Method for labeling specifically living bacteria comprising the use of modified non endogenous monosaccharide compounds

Abstract

The invention relates to a method for labeling specifically living bacteria, comprising the steps of: a) incubating said bacteria of said sample with at least one modified monosaccharide compound comprising a first reactive chemical group capable to chemically react with a second reactive group, so that a residue bearing said first reactive group is incorporated into the envelope of such bacteria, and b) contacting said modified monosaccharide residue incorporated within the envelope of the bacteria with a labeling molecule comprising a said second reactive group, for generating the chemical reaction of said first reactive group with said second reactive group, characterized in that said modified monosaccharide compound has the following formula (I), or a salt thereof: Wherein —A, B and C can be independently H, OH, NH.sub.2, OH and NH.sub.2 being substituted or not by protecting groups thereof and —D is an alkyl chain in C.sub.2 to C.sub.4, each carbon being substituted or not by OH or NH.sub.2, OH and NH.sub.2 being substituted or not by protecting groups thereof and —at least one of A, B, C or D groups is substituted by a said first reactive group. ##STR00001##

Claims

1. A method for labeling specifically living bacteria in a sample comprising bacteria, wherein the method comprises the steps of: a) incubating said bacteria of said sample with at least one modified monosaccharide compound comprising a first reactive group Ra, which is capable to chemically react with a second reactive group, so that a residue bearing said first reactive group is incorporated into the envelope of said living bacteria, and b) contacting said residue incorporated into the envelope of said living bacteria with a labeling molecule comprising said second reactive group, for generating the chemical reaction of said first reactive group of said residue incorporated within said living bacteria with said second reactive group of said labeling molecule, resulting in a covalent link, wherein said residue is not an endogenous ulosonic acid residue of the envelope of said bacteria, said modified monosaccharide compound having the following formula (I), or a salt thereof: ##STR00016## wherein A is H, OH, or NH.sub.2, wherein OH and NH.sub.2 being substituted or not by a protecting group selected from the group consisting of alkyl, hydroxyalkyl, acyl, formyl and imidoyl groups, B is OH or NH.sub.2, wherein OH and NH.sub.2 being substituted or not by a protecting group selected from the group consisting of alkyl, hydroxyalkyl, acyl, formyl and imidoyl groups and C is OH or NH.sub.2, OH and NH.sub.2 being substituted or not by a protecting group selected in the group consisting of alkyl, hydroxyalkyl, acyl, formyl and imidoyl groups, and D is a C.sub.2 or C.sub.4 alkyl, each carbon being substituted or not by OH or NH.sub.2, wherein the OH and NH.sub.2 are each substituted or not by a protecting group selected from the group consisting of alkyl, hydroxyalkyl, acyl, formyl and imidoyl groups, and the D group is substituted by said first reactive group, wherein the first reactive group Ra is selected from the group consisting of -N.sub.3, a group bearing an azido, an alkyne, and a group bearing an alkyne, and said second reactive group is selected from the group consisting of -N.sub.3, a group bearing an azido, an alkyne, and a group bearing an alkyne, wherein said first reactive group is capable of reacting with said second reactive group via an azide alkyne cycloaddition.

2. The method according to claim 1, wherein said modified monosaccharide is a compound of formula (I) wherein D=-CHOH—CH.sub.2—OH, A=H, B=OH, C═OH, wherein the OH of B, C and D is substituted or not by a protecting group and at least one OH of D group is substituted by said first reactive group Ra.

3. The method according to claim 1, wherein said modified monosaccharide compound is a compound of formula (I) wherein D=-CHOH—CH.sub.2—Ra, A=H, B=OH, C═OH, wherein the OH of B, C and D is substituted or not by a protecting group.

4. The method according to claim 1, wherein Ra is —N.sub.3 or —C≡CH.

5. The method according to claim 1, wherein said modified monosaccharide compound is a compound having the following stereoisomer formula (Ia), or a salt thereof: ##STR00017##

6. The method according to claim 1, wherein said bacteria are Gram positive bacteria.

7. The method according to claim 1, for labeling one or more species bacteria selected from the group consisting of Bacillus cereus, Bacillus cereus paris, Enterococcus durans, Enterococcus faecalis, Escherichia coli, Klebsiella pneumonia, Legionella pneumophila, Listeria monocytogenes, Micrococcus luteus, Neisseria gonorrhoeae, Neisseria meningitides, Proteus mirabilis, Providencia stuartii, Pseudomonas fluorescens Migula, Salmonella typhimurium, Serratia marcescens, Staphylococcus aureus, Staphylococcus aureus aureus, Staphylococcus epidermis, Staphylococcus saprophyticus, Stenotrophomonas maltophilia, Streptococcus agalactiae and Vibrio cholerae.

8. The method according to claim 1, wherein said method comprises the further step of: c) detecting living bacteria which comprises detecting whether said bacteria comprise said labeling molecule bound to their envelope and/or immobilizing said living bacteria bearing said labeling molecule onto a solid substrate, wherein said labeling molecule is a molecule comprising a detectable substance or capable to react or to be bound to a detectable substance or said labeling molecule is a first molecule bearing said second reactive group, said first molecule being capable to react or to be bound to a second molecule and/or to a solid substrate, said second molecule comprising a detectable substance and/or said second molecule being bound or capable to be bound to said solid substrate.

9. The method according to claim 8 for specifically detecting living bacteria of said bacteria in a sample comprising bacteria, wherein said labeling molecule is a detectable molecule comprising a detectable substance, wherein in the step c), detecting living bacteria comprises detecting whether said bacteria comprise said detectable molecule bound to the glycans of their outer membrane.

10. The method according to claim 8, wherein said labeling molecule is a first ligand or first binding protein bearing said second reactive group and in step c) said living bacteria coupled to said first ligand or first binding protein is detected and/or immobilized by contacting said first ligand or first binding protein with a second ligand or second binding protein reacting or binding specifically to said first ligand or first binding protein.

11. The method according to claim 8, wherein said labeling molecule is a first ligand, bearing said second reactive group, and in step c) said living bacteria coupled to said first ligand are detected by reaction of said bacteria with an antibody specific to said first ligand, said antibody bearing a detectable substance.

12. A kit for carrying out the method of claim 1, which comprises: said modified monosaccharide compound of formula (I), and said labeling molecule comprising said second reactive group capable of reacting with said first reactive group, and optionally reactants for generating the reaction of said first reactive group with said second reactive group of said labeling molecule.

13. The kit according to claim 12, which further comprises: a detectable molecule or second molecule bearing a detectable substance comprising a fluorochrome or luminescent molecule or an enzyme, and/or a solid substrate bearing said second molecule capable of specifically reacting or binding with said labeling molecule, and optionally a culture or incubation medium allowing the growth of said bacteria.

14. The method according to claim 1, wherein said residue is a not naturally occurring stereoisomer of an endogenous ulosonic acid residue of the envelope of said bacteria.

15. The method according to claim 1, wherein the protecting group is an alkyl, hydroxyalkyl, acyl, or formyl group.

16. The method according to claim 2, wherein the protecting group is an alkyl, hydroxyalkyl, acyl, or formyl group.

17. The method according to claim 1, wherein the first reactive group is —N.sub.3 or a group bearing an azido, and the second reactive group is an alkyne or a group bearing an alkyne.

18. The method according to claim 11, wherein the detectable substance is a fluorochrome or luminescent molecule or an enzyme.

19. The method according to claim 11, wherein the first ligand is biotin.

20. The method according to claim 1, for labeling one or more bacteria selected from the group consisting of Neisseria gonorrhoeae, Enterococcus faecalis, Listeria monocytogenes, Micrococcus luteus, Staphylococcus aureus, Staphylococcus aureus aureus, and Staphylococcus epidermis.

Description

EXAMPLE 1: SYNTHESIS OF COMPOUND (IA): 4EKDO-N.SUB.3 .(6)

(1) In the synthesis the following reagents and conditions have been used: (i) TsCI, pyridine. (ii) pyridine, Ac.sub.20. (iii) sodium azide. DMF. (iv) CH.sub.3ONa, CH.sub.3OH. (v) oxaloacetic acid, NaOH, H.sub.20. (vi) Dowex® 50 (H.sup.+). (viii) NH.sub.4OH.

(2) Thin layer chromatography was performed over Merck 60 F.sub.254 with detection by UV, and/or by charring with sulphuric acid or KMn0.sub.4 or phosphomolybdic acid solutions. Silica gel 60 40-63 μm was used for flash column chromatography.

(3) NMR spectra were taken on Bruker Avance 300 or 500 MHz spectrometers, using the residual protonated solvent as internal standard. Chemical shifts δ 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).

(4) Mass spectra were taken on a Thermo Scientific TSQ or on a Bruker micrOTOFq or on a Waters LCT Premier XE (ToF), with electrospray ionization in the positive (ESI+) mode of detection.

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

(6) Compound (Ia or 6) was prepared as follows with a final purification of epimer (Ia or 6) from the mixture (Ia or 6)/(I-1 or 6′).

(7) The following scheme 1 shows the various compounds involved in the steps of the synthesis.

(8) ##STR00011##

(9) The reagents and conditions in the various steps are: (i) TsCl (1.1 eq.), pyridine (1.0 M), 100° C.->r. t., 18 h. (ii) pyridine/Ac.sub.2O (2:1, 0.7 M), 18 h. (iii) NaN.sub.3 (2.0 eq.), DMF (0.4 M), 80° C. 20 h, 15% over 3 steps, (iv) CH.sub.3ONa (0.1 eq.). CH.sub.3OH (0.2 M), r. t., 3 h, 99%. (v) Oxaloacetic acid (1.6 eq.), NaOH (pH 11), H.sub.2O (0.5 M), r. t., 2 h. (vi) Dowex® 50 (H.sup.+). (vii) 80° C., 20 min. (viii) NH.sub.4OH. 1% of isolated 4eKdo-N.sub.3 over 4 steps.

1) Preparation of 5-azido-5-deoxy-1,2,3-tri-O-acetyl-D-arabinofuranose (4)

(10) ##STR00012##

(11) Commercial D-arabinose (1) (6.00 g, 40 mmol) was heated at 100° C. for 2 hours in pyridine (40 mL). The solution was allowed to cool down, further treated with tosyl chloride (8.38 g, 44 mmol, 1.1 equiv.), and stirred for 16 hours at room temperature ((2), not isolated). Acetic anhydride (20 mL) was then added. After complete acetylation, as determined by TLC, solvents were evaporated, and residual traces were co-evaporated several times with toluene ((3), not isolated). The residue was dissolved in DMF (100 mL), sodium azide (5.20 g, 80 mmol, 2.0 eq.) was added, and the suspension was heated at 80° C. for 20 hours. After dilution with ethyl acetate and washing with water, the organic layer was dried over anhydrous magnesium sulfate and concentrated. The residue was purified by flash chromatography (petroleum ether/ethyl acetate 7:3). The first eluted product was determined to be the expected 5-azido-1,2,3-tri-O-acetyl-D-arabinofuranose (4) (1.83 g, 15%, α/β˜2:1).

(12) .sup.1H-NMR (360 MHz, CDCI.sub.3) δ (ppm): 6.41 (d, 0.33H, J.sub.1,2 3.5 Hz, H-1β); 6.23 (d, 0.67H, J.sub.1,2˜1 Hz, H-1α); 5.40-5.37 (m, 1.34H, H-2β, H-3β); 5.23 (d, 0.67H, J.sub.1,2˜1 Hz, H-2α); 5.06 (d, 0.67H, J.sub.3,4 4.6 Hz, H-3α); 4.30 (ddd, 0.67H, H-4α); 4.16-4.10 (m, 0.33H, H-4β): 3.69 (dd, 0.67H, J.sub.5a,5b 13.5, J.sub.4,5a 3.1 Hz, H-5aα); 3.61 (dd, 0.33H, J.sub.5A,f5b13.1, J.sub.4,5a 3.6 Hz. H-5aβ); 3.51-3.43 (m, 1H, H-5bα, H-5bβ); 2.15, 2.13, 2.12, 2.11, 2.11, 2.09 (6s, 18H, 6 CH.sub.3CO).

(13) .sup.13C-NMR (62.5 MHz, CDCI.sub.3) δ (ppm): 170.3, 170.0, 169.1 (3 C-0); 99.2 (C-1α); 93.5 (C-1β); 84.1 (C-4α); 80.8 (C-4β); 80.6 (C-3α); 77.4 (C-2α); 75.1 (C-2β); 74.8 (C-3β); 53.0 (C-5β); 51.3 (C-5α): 20.9, 20.6, 20.3 (3 CH.sub.3).

(14) LRMS (ESI+): [M+H].sup.+ 324.0.

(15) HRMS (ESI+): [M+H].sup.+ (C.sub.11H.sub.15N.sub.3NaO.sub.7) Calc. m/z: 324.0802, found: 324.0802.

2) Preparation of 5-azido-5-deoxy-D-arabinofuranose (5)

(16) ##STR00013##

(17) Protected 5-azido-1,2,3-tri-O-acetyl-D-arabinose (4) was then dissolved into anhydrous methanol (30 mL), treated with a methanolic solution of CH.sub.3ONa (0.2 mol.Math.L.sup.−1, 3 mL) and stirred at room temperature for 3 hours under an argon atmosphere. After neutralization (Dowex® 50 (H.sup.+)) filtration, and concentration, 5-azido-5-deoxy-D-arabinofuranose (5) was obtained in 99% yield (1.03 g).

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

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

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

(21) Anomer alpha (5α):

(22) .sup.1H-NMR (500 MHz, D.sub.2O) δ (ppm): 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).

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

(24) Anomer beta (5β):

(25) .sup.1H-NMR (500 MHz, D.sub.2O) δ (ppm): 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).

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

3) Preparation of Ammonium 8-azido-3,8-dideoxy-D-gluco-octulosonate (4eKdo-N.SUB.3.) (6)

(27) ##STR00014##

(28) A cool (4° C.) solution of 5-azido-5-deoxy-D-arabinofuranose (5) (437 mg, 2.5 mmol) in water (2.1 mL) was added to a solution of oxaloacetic acid (528 mg, 4.0 mmol) in water (2.5 mL), the pH of which has been adjusted to ˜11 by addition of aqueous NaOH (10M). After being stirred for two hours at room temperature, the solution was neutralized (Dowex® 50 (H.sup.+)), filtrated, and heated 20 min at 80° C. After its pH had been adjusted to ˜7 with AcOH (0.5M), the resulting Kdo-N.sub.3/4eKdo-N.sub.3 mixture was purified by anion exchange chromatography (Dowex® 1X8 (HC0.sub.2.sup.−″)). Initial elution with water gave unreacted (5) (150 mg, 34%). Further elution with a concentration gradient of formic acid (0.5 mol.Math.L.sup.−1 to 2.0 mol.Math.L.sup.−1), freeze-drying, treatment with a Dowex® 50 (H) resin, and neutralization by ammonia (0.2 mol.Math.L.sup.−1), gave after concentration, a mixture of Kdo-N. and 4eKdo-N.sub.3. This mixture of (6) (1a) and (6′) (I-1) was again purified by anion exchange chromatography (Dowex® 1X8 (HC0.sub.2.sup.−)) by elution with a slow concentration gradient of formic acid (1.5 mol.Math.L.sup.−1 to 2.0 mol.Math.L.sup.−1), freeze-drying, treatment with a Dowex® 50 (H.sup.+) resin, and neutralization by ammonia (0.2 mol.Math.L.sup.−1), gave after concentration, the ammonium of the specific minor epimer 8-azido-3,8-dideoxy-D-gluco-octulosonate (6 or Ia), 7 mg.

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

(30) Rf (isopropyl alcohol/water 9:1): 0.38.

(31) IR (cm.sup.−1): 3296, 2931, 2104 (N.sub.3), 1612, 1384, 1283, 1072, 805.

(32) LRMS (ESI): 262.1.

(33) HRMS (ESI): [M−H]″ (C.sub.8H.sub.12N.sub.30.sub.7) Calc. m/z: 262.0681, found: 262.0669.

(34) Major conformer pyranose form (NMR 7.5):

(35) .sup.1H-NMR (600 MHz, D.sub.2O) δ: 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.7,8a 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).

(36) .sup.13C-NMR (150 MHz, D.sub.2O) δ: 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).

(37) Major conformer furanose form (NMR 3.0):

(38) .sup.1H-NMR (600 MHz, D.sub.2O) δ: 4.61 (ddd, 1H, J.sub.4,5 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.7,8b 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).

(39) .sup.13C-NMR (150 MHz, D.sub.2O) δ: 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).

(40) Minor conformer pyranose form (NMR 1.5):

(41) .sup.1H-NMR (600 MHz, D.sub.2O) δ: 4.01 (ddd, 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.7,8α 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).

(42) .sup.13C-NMR (150 MHz, D.sub.2O) δ: 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).

(43) Minor conformer furanose form (NMR 1.0):

(44) .sup.1H-NMR (600 MHz, D.sub.2O) δ: 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).

(45) .sup.13C-NMR (150 MHz, D.sub.2O) δ: 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).

EXAMPLE 2

(46) Comparison of labeling of living bacteria with compounds 4eKdo-N.sub.3(Ia) of the present invention and compound Kdo-N.sub.3 (I-1). 1) Material and Methods.

(47) 1.1) Bacterial Strains and Growth Conditions.

(48) The 28 bacterial strains listed in Table 1 are grown in the culture media and conditions listed in tables 1 and 2. All strains were grown in a rotary shaker (160 rpm) at 30 or 37° C.

(49) TABLE-US-00001 TABLE 1 microorganisms Growth BACTERIA REFERENCES conditions Bacillus cereus Laboratory strain TSB-24 H-37° C. (FIEROBE team LCB/CNRS) Bacillus cereus CIP 66.24T TSB-24 H-37° C. Bacillus cereus paris Laboratory strain TSB-24 H-30° C. (HENRY team/CNRS) Enterococcus durans Laboratory strain TSB-24 H-37° C. (FIEROBE team LCB/CNRS) Enterococcus faecalis Laboratory strain TSB-24 H-37° C. (FIEROBE team LCB/CNRS) Escherichia coli K12 MG1655 LB-24 H-37° C. Escherichia coli O86 Laboratory strain LB-24 H-37° C. (DENAMUR team/INSERM) Stenotrophomonas Clinical strain TSB-24 H-37° C. maltophilia (LASCOLA/Marseille, La Timone) Streptococcus Clinical strain TSB-24 H-37° C. agalactiae (LASCOLA/Marseille, La Timone) Vibrio cholerae CIP 104151 TSB-24 H-37° C. Klebsiella pnuemoniae CIP 101114 TSB + 5% sheep blood-24 H-30° C. Klebsiella pnuemoniae Clinical strain (LASCOLA/ TSB-24 H-37° C. Marsellie, La Timone) Legionella LG 0846 3022 (CNRL, YEC + sup. pneumophila sg6 environmental strain) Legionella 10%- 24 H-37° C. Listerla monocytogenes CIP 82.110T BHI-24 H-37° C. 1/2 Listerla monocytogenes CIP 100607 BHI-24 H-37° C. 1/2a Micrococcus luteus Laboratory strain TSB-24 H-30° C. (FIEROBE team LCB/CNRS) Neisseria gonorrhoeae CIP 79.16T TSB + 10% Horse blood-24 H-37° C. Neisseria meningitidis CIP 107858 TSB + 10% Horse blood-24 H-37° C. Proteus mirabilis Clinical strain (LASCOLA/ TSB-24 H-37° C. Marseille, La Timone) Providencia stuartii Clinical strain (LASCOLA/ TSB-24 H-37° C. Marseille, La Timone) Pseudomonas ATOC 4927 TSB-24 H-37° C. fluorescens Migula Pseudomonas Laboratory strain (HENRY TSB-24 H-37° C. fluorescens Paris team/CNRS) Salmonella Laboratory strain (BARRAS TSB-24 H-37° C. typhimurium 12023 team/CNRS) Serratia marcescens CIP102446 TSB-24 H-30° C. Serratia marcescens Clinical strain (LASCOLA/ TSB-24 H-37° C. Marseille, La Timone) Staphyloccus aureus Clinical strain (LASCOLA/ TSB-24 H-37° C. Marseille, La Timone) Staphyloccus aureus CIP 53-156 TSB-24 H-37° C. aureus Staphyloccus epidermis Laboratory strain TSB-24 H-37° C. (FIEROBE team LCB/CNRS) Staphylococcus Clinical strain (LASCOLA/ TSB-24 H-37° C. saprophyticus Marseille, La Timone) Stenotrophomonas Clinical strain (LASCOLA/ TSB-24 H-37° C. maltophilia Marseille, La Timone) Streptococcus Clinical strain (LASCOLA/ TSB-24 H-37° C. agalactiae Marseille, La Timone) Vibrio cholerae CIP 104151 TSB-24 H-37° C.

(50) TABLE-US-00002 TABLE 2 Composition Provider Reference Tryptic casein peptone (pancreatic) 17 g/l + Sigma  22092 Soy Soya peptone (papain digest.) 3 g/l + Aldrich Broth Sodium chloride 5 g/l + Dipotassium (USA) (TSB) hydrogen phosphate 2.5 g/l + Glucose 2.5 g/l pH 7.3 Brain brain infusion solids 12.5 g/l + beef Oxoid CM1135 Heart heart infusion solids 5 g/l + proteose (GB) Infusion peptone 10 g/l + glucose 2 g/l + (BHI) sodium chloride 5 g/l + di sodium phosphate 2.5 g/l YEC yeast extract 10 g/l + Casamino acid Becton Bacto Dickinson 212720 (USA) Luria Bactotryptone 10 g/l+ Becton 211699 Bertani Dickinson (LB) (USA) Bacto yeast extract 5 g/l+ Becton 212720 Dickinson (USA) Sodium chloride+ Sigma  55886 Aldrich (USA) pastagar 15 g/l Biorad  64946 (USA) pH 7.2

(51) 1.2) Copper Catalyzed Click Chemistry

(52) Overnight cultures were diluted 100 times in fresh medium (final volume 100 μI) containing Kdo-N.sub.3 (1-1) or 4eKdo-N.sub.3 (Ia) (10 mM). Bacteria were incubated at 30 or 37° C. for 24 hours and then washed 3 times with phosphate buffer (0.05 M, pH 7.5) by centrifugation at 13.000×g for 2 min at room temperature.

(53) Two fluorochrome-alkyne probes of following formula A488-yne (7a) and A594-yne (7b) were used:

(54) ##STR00015##

(55) CuSO.sub.4 and TGTA, at a final concentration of 2 mM and 4 mM respectively, were mixed overnight in phosphate buffer (0.05 M, pH 7.5) at 37° C. under vigorous shaking. Next, aminoguanidine, sodium ascorbate and fluorochrome-alkyne (7a) or (7b) at a final concentration of 4 mM, 5 mM and 1 mM respectively were added to CuSO.sub.4/TGTA overnight mix. Finally, bacteria were resuspended in this solution and incubated for 30 minutes at 37° C. Cells were then washed 3 times with phosphate buffer by centrifugation at 13,000×g for 2 min at room temperature and analyzed by microscopy.

(56) 1.3) Fluorescence Microscopy.

(57) Bacteria were inoculated onto glass cover slips and covered with a thin (1 mm of thickness) semisolid 1% agar pad made with dilute LB (1/10 in phosphate buffer (0.05 M, pH 7.5)). Images were recorded with epifluorescence automated microscope (Nikon TE2000-E-PFS, Nikon, France) equipped with a CoolSNAP HQ 2 camera (Roper Scientific, Roper Scientific SARL, France) and a 100×/1.4 DLL objective. Excitation light was emitted by a 120 W metal halide light and signal was monitored using appropriate filters. Digital analysis and image processing were conducted by a custom automation script (Visual Basic) under Metamorph 7.5 (Molecular Devices, Molecular Devices France, France).

(58) 2) Results

(59) 21.) 28 different strains of bacteria have been tested with both compounds 4eKdo-N.sub.3 (Ia) and Kdo-N.sub.3 (I-1) in comparison.

(60) These strains were grown first in the presence of compound (Ia) or (I-1) and the incorporation of the azido chemical reporter into the bacteria was monitored in a subsequent step, using copper-catalysed azide-alkyne cycloaddition in the conditions previously described, with copper sulfate, sodium ascorbate, TGTA, a water-soluble tris (triazolyl) ligand for copper (I), and a fluorochrome-alkyne probe of above formula (7a) or (7b), for 30 minutes as above disclosed.

(61) In these experiments, strains showing highly distinctive fluorescence on their membrane, indicative of an effective metabolic incorporation of the chemical reporter have been marked “+” in the table 3 below, the absence of labeling has been marked “−” in table 3, and the not tested bacteria have been marked “NT” in table 3.

(62) Table 3:

(63) TABLE-US-00003 TABLE 3 Strain Kdo-N.sub.3 4eKdo-N.sub.3 Bacillus cereus NT + Bacillus cereus NT + Bacillus cereus paris NT + Enterococcus durans NT + Enterococcus faecalis − + Escherichia coli K12 + + Escherichia coli O86 + + Klebsiella pneumoniae + + Klebsiella pneumoniae + + Legionella pneumophila sg6 + + Listeria monocytogenes ½ − + Listeria monocytogenes ½a − + Micrococcus luteus − + Neisseria gonorrhoeae − + Neisseria meningitidis + + Proteus mirabilis + + Providenda stuartii + + Pseudomonas fluorescens Migula + + Salmonella Typhimurium 12023 + + Serratia marcescens + + Serratia marcescens + + Staphylococcus aureus − + Staphylococcus aureus aureus − + Staphylococcus epidermis − + Staphylococcus saprophyticus NT + Stenotrophomonas maltophilia + + Streptococcus agalactiae NT + Vibrio cholerae + +

(64) These experiments shows that compound (Ia) of the present invention is assimilated by a broad range of bacteria and interestingly, compound (Ia) is assimilated by the following Gram positive bacteria which did not assimilate compound (I-1): Enterococcus faecalis. Listeria monocytogenes, Micrococcus luteus, Staphylococcus aureus, Staphylococcus aureus aureus and Staphylococcus epidermis as well as the Gram negative Neisseria gonorrhoeae.