found: 453.1726.

Invention Example 3

Synthesis of 2:3-isopropylidene-5-O-methanesulfonyl-D-arabinose O-methyloxime (4)

(38) ##STR00039##

(39) To a solution of 2:3-isopropylidene-D-arabinose O-methyloxime (3) (3E/3Z) (100 mg, 0.46 mmol, 1.0 eq.) in dry pyridine (2.0 mL) at 20 C., mesyl chloride (0.10 mL, 1.37 mmol, 3.0 eq.) was added and the reaction mixture was stirred for 1.5 hours at 20 C. After quenching the reaction with CH.sub.3OH (0.3 mL), solvents were removed under vacuum. The resulting residue was purified by silica flash column chromatography (cyclohexane/ethyl acetate 6:4) to yield a mixture of 2:3-isopropylidene-5-O-methanesulfonyl-D-arabinose O-methyloxime (4E/4Z) (NMR ratio 4:1, 110 mg, 81%) as colorless oil. An aliquot of pure (4E) isomer was obtained by flash column chromatography (dichloromethane/diethyl ether 9:1) and characterized. Purity of more than 95% by NMR.

(40) Rf (cyclohexane/ethyl acetate 6:4): 0.24.

(41) IR (cm.sup.1): 3500, 2989, 2941, 2824, 1631, 1458, 1350, 1215, 1170, 1067, 1033, 959, 887, 863, 833.

(42) HRMS (ESI.sup.+): [M+H].sup.+ (C.sub.10H.sub.20NO.sub.7S.sup.+) Calc. m/z: 298.0955, found: 298.0947.

(43) Isomer (4E):

(44) Rf (cyclohexane/ethyl acetate 6:4): 0.20.

(45) .sup.1H-NMR (500 MHz, CDCl.sub.3) : 7.42 (d, 1H, J.sub.1,2 5.6 Hz, H-1); 4.56 (dd, 1H, J.sub.2,3 6.8, J.sub.1,2 5.6 Hz, H-2); 4.41 (dd, 1H, J.sub.5a,5b 11.0, J.sub.4,5a 2.7 Hz, H-5a); 4.28 (dd, 1H, J.sub.5a,5b 11.0, J.sub.4,5b 5.7 Hz, H-5b); 4.03 (ddd, 1H, J.sub.3,4 7.0, J.sub.4,5b 5.7, J.sub.4,5a 2.7 Hz, H-4); 4.01 (dd, 1H, J.sub.2,3 6.8, J.sub.3,4 7.0 Hz, H-3); 3.85 (s, 3H, CH.sub.3O); 3.06 (s, 3H, CH.sub.3S); 1.41 (s, 3H, CH.sub.3C); 1.39 (s, 3H, CH.sub.3C).

(46) .sup.13C-NMR (125 MHz, CDCl.sub.3) : 148.2 (C-1); 110.9 (C-6); 77.9 (C-3); 76.2 (C-2); 70.9 (C-5); 70.8 (C-4); 62.3 (CH.sub.3O); 37.8 (CH.sub.3S); 27.0, 26.9 (2 CH.sub.3C).

(47) Isomer (4Z):

(48) .sup.1H-NMR (300 MHz, CDCl.sub.3) : 6.87 (d, 1H, J.sub.1,2 5.9 Hz, H-1); 4.96 (dd, 1H, J.sub.2,3 7.3, J.sub.1,2 5.9 Hz, H-2); 4.45 (dd, 1H, J.sub.5a,5b 11.4, J.sub.4,5a 2.4 Hz, H-5a); 4.29 (dd, 1H, J.sub.5a,5b 11.4, J.sub.4,5b 7.9 Hz, H-5b); 3.98 (ddd, 1H, J.sub.4,5b 7.9, J.sub.4,3 7.5, J.sub.4,5a 2.4 Hz, H-4); 3.93 (s, 3H, CH.sub.3O); 3.84 (dd, 1H, J.sub.3,4 7.5, J.sub.3,2 7.3 Hz, H-3); 3.06 (s, 3H, CH.sub.3S); 1.40 (s, 3H, CH.sub.3C); 1.39 (s, 3H, CH.sub.3C).

(49) .sup.13C-NMR (75 MHz, CDCl.sub.3) : 150.7 (C-1); 111.2 (C-6); 79.1 (C-3); 72.9 (C-2); 71.3 (C-5); 70.9 (C-4); 62.9 (CH.sub.3O); 37.9 (CH.sub.3S); 27.0, 26.6 (2 CH.sub.3C).

Invention Example 4

Synthesis of 2:3-isopropylidene-D-arabinose O-methyloxime (3)

(50) ##STR00040##

(51) A solution of 2:3,4:5-diisopropylidene-D-arabinose O-methyloxime (2) (2E/2Z) and impurity (1.50 g) in 80% (v/v) aqueous acetic acid (30 mL) was heated to 40 C. at 200 mbar of pressure on a rotavap. After 2.5 hours solvents were removed under reduced pressure and the residue was co-evaporated with toluene. A mixture of isomers 2:3-isopropylidene-D-arabinose O-methyloxime (3E/3Z) (NMR ratio 4:1, 876 mg, 58% over 3 steps) were obtained after silica gel flash column chromatography (cyclohexane/ethyl acetate 1:1) as colorless oil. Purity of more than 95% by NMR.

(52) Rf (cyclohexane/ethyl acetate 1:1): 0.24.

(53) IR (cm.sup.1): 3409, 2939, 1373, 1216, 1040, 885.

(54) HRMS (ESI.sup.+): [M+H].sup.+ (C.sub.9H.sub.18NO.sub.5.sup.+) Calc. m/z: 220.1179, found: 220.1184.

(55) Isomer (3E):

(56) .sup.1H-NMR (500 MHz, CDCl.sub.3) : 7.44 (d, 1H, J.sub.1,2 5.5 Hz, H-1); 4.56 (dd, 1H, J.sub.2,3 7.4, J.sub.1,2 5.5 Hz, H-2); 4.07 (dd, 1H, J.sub.2,3 7.4, J.sub.3,4 5.6 Hz, H-3); 4.13 (ddd, 1H, J.sub.3,4 5.6, J.sub.4,5 5.1, J.sub.4,5 4.7 Hz, H-4); 3.84 (s, 3H, CH.sub.3O); 3.72-3.68 (m, 2H, 2H-5); 1.42 (s, 3H, CH.sub.3C); 1.38 (s, 3H, CH.sub.3C).

(57) .sup.13C-NMR (125 MHz, CDCl.sub.3) : 149.1 (C-1); 110.3 (C-6); 79.4 (C-3); 75.0 (C-2); 71.6 (C-4); 63.4 (C-5); 62.2 (CH.sub.3O); 26.9, 26.7 (2 CH.sub.3C).

(58) Isomer (3Z):

(59) .sup.1H-NMR (500 MHz, CDCl.sub.3) : 6.86 (d, 1H, J.sub.1,2 5.9 Hz, H-1); 4.95 (dd, 1H, J.sub.2,3 7.7, J.sub.1,2 5.9 Hz, H-2); 3.92 (s, 3H, CH.sub.3O); 3.87 (dd, 1H, J.sub.2,3 7.7, J.sub.3,4 6.9 Hz, H-3); 3.82-3.75 (m, 2H, H-4, H-5a); 3.72-3.68 (m, 1H, H-5b); 1.40 (2s, 6H, 2 CH.sub.3C).

(60) .sup.13C-NMR (125 MHz, CDCl.sub.3) : 151.0 (C-1); 110.9 (C-6); 80.4 (C-3); 72.9 (C-2); 72.7 (C-4); 63.5 (C-5); 62.8 (CH.sub.3O); 27.0, 26.5 (2 CH.sub.3C).

Invention Example 5

Synthesis of 2:3,4:5-diisopropylidene-D-arabinose O-methyloxime (2) from D-()-arabinose

(61) ##STR00041##

(62) To a solution of D-()-arabinose (4.00 g, 26.6 mmol, 1.0 eq.) in dry pyridine (90 mL) was added methoxyamine hydrochloride (2.72 g, 32.0 mmol, 1.2 eq.) and the mixture was stirred at room temperature for 15 hours. Solvents were removed under reduced pressure and the residue was co-evaporated with toluene three times. The residue was resuspended in 2,2-dimethoxypropane (100 mL) and p-toluenesulfonic acid (1.01 g, 5.33 mmol, 0.2 eq.) was added and the suspension was heated to reflux for 4 hours followed by further 15 hours of stirring at room temperature. The reaction mixture was filtered over Celite and solvents were evaporated. The residue was dissolved in ethyl acetate (200 mL) and was washed with saturated aq. NaCl solution (2150 mL). Purification by silica flash column chromatography (cyclohexane/ethyl acetate 9:1) yields a mixture of isomers 2:3,4:5-diisopropylidene-D-arabinose O-methyloxime (2E/2Z) and an unknown impurity (NMR ratio 6:1:0.4, 5.83 g) as colorless oil. This mixture was used without further purification in the next step. An aliquot of pure (2E) was obtained by a second flash column chromatography (dichloromethane/MTBE 98:2) and characterized.

(63) Isomer (2E):

(64) Rf (CH.sub.2Cl.sub.2/MTBE 98:2): 0.35.

(65) IR (cm.sup.1): 2987, 2939, 2900, 2821, 1631, 1456, 1381, 1371, 1241, 1212, 1150, 1065, 1038, 887, 842.

(66) .sup.1H-NMR (500 MHz, CDCl.sub.3) : 7.35 (d, 1H, J.sub.1,2 6.3 Hz, H-1); 4.46 (dd, 1H, J.sub.2,3 7.1, J.sub.1,2 6.3 Hz, H-2); 4.13 (ddd, 1H, J.sub.3,4 6.9, J.sub.4,5a 6.1, J.sub.4,5b 4.8 Hz, H-4); 4.08 (dd, 1H, J.sub.5a,5b 8.5, J.sub.4,5a 6.1 Hz, H-5a); 3.97 (d, 1H, J.sub.2,3 7.1, J.sub.3,4 6.9 Hz, H-3); 3.94 (dd, 1H, J.sub.5a,5b 8.5, J.sub.4,5b 4.8 Hz, H-5b); 3.85 (s, 3H, CH.sub.3O); 1.40 (s, 3H, CH.sub.3C); 1.38 (s, 6H, 2 CH.sub.3C); 1.32 (s, 3H, CH.sub.3C).

(67) .sup.13C-NMR (125 MHz, CDCl.sub.3) : 147.8 (C-1); 110.8 (C-6); 110.0 (C-7); 79.4 (C-3); 76.7 (C-2); 76.6 (C-4); 67.1 (C-5); 62.1 (CH.sub.3O); 27.1, 27.0, 26.9, 25.4 (4 CH.sub.3C).

(68) HRMS (ESI.sup.+): [M+H].sup.+ (C.sub.12H.sub.22NO.sub.5.sup.+) Calc. m/z: 260.1492, found: 260.1502.

Invention Example 6

Use of 5-azido-5-deoxy-2:3-isopropylidene-D-arabinose for the synthesis of 5-azido-5-deoxy-D-arabinofuranose or Ara-N.SUB.3 .(7)

(69) ##STR00042##

(70) To a solution of crude 5-azido-5-deoxy-2:3-isopropylidene-D-arabinose (6) (100 mg), obtained in invention Example 2 above, in a mixture of CH.sub.2Cl.sub.2/H.sub.2O (20:1, 21 mL), trifluoroacetic acid (1 mL) was added and the mixture was stirred at room temperature for 1 hour. Solvents were then evaporated, the crude residue was resuspended in water and lyophilized. After silica flash column chromatography (dichloromethane/methanol 92:8), the compound 5-azido-5-deoxy-D-arabinofuranose or Ara-N.sub.3 (7) (50 mg, 58% over 2 steps from compound (5)) was obtained as a mixture of / anomers (NMR ratio 55:45) as a colorless oil. Purity of more than 95% by NMR.

(71) Rf (dichloromethane/methanol 92:8): 0.28.

(72) IR (cm.sup.1): 3367, 2106, 1281, 1040.

(73) HRMS (ESI.sup.+): [M+H-N.sub.2]+(C.sub.5H.sub.10NO.sub.4.sup.+) Calc. m/z: 148.0604, found: 148.0610.

(74) Anomer Alpha (7):

(75) .sup.1H-NMR (500 MHz, D.sub.2O) : 5.24 (d, 1H, J.sub.1,2 2.9 Hz, H-1); 4.17 (ddd, 1H, J.sub.3,4 6.4, J.sub.4,5b 5.8, J.sub.4,5a 3.5 Hz, H-4); 4.01 (dd, 1H, J.sub.2,3 4.6, J.sub.1,2.2.9 Hz, H-2); 3.97 (dd, 1H, J.sub.3,4 6.4, J.sub.3,2 4.6 Hz, H-3); 3.64 (dd, 1H, J.sub.5a,5b 13.6, J.sub.4,5a 3.5 Hz, H-5a); 3.44 (dd, 1H, J.sub.5a,5b 13.6, J.sub.4,5b 5.8 Hz, H-5b).

(76) .sup.13C-NMR (125 MHz, D.sub.2O) : 101.0 (C-1); 81.3 (C-4); 81.2 (C-2); 76.3 (C-3); 51.5 (C-5).

(77) Anomer Beta (7):

(78) .sup.1H-NMR (500 MHz, D.sub.2O) : 5.28 (br d, 1H, J.sub.1,2 3.1 Hz, H-1); 4.10-4.05 (m, 2H, H-2, H-3); 3.89 (ddd, 1H, J.sub.3,4-7.1, J.sub.4,5b 6.5, J.sub.4,5a 3.5 Hz, H-4); 3.59 (dd, 1H, J.sub.5a,5b 13.3, J.sub.4,5a 3.5 Hz, H-5a); 3.42 (dd, 1H, J.sub.5a,5b 13.3, J.sub.4,5b 6.5 Hz, H-5b).

(79) .sup.13C-NMR (125 MHz, D.sub.2O) : 95.2 (C-1); 79.6 (C-4); 75.8 (C-2); 74.7 (C-3); 52.6 (C-5).

Invention Example 7

Use of 5-azido-5-deoxy-2:3-isopropylidene-D-arabinose for the synthesis of ethyl (4R)-4-4-{(4R,5R)-5-[(R)-2-azido-1-hydroxyethyl]-2,2-dimethyl-1,3-dioxolan-4-yl)-4-hydroxy-2-methylenebutanoate (8a)

(80) ##STR00043##

(81) To a solution of 5-azido-5-deoxy-2:3-isopropylidene-D-arabinose (6) (40 mg), obtained in invention Example 2 above, in CH.sub.3CN/H.sub.2O (1:1, 18.0 mL, 0.01 M) at 0 C., ethyl 2-(bromomethyl) acrylate (0.085 mL, 0.61 mmol, 3.3 eq.) and aqueous formic acid (10%, 0.19 mL) were added and the reaction mixture was allowed to stir for 10 min at 0 C. Indium powder (23 mg, 0.20 mmol, 1.1 eq.) was then added and the reaction mixture was stirred for 3 hours while temperature was maintained between 0-5 C. After filtration, solvents were evaporated under reduced pressure and the resulting mixture was purified by flash column chromatography (cyclohexane/ethyl acetate 75:25) to afford compound (8a) (33 mg, 54%) as colorless oil. Purity of more than 95% by NMR.

(82) Rf (cyclohexane/ethyl acetate 7:3): 0.22.

(83) IR (cm.sup.1): 3397, 2986, 2935, 2101, 1710, 1629, 1444, 1371, 1211, 1162, 1068, 1026, 949, 872.

(84) .sup.1H-NMR (500 MHz, CDCl.sub.3) : 6.29 (d, 1H, J.sub.10a,10b 1.1 Hz, H-10a); 5.79 (d, 1H, J.sub.10a,10b 1.1 Hz, H-10b); 4.92 (br s, 1H, OH); 3.38 (br s, 1H, OH); 4.24 (q, 2H, .sup.3J 7.2 Hz, OCH.sub.2CH.sub.3); 3.83 (dd, 1H, J.sub.6,7 8.4, J.sub.5,6 7.4 Hz, H-6); 3.72 (ddd, 1H, J.sub.6,7 8.4, J.sub.7,8b 5.3, J.sub.7,8a 2.7 Hz, H-7); 3.68 (ddd, 1H, J.sub.4,5 8.9, J.sub.4,3b 7.5, J.sub.4,3a 2.2 Hz, H-4); 3.60 (dd, 1H, J.sub.5,4 8.9, J.sub.5,6 7.4 Hz, H-5); 3.52 (dd, 1H, J.sub.8a,8b 12.6, J.sub.7,8a 2.7 Hz, H-8a); 3.36 (dd, 1H, J.sub.8a,8b 12.6, J.sub.7,8b 5.3 Hz, H-8b); 2.87 (dd, 1H, J.sub.3a,3b 14.4, J.sub.4,3a 2.2 Hz, H-3a); 2.49 (dd, 1H, J.sub.3a,3b 14.4, J.sub.4,3b 7.5 Hz, H-3b); 1.35 (s, 3H, CH.sub.3C); 1.33 (s, 3H, CH.sub.3C); 1.31 (t, 3H, .sup.3J7.2 Hz, OCH.sub.2CH.sub.3).

(85) .sup.13C-NMR (75 MHz, CDCl.sub.3) : 170.0 (C-1); 136.8 (C-2); 129.6 (C-10); 109.4 (C-9); 82.3 (C-5); 80.7 (C-6); 73.1 (C-4); 72.7 (C-7); 62.1 (OCH.sub.2CH.sub.3); 54.3 (C-8); 37.4 (C-3); 27.0 (2 CH.sub.3); 14.3 (OCH.sub.2CH.sub.3).

(86) HRMS (ESI.sup.+): [M+H].sup.+ (C.sub.14H.sub.24N.sub.3O.sub.6.sup.+) Calc. m/z: 330.1660, found: 330.1670.

(87) [].sub.D=+18.0 (c 1.0, CHCl.sub.3).

Invention Example 8

Use of 5-azido-5-deoxy-2:3-isopropylidene-D-arabinose for the synthesis of ethyl (4S)-4-{(4R,5R)-5-[(R)-2-azido-1-hydroxyethyl]-2,2-dimethyl-1,3-dioxolan-4-yl)-4-hydroxy-2-methylenebutanoate (8b)

(88) ##STR00044##

(89) To a solution of 5-azido-5-deoxy-2:3-isopropylidene-D-arabinose (6) (40 mg) in CH.sub.3CN/H.sub.2O (1:1, 18.0 mL, 0.01 M) at 0 C., ethyl 2-(bromomethyl) acrylate (0.085 mL, 0.61 mmol, 3.3 eq.) and aqueous formic acid (10%, 0.19 mL) were added and the reaction mixture was allowed to stir for 10 min at 0 C. Indium powder (23 mg, 0.20 mmol, 1.1 eq.) was then added and the reaction mixture was stirred for 3 hours while temperature was maintained between 0-5 C. After filtration, solvents were evaporated under reduced pressure and the resulting mixture was purified by flash column chromatography (cyclohexane/ethyl acetate 75:25) to afford ethyl (4S)-4-{(4R,5R)-5-[(R)-2-azido-1-hydroxyethyl]-2,2-dimethyl-1,3-dioxolan-4-yl)-4-hydroxy-2-methylenebutanoate (8b) (17 mg, 28%) as colorless oil. Purity of more than 95% by NMR.

(90) Rf (cyclohexane/ethyl acetate 7:3): 0.19.

(91) IR (cm.sup.1): 3397, 2986, 2935, 2101, 1710, 1629, 1444, 1371, 1211, 1162, 1068, 1026, 949, 872.

(92) .sup.1H-NMR (500 MHz, CDCl.sub.3) : 6.24 (d, 1H, J.sub.10a,10b 0.9 Hz, H-10a); 5.71 (d, 1H, J.sub.10a,10b 0.9 Hz, H-10b); 4.21 (q, 2H, 317.1 Hz, OCH.sub.2CH.sub.3); 4.00 (dd, 1H, J.sub.5,6 7.8, J.sub.4,5 3.0 Hz, H-5); 3.93 (dddd, 1H, J.sub.3b,4 9.0, J.sub.4,OH 5.6, J.sub.4,5 3.0, J.sub.4,3a 3.0 Hz, H-4); 3.89 (dd, 1H, J.sub.6,7 7.9, J.sub.5,6 7.8 Hz, H-6); 3.75 (dddd, 1H, J.sub.6,7 7.9, J.sub.7,8b 6.1, J.sub.7,8a 2.8, J.sub.7,OH 2.5 Hz, H-7); 3.55 (dd, 1H, J.sub.8a,8b 12.6, J.sub.7b,8a 2.8 Hz, H-8a); 3.46 (br d, 1H, J.sub.4,OH 5.6 Hz, OH(4)); 3.40 (dd, 1H, J.sub.8a,8b 12.6, J.sub.7,8b 6.1 Hz, H-8b); 3.38 (br d, 1H, J.sub.4,OH 2.5 Hz, OH(7)); 2.66 (dd, 1H, J.sub.3a,3b 14.4, J.sub.4,3a 3.0 Hz, H-3a); 2.50 (dd, 1H, J.sub.3a,3b 14.4, J.sub.4,3b 9.0 Hz, H-3b); 1.40 (s, 3H, CH.sub.3C); 1.35 (s, 3H, CH.sub.3C); 1.29 (t, 3H, .sup.3J7.1 Hz, OCH.sub.2CH.sub.3).

(93) .sup.13C-NMR (75 MHz, CDCl.sub.3) : 168.4 (C-1); 137.7 (C-2); 128.3 (C-10); 109.7 (C-9); 81.8 (C-5); 76.8 (C-6); 72.6 (C-7); 69.9 (C-4); 61.5 (OCH.sub.2CH.sub.3); 54.4 (C-8); 37.0 (C-3); 27.2 (2 CH.sub.3); 14.3 (OCH.sub.2CH.sub.3).

(94) HRMS (ESI.sup.+): [M+H].sup.+ (C.sub.14H.sub.24N.sub.3O.sub.6.sup.+) Calc. m/z: 330.1660, found: 330.1670.

(95) [].sub.D=+7.7 (c 1.0, CHCl.sub.3).

Invention Example 9

Use of ethyl (4R)-4-{(4R,5R)-5-[(R)-2-azido-1-hydroxyethyl]-2,2-dimethyl-1,3-dioxolan-4-yl)-4-hydroxy-2-methylenebutanoate (8a) for the synthesis of ethyl (4R)-4-{(4R,5R)-5-[(R)-2-azido-1-hydroxyethyl]-2,2-dimethyl-1,3-dioxolan-4-yl}-4-hydroxy-2-oxobutanoate (9a)

(96) ##STR00045##

(97) A solution of ethyl (4R)-4-{(4R,5R)-5-[(R)-2-azido-1-hydroxyethyl]-2,2-dimethyl-1,3-dioxolan-4-yl)-4-hydroxy-2-methylenebutanoate (8a) (120 mg, 0.36 mmol, 1.0 eq.) in CH.sub.3OH (18 mL, 0.02 M), as obtained from invention Example 7 above, at 78 C. was bubbled with ozone (O.sub.3) for 15 min. The solution was purged with oxygen for 2 min until the blue color disappeared and then was degassed with argon for 5 min. An excess of dimethyl sulfide (0.1 mL, 1.36 mmol, 3.8 eq.) was added and the mixture was stirred at room temperature for 2 hours. After solvents evaporation, the residue was extracted with Et.sub.2O (310 mL) and the organic phases were washed with brine (210 mL), dried over anhydrous MgSO.sub.4 and concentrated under reduced pressure. Ethyl (4R)-4-{(4R,5R)-5-[(R)-2-azido-1-hydroxyethyl]-2,2-dimethyl-1,3-dioxolan-4-yl}-4-hydroxy-2-oxobutanoate (9a) (97 mg) was used for the next step without further purification.

(98) Colourless Oil

(99) Rf (cyclohexane/ethyl acetate 1:1): 0.40

(100) IR (cm.sup.1): 3377, 2988, 2103, 1729, 1443, 1373, 1257, 1164, 871.

(101) .sup.1H-NMR (500 MHz, CDCl.sub.3) : 4.32 (q, 2H, .sup.3J7.2 Hz, OCH.sub.2CH.sub.3); 4.15 (ddd, 1H, J.sub.3b,4 8.8, J.sub.4,5 8.6, J.sub.4,3a 3.1 Hz, H-4); 3.84 (dd, 1H, J.sub.6,7 8.2, J.sub.5,6 7.3 Hz, H-6); 3.74 (dd, 1H, J.sub.4,5 8.6, J.sub.5,6 7.3 Hz, H-5); 3.74 (ddd, 1H, J.sub.6,7 8.2, J.sub.7,8b 5.7, J.sub.7,8a 2.8 Hz, H-7); 3.59 (dd, 1H, J.sub.8a,8b 12.6, J.sub.7,8a 2.68 Hz, H-8a); 3.40 (dd, 1H, J.sub.8a,8b 12.6, J.sub.7,8b 5.7 Hz, H-8b); 3.40 (dd, 1H, J.sub.3a,3b 18.2, J.sub.4,3a 3.1 Hz, H-3a); 3.01 (dd, 1H, J.sub.3a,3b 18.2, J.sub.4,3b 8.8 Hz, H-3b); 1.36 (t, 3H, .sup.3J7.2 Hz, OCH.sub.2CH.sub.3); 1.34 (s, 3H, CH.sub.3); 1.33 (s, 3H, CH.sub.3).

(102) .sup.13C-NMR (125 MHz, CDCl.sub.3) : 194.0 (C-2); 160.7 (C-1); 110.1 (C-9); 82.1 (C-5); 80.6 (C-6); 72.6 (C-7); 69.6 (C-4); 63.1 (OCH.sub.2CH.sub.3); 54.3 (C-8); 44.2 (C-3); 27.0, 26.9 (2 CH.sub.3); 14.2 (OCH.sub.2CH.sub.3).

(103) HRMS (ESI.sup.+): [2M+Na].sup.+ (C.sub.26H.sub.42N.sub.6NaO.sub.14.sup.+) Calc. m/z: 685.2651, found: 685.2656.

(104) [].sub.D=+39.5 (c 1.0, CHCl.sub.3).

Invention Example 10

Use of ethyl (4S)-4-4-{(4R,5R)-5-[(R)-2-azido-1-hydroxyethyl]-2,2-dimethyl-1,3-dioxolan-4-yl)-4-hydroxy-2-methylenebutanoate (8b) for the synthesis of ethyl (4S)-4-{(4R,5R)-5-[(R)-2-azido-1-hydroxyethyl]-2,2-dimethyl-1,3-dioxolan-4-yl}-4-hydroxy-2-oxobutanoate (9b)

(105) ##STR00046##

(106) A solution of ethyl (4S)-4-{(4R,5R)-5-[(R)-2-azido-1-hydroxyethyl]-2,2-dimethyl-1,3-dioxolan-4-yl)-4-hydroxy-2-methylenebutanoate (8b) (70 mg, 0.21 mmol, 1.0 eq.), as obtained from invention Example 8, in CH.sub.3OH (10.0 mL, 0.02 M) at 78 C. was bubbled with ozone (O.sub.3) for 15 min. The solution was purged with oxygen for 2 min until the blue color disappeared and then was degassed with argon for 5 min. An excess of dimethyl sulfide (0.1 mL, 1.36 mmol, 6.5 eq.) was added and the mixture was stirred at room temperature for 2 hours. After solvents evaporation, the residue was extracted with Et.sub.2O (35 mL) and the organic phases were washed with brine (25 mL), dried over anhydrous MgSO.sub.4 and concentrated under reduced pressure. Ethyl (4S)-4-{(4R,5R)-5-[(R)-2-azido-1-hydroxyethyl]-2,2-dimethyl-1,3-dioxolan-4-yl}-4-hydroxy-2-oxobutanoate (9b) (45 mg) was used for the next step without further purification.

(107) Colourless Oil

(108) Rf (cyclohexane/ethyl acetate 1:1): 0.41.

(109) IR (cm.sup.1): 3443, 2989, 2104, 1731, 1373, 1256, 1069, 870.

(110) .sup.1H-NMR (500 MHz, CDCl.sub.3) : 4.38 (ddd, 1H, J.sub.3a,4 8.2, J.sub.4,3b 4.2, J.sub.4,5 3.1 Hz, H-4); 4.32 (q, 2H, .sup.3J7.2 Hz, OCH.sub.2CH.sub.3); 4.01 (dd, 1H, J.sub.5,6 7.7, J.sub.4,5 3.1 Hz, H-5); 3.90 (dd, 1H, J.sub.6,7 8.0, J.sub.5,6 7.7 Hz, H-6); 3.76 (ddd, 1H, J.sub.6,7 8.0, J.sub.7,8b 6.3, J.sub.7,8a 3.0 Hz, H-7); 3.59 (dd, 1H, J.sub.8a,8b 12.6, J.sub.7,8a 3.0 Hz, H-8a); 3.43 (dd, 1H, J.sub.8a,8b 12.6, J.sub.7,8b 6.3 Hz, H-8b); 3.20 (dd, 1H, J.sub.3a,3b 17.4, J.sub.4,3a 8.2 Hz, H-3a); 3.11 (dd, 1H, J.sub.3a,3b 17.4, J.sub.4,3b 4.2 Hz, H-3b); 1.39 (s, 3H, CH.sub.3C); 1.36 (t, 3H, .sup.3J7.2 Hz, OCH.sub.2CH.sub.3); 1.35 (s, 3H, CH.sub.3C).

(111) .sup.13C-NMR (125 MHz, CDCl.sub.3) : 193.4 (C-2); 160.8 (C-1); 110.0 (C-9); 81.6 (C-5); 76.6 (C-6); 72.6 (C-7); 66.6 (C-4); 63.0 (OCH.sub.2CH.sub.3); 54.5 (C-8); 43.2 (C-3); 27.2, 27.0 (2 CH.sub.3); 14.2 (OCH.sub.2CH.sub.3).

(112) HRMS (ESI.sup.+): [2M+Na].sup.+ (C.sub.26H.sub.42N.sub.6O.sub.14Na.sup.+) Calc. m/z: 685.2651, found: 685.2672.

(113) [].sub.D=0.6 (c 1.0, CHCl.sub.3).

Invention Example 11

Use of ethyl (4R)-4-4-{(4R,5R)-5-[(R)-2-azido-1-hydroxyethyl]-2,2-dimethyl-1,3-dioxolan-4-yl}-4-hydroxy-2-oxobutanoate (9a) for the synthesis of ammonium 8-azido-3,8-dideoxy-D-manno-oct-2-ulosonate (Kdo-N.SUB.3.) (10a)

(114) ##STR00047##

(115) To a solution of crude of ethyl (4R)-4-{(4R,5R)-5-[(R)-2-azido-1-hydroxyethyl]-2,2-dimethyl-1,3-dioxolan-4-yl}-4-hydroxy-2-oxobutanoate (9a) (90 mg) in water (22.5 mL) as obtained from invention Example 9, trifluoroacetic acid (2.5 mL) was added and the mixture was stirred at 40 C. for 2 hours. Solvents were removed under reduced pressure without warming the bath and then co-evaporated twice with toluene to assure complete removal of acid. The residue was neutralized with ammonia (0.2 mol.Math.L.sup.1) and then concentrated. Purification by flash column chromatography (ethyl acetate/ethanol/water 65:30:5) gave pure ammonium 8-azido-3,8-dideoxy-D-manno-oct-2-ulosonate or Kdo-N.sub.3 (10a) (67 mg, 71% starting from (8a)) as yellowish foam. Purity of more than 95% by NMR.

(116) Rf (ethyl acetate/ethanol/water 65:30:5): 0.20.

(117) IR (cm.sup.1): 3297, 2934, 2107, 1610, 1417, 1284, 1076, 756.

(118) HRMS (ESI.sup.): [M-NH.sub.4].sup. (C.sub.8H.sub.12N.sub.3O.sub.7.sup.) Calc. m/z: 262.0681, found: 262.0685.

(119) Major Conformer Alpha Pyranose Form (NMR 16):

(120) .sup.1H-NMR (600 MHz, D.sub.2O) : 4.06 (ddd, 1H, J.sub.4,3a 12.2, J.sub.4,3b 5.2, J.sub.4,5 3.1 Hz, H-4); 4.03 (ddd, 1H, J.sub.6,7 9.3, J.sub.7,8b 6.1, J.sub.7,8a 2.8 Hz, H-7); 4.02 (dd, 1H, J.sub.4,5 3.1, J.sub.5,6 0.8 Hz, H-5); 3.82 (dd, 1H, J.sub.6,7 9.3, J.sub.5,6 0.8 Hz, H-6); 3.60 (dd, 1H, J.sub.8a,8b 13.1, J.sub.7,8a 2.8 Hz, H-8a); 3.43 (dd, 1H, J.sub.8a,8b 13.1, J.sub.7,8b 6.1 Hz, H-8b); 1.98 (dd, 1H, J.sub.3a,3b 12.9, J.sub.4,3a 12.2 Hz, H-3a); 1.88 (dd, 1H, J.sub.3a,3b 12.9, J.sub.4,3b 5.2 Hz, H-3b).

(121) .sup.13C-NMR (150 MHz, D.sub.2O) : 177.7 (C-1); 97.5 (C-2); 72.7 (C-6); 69.2 (C-7); 37.5 (C-5); 67.2 (C-4); 54.8 (C-8); 34.7 (C-3).

(122) Major Conformer Alpha Furanose Form (NMR 5.5):

(123) .sup.1H-NMR (600 MHz, D.sub.2O) : 4.50 (ddd, 1H, J.sub.4,3a 7.1, J.sub.4,5 3.5, J.sub.4,3b 3.2 Hz, H-4); 4.46 (dd, 1H, J.sub.4,5 3.5, J.sub.5,6 1.2 Hz, H-5); 3.85 (ddd, 1H, J.sub.6,7 9.0, J.sub.7,8b 5.9, J.sub.7,8a 2.7 Hz, H-7); 3.63 (dd, 1H, J.sub.8a,8b 13.2, J.sub.7,8a 2.7 Hz, H-8a); 3.63 (dd, 1H, J.sub.6,7 9.0, J.sub.5,6 1.2 Hz, H-6); 3.51 (dd, 1H, J.sub.8a,8b 13.2, J.sub.7,8b 5.9 Hz, H-8b); 2.59 (dd, 1H, J.sub.3a,3b 14.3, J.sub.4,3a 7.1 Hz, H-3a); 2.08 (dd, 1H, J.sub.3a,3b 14.3, J.sub.4,3b 3.2 Hz, H-3b).

(124) .sup.13C-NMR (150 MHz, D.sub.2O) : 178.6 (C-1); 105.3 (C-2); 86.5 (C-5); 73.6 (C-4); 72.5 (C-6); 71.4 (C-7); 54.8 (C-8); 45.8 (C-3).

(125) Minor Conformer Beta Furanose Form (NMR 2.5):

(126) .sup.1H-NMR (600 MHz, D.sub.2O) : 4.52 (ddd, 1H, J.sub.4,3b 7.5, J.sub.4,3a 7.0, J.sub.4,5 6.0 Hz, H-4); 4.18 (dd, 1H, J.sub.5,4 6.0, J.sub.5,6 2.2 Hz, H-5); 3.86 (ddd, 1H, J.sub.6,7 8.9, J.sub.7,8b 6.1, J.sub.7,8a 2.4 Hz, H-7); 3.66 (dd, 1H, J.sub.6,7 8.9, J.sub.5,6 2.2 Hz, H-6); 3.64 (dd, 1H, J.sub.8a,8b 13.3, J.sub.7,8a 2.4 Hz, H-8a); 3.52 (dd, 1H, J.sub.8a,8b 13.3, J.sub.7,8b 6.1 Hz, H-8b); 2.37 (dd, 1H, J.sub.3a,3b 13.4, J.sub.4,3a 7.0 Hz, H-3a); 2.30 (dd, 1H, J.sub.3a,3b 13.4, J.sub.4,3b 7.5 Hz, H-3b).

(127) .sup.13C-NMR (150 MHz, D.sub.2O) : 177.9 (C-1); 104.1 (C-2); 86.5 (C-5); 71.8 (C-4); 71.4 (C-7); 71.2 (C-6); 54.6 (C-8); 44.7 (C-3).

(128) Minor Conformer Beta Pyranose Form (NMR 1.0):

(129) .sup.1H-NMR (600 MHz, D.sub.2O) : 4.06-4.01 (m, 1H, H-4); 3.97 (ddd, 1H, J.sub.6,7 9.2, J.sub.7,8b 6.7, J.sub.7,8a 2.7 Hz, H-7); 3.96 (dd, 1H, J.sub.4,5 3.3, J.sub.5,6 1.1 Hz, H-5); 3.67 (dd, 1H, J.sub.8a,8b 13.2, J.sub.7,8a 2.7 Hz, H-8a); 3.53 (dd, 1H, J.sub.6,7 9.2, J.sub.5,6 1.1 Hz, H-6); 3.45 (dd, 1H, J.sub.8a,8b 13.2, J.sub.7,8b 6.7 Hz, H-8b); 2.33-2.27 (m, 1H, H-3a); 1.76 (dd, 1H, J.sub.3a,3b 12.6, J.sub.4,3b 12.1 Hz, H-3b).

(130) .sup.13C-NMR (150 MHz, D.sub.2O) : 176.1 (C-1); 98.2 (C-2); 75.3 (C-6); 69.5 (C-7); 68.6 (C-4); 66.5 (C-5); 55.0 (C-8); 36.2 (C-3).

Invention Example 12

Use of ethyl (4S)-4-{(4R,5R)-5-[(R)-2-azido-1-hydroxyethyl]-2,2-dimethyl-1,3-dioxolan-4-yl}-4-hydroxy-2-oxobutanoate (9b) for the synthesis of ammonium 8-azido-3,8-dideoxy-D-gluco-oct-2-ulosonate (4eKdo-N.SUB.3.) (10b)

(131) ##STR00048##

(132) To a solution of crude ethyl (4S)-4-{(4R,5R)-5-[(R)-2-azido-1-hydroxyethyl]-2,2-dimethyl-1,3-dioxolan-4-yl}-4-hydroxy-2-oxobutanoate (9b) (40 mg), as obtained from invention Example 10, in water (10 mL), trifluoroacetic acid (1 mL) was added and the mixture was stirred at 40 C. for 2 hours. Solvents were removed under reduced pressure without warming the bath and then co-evaporated twice with toluene to assure complete removal of acid. The residue was neutralized with ammonia (0.2 mol.Math.L.sup.1) and then concentrated. Purification by flash column chromatography (ethyl acetate/ethanol/water 65:30:5) gave pure ammonium 8-azido-3,8-dideoxy-D-gluco-oct-2-ulosonate or 4eKdo-N.sub.3 (10b) (24 mg, 46% starting from (8b)) as yellowish foam. Purity of more than 95% by NMR.

(133) Rf (ethyl acetate/ethanol/water 65:30:5): 0.22.

(134) IR (cm.sup.1): 3296, 2931, 2104, 1612, 1384, 1283, 1072, 805.

(135) HRMS (ESI.sup.): [M-NH.sub.4].sup. (C.sub.8H.sub.12N.sub.3O.sub.7.sup.) Calc. m/z: 262.0681, found: 262.0669.

(136) Major Conformer Pyranose Form (NMR 7.5):

(137) .sup.1H-NMR (600 MHz, D.sub.2O) : 4.15 (dd, 1H, J.sub.6,7 9.1, J.sub.5,6 1.0 Hz, H-6); 4.10 (ddd, 1H, J.sub.4,5 4.1, J.sub.4,3a 3.7, J.sub.4,3b 2.6 Hz, H-4); 4.03 (ddd, 1H, J.sub.6,7 9.1, J.sub.7,8b 6.3, J.sub.7,8a 2.8 Hz, H-7); 3.82 (dd, 1H, J.sub.4,5 4.1, J.sub.5,6 1.0 Hz, H-5); 3.63 (dd, 1H, J.sub.8a,8b 13.1, J.sub.7,8a 2.8 Hz, H-8a); 3.47 (dd, 1H, J.sub.8a,8b 13.1, J.sub.7,8b 6.3 Hz, H-8b); 2.23 (dd, 1H, J.sub.3a,3b 15.0, J.sub.4,3a 3.7 Hz, H-3a); 1.84 (dd, 1H, J.sub.3a,3b 15.0, J.sub.4,3b 2.6 Hz, H-3b).

(138) .sup.13C-NMR (150 MHz, D.sub.2O) : 177.9 (C-1); 97.1 (C-2); 69.3 (C-7); 68.6 (C-6); 68.5 (C-4); 66.8 (C-5); 54.7 (C-8); 32.7 (C-3).

(139) Major Conformer Furanose Form (NMR 3.0):

(140) .sup.1H-NMR (600 MHz, D.sub.2O) : 4.61 (ddd, 1H, J.sub.4,3b 6.0, J.sub.4,3a 5.0, J.sub.4,5 4.8 Hz, H-4); 4.37 (dd, 1H, J.sub.4,5 4.8, J.sub.5,6 3.5 Hz, H-5); 3.91 (dd, 1H, J.sub.6,7 8.2, J.sub.5,6 3.5 Hz, H-6); 3.90 (ddd, 1H, J.sub.6,7 8.2, J.sub.7,8b 6.7, J.sub.7,8a 2.6 Hz, H-7); 3.59 (dd, 1H, J.sub.8a,8b 13.0, J.sub.7,8a 2.6 Hz, H-8a); 3.51 (dd, 1H, J.sub.8a,8b 13.0, J.sub.7,8b 6.7 Hz, H-8b); 2.40 (dd, 1H, J.sub.3a,3b 13.9, J.sub.4,3a 5.0 Hz, H-3a); 2.37 (dd, 1H, J.sub.3a,3b 13.9, J.sub.4,3b 6.0 Hz, H-3b).

(141) .sup.13C-NMR (150 MHz, D.sub.2O) : 178.4 (C-1); 103.8 (C-2); 82.0 (C-5); 72.7 (C-4); 71.8, 71.6 (C-6, C-7); 54.1 (C-8); 45.6 (C-3).

(142) Minor Conformer Pyranose Form (NMR 1.5):

(143) .sup.1H-NMR (600 MHz, D.sub.2O) : 4.01 (ddd, 1H, J.sub.4,5 4.2, J.sub.4,3a 4.0, J.sub.4,3b 3.1 Hz, H-4); 3.96 (ddd, 1H, J.sub.6,7 8.9, J.sub.7,8b 6.2, J.sub.7,8a 2.7 Hz, H-7); 3.91 (dd, 1H, J.sub.6,7 8.9, J.sub.5,6 1.2 Hz, H-6); 3.76 (dd, 1H, J.sub.4,5 4.2, J.sub.5,6 1.2 Hz, H-5); 3.62 (dd, 1H, J.sub.8a,8b 13.2, J.sub.7,8a 2.7 Hz, H-8a); 3.45 (dd, 1H, J.sub.8a,8b 13.2, J.sub.7,8b 6.2 Hz, H-8b); 2.16 (dd, 1H, J.sub.3a,3b 15.1, J.sub.4,3a 4.0 Hz, H-3a); 2.12 (dd, 1H, J.sub.3a,3b 15.1, J.sub.4,3b 3.1 Hz, H-3b).

(144) .sup.13C-NMR (150 MHz, D.sub.2O) : 176.7 (C-1); 96.8 (C-2); 69.4 (C-7); 70.9 (C-6); 68.5 (C-4); 66.9 (C-5); 54.8 (C-8); 34.2 (C-3).

(145) Minor Conformer Furanose Form (NMR 1.0):

(146) .sup.1H-NMR (600 MHz, D.sub.2O) : 4.58 (ddd, 1H, J.sub.4,3a 5.6, J.sub.4,5 4.1, J.sub.4,3b 1.7 Hz, H-4); 4.24 (dd, 1H, J.sub.5,6 4.5, J.sub.4,5 4.1 Hz, H-5); 3.97 (dd, 1H, J.sub.6,7 7.5, J.sub.5,6 4.5 Hz, H-6); 3.91 (ddd, 1H, J.sub.6,7 7.5, J.sub.7,8b 6.6, J.sub.7,8a 3.1 Hz, H-7); 3.60 (dd, 1H, J.sub.8a,8b 13.2, J.sub.7,8a 3.1 Hz, H-8a); 3.51 (dd, 1H, J.sub.8a,8b 13.2, J.sub.7,8b 6.6 Hz, H-8b); 2.55 (dd, 1H, J.sub.3a,3b 14.3, J.sub.4,3a 5.6 Hz, H-3a); 2.15 (dd, 1H, J.sub.3a,3b 14.3, J.sub.4,3b 1.7 Hz, H-3b).

(147) .sup.13C-NMR (150 MHz, D.sub.2O) : 178.3 (C-1); 104.3 (C-2); 83.7 (C-5); 73.0 (C-4); 71.9 (C-6); 70.9 (C-7); 54.5 (C-8); 45.6 (C-3).

Invention Example 13

Use of Synthesis Intermediates (8a) or (8b) to Detect Gram Negative Bacteria

Material and Methods

(148) 1) Bacterial Strains and Growth Conditions.

(149) Gram negative bacteria strains comprising Legionella pneumophila serogroup I, Legionella gormanii (Fluoribacter gormanii), Escherichia coli K12 and Escherichia coli O13 were grown in their specific culture medium. Legionella pneumophila serogroup 1 and Legionella gormanii (Fluoribacter gormanii) were grown in Yeast Extract medium supplemented with L-Cysteine, ferric pyrophosphate and -ketoglutarate (YEC). Escherichia coli K12 and Escherichia coli O13 were grown in 2YT medium (Yeast extract, tryptone and NaCl). All strains were grown in a rotary shaker (180 rpm) at 37 C.

(150) 2) Copper Catalyzed Click Chemistry Protocol.

(151) Overnight cultures were diluted 100 times in fresh liquid medium with 1% of DMSO (final volume 300 l) containing synthesis intermediates (8a) or (8b) (10 mM). Bacteria were incubated at 37 C. for 18 hours for E. coli strains and 24 hours for Legionella strains, and then aliquots of 200 L were washed 2 times with PBS/DMSO (95:5) buffer (200 L) and 1 time with PBS buffer (1X, 200 L) by centrifugation at 12000g for 1 min at room temperature.

(152) The pellet was re-suspended in 200 L of the click solution (CR110-CCH (0.10 mM), sodium ascorbate (5.0 mM), TGTA (4.0 mM) and copper sulfate pentahydrate (2.0 mM) in a mixture of PBS buffer and DMSO (99:1)) and transferred in a 2.0 mL microtube for a better agitation. This suspension was vigorously agitated in the dark for 30 min at room temperature, then the suspension was transferred back in a 1.5 mL microtube. Bacteria were washed 2 times with PBS/DMSO (95:5) buffer (200 L) and 1 time with PBS buffer (200 L, 12 000 rpm, 1 min, r.t.). The pellet was re-suspended in PBS buffer (200 L) and kept at 4 C. in the dark. Bacteria were further analyzed by confocal microscopy.

(153) ##STR00049##

Results

(154) Some of the Gram negative bacteria, including E. coli (K12 and O13), Legionella (pneumophila serogroup 1 and gormanii), were detected with synthesis intermediate compounds (8a) and (8b).

(155) In conclusion, synthesis intermediate compounds (8a) and (8b) result in a clear labelling signal on the 4 examples of studied bacteria Gram negative.

(156) ##STR00050##

(157) The synthesis starts from the commercially available D-arabinose and is providing an overall yield of 17 mol % for Kdo-N.sub.3 with a purity of more than 95%, an overall yield of 6 mol % for 4eKdo-N.sub.3 with a purity of more than 95% and an overall yield of 26 mol % for Ara-N.sub.3 with a purity of more than 95%.

(158) One skilled in the art will understand that various variations of the conditions of reaction of the invention can be made without departing from the core of the invention, including variations of the concentrations, nature of solvents, temperature, pressure, duration of reaction and stirring. Therefore, the invention covers all technical equivalents of the invention defined by the claims.

REFERENCES CITED

(159) [1] Gao, J., Hrter, R., Gordon, D. M., Whitesides, G. M., J. Org. Chem. 1994, 59, 3714-3715. [2] Gillingham, D. G., Stallforth, P., Adibekian, A., Seeberger, P. H., Hilvert, D., Nat. Chem. 2010, 2, 102-105. [3] Bird, J. W., Diaper, D. G. M., Can. J. Chem. 1969, 47, 145-150. [4] Lee, J. G., Kwak, K. H., Hwang, J. P., Tetrahedron Lett. 1990, 31, 6677-6680. [5] Chrest, M., Felkin, H., Prudent, N., Tetrahedron Lett. 1968, 18, 2199-2204; Anh, N. T., Top. Curr. Chem. 1980, 88, 145-162. [6] Dumont, A., Malleron, A., Awwad, M., Dukan, S., Vauzeilles, B., Angew. Chem. Int. Ed. 2012, 51, 3143-3146. [7] McNicholas, P. A., Batley, M., Redmond, J. W., Carbohydrate Research, 1987, 165, 17-22.