Methods for Chemical Synthesis of Biologically Active Compounds Using Supramolecular Protective Groups and Novel Compounds Obtainable Thereby

Abstract

The invention relates to drug development and synthetic chemistry, in particular to the manufacture of biologically active compounds based on naturally occurring molecules. It also relates to novel biologically active compounds, for example aminoglycoside antibiotics, in a substantially pure regioisomeric form. More particularly, the present invention relates to methods for the chemo- or regioselective derivatization of a target compound with multiple reactive groups, some of which may be derivatezed, and other which will not be derivatized.

Claims

1. A method for the chemo- and/or regio selective derivatization of a target compound comprising multiple chemically equivalent reactive groups, wherein at least one reactive group is to be derivatized and wherein at least one reactive group is not to be derivatized, the method comprising the steps of a. contacting the target compound with at least one non-covalent protective group under conditions allowing for the formation of a regioselective host-guest complex, wherein the protective group is an oligonucleotide or oligopeptide aptamer having a selective affinity for the at least one reactive group not to be modified; followed and/or accompanied by b. chemical derivatization of the target compound.

2. Method according to claim 1, wherein the target compound comprises at least three chemically equivalent reactive groups.

3. Method according to claim 1 or 2, wherein the chemically equivalent reactive groups are amines, hydroxyls, hydroxylamines, carboxylic acids, thiols, aldehydes, ketones, enamines, CC double bonds or CC triple bonds.

4. Method according to any one of the preceding claims, wherein the target compound is a biologically active compound.

5. Method according to any one of the preceding claims, wherein the target compound is a proteinaceous substance.

6. Method according to any one of claims 1 to 4, wherein the target compound is a saccharide or derivative thereof.

7. Method according to claim 6, wherein the target compound is a monosaccharide, oligosaccharide, polysaccharides, or derivative thereof.

8. Method according to claim 7, wherein the derivative is a glycoside, preferably an O-glycoside, N-glycoside, S-glycoside, C-glycoside or halogen-glycoside.

9. Method according to claim 8, wherein the target compound is an aminoglycoside antibiotic, preferably an aminoglycoside based on a neamine scaffold.

10. Method according to claim 9, wherein the aminoglycoside antibiotic is selected from the group consisting of neomycin, paromomycin, ribostamycin, kanamycin and streptomycin.

11. Method according to any one of the preceding claims, wherein the oligonucleotide aptamer consists of from 8 to about 60 nucleotides, preferably 15-40.

12. Method according to any one of the preceding claims, wherein the protective group is an RNA or DNA aptamer.

13. Method according to claim 11, wherein the RNA or DNA aptamer is obtained by a screening process comprises the steps of: (1) constructing a random single-stranded DNA (ssDNA) library and preparing a primer; (2) preparing the random single-stranded DNA (ssDNA) library by PCR amplification (for DNA SELEX) or an RNA library by transcription (for RNA SELEX); (3) carrying out multiple rounds of SELEX screening; (4) detecting the appetency; (5) cloning and sequencing DNA.

14. Method according to claim 12, wherein the RNA aptamer is 5-GGA CUG GOC GAG AAG UUU AGU CC-3, 5-CUG CAG UCC GAA AAG GGC CAG-3, 5-UGU GUA GGG CGA AAA GUU UUA-3 or 5-GGC ACG AGG UUU AGC UAC ACU CGU GCC-3.

15. Method according to any one of claims 1-11, wherein the protective group is an oligopeptide aptamer, preferably wherein the oligopeptide aptamer consists of from 8-18 amino acids, preferably 10-13 amino acids.

16. Method according to claim 15, wherein the oligopeptide aptamer is obtained by expressing a library of candidate oligopeptide aptamers in a recombinant host cell by infection with phages, selecting at least one host cell expressing an oligopeptide aptamer, and identifying the oligopeptide aptamer.

17. Method according to claim 15, wherein oligopeptide aptamer is selected from the group consisting of VNRSSDHWNLTT, DYDTLRTVAPTR, NGSLQRSFVISH, HVRIYVDTIEIR, GAMHLPWHMGTL and GAMIIPPRIIMGPL.

18. Method according to any one of the preceding claims, wherein the chemical derivatization comprises acylation, alkylation, oxidation, PEGylation, reductive amination, aza-Michael reaction or urea bond formation.

19. Method according to any one of the preceding claims, wherein the host-guest complex is formed while the protective group is in solution.

20. Method according to any one of the preceding claims, wherein the host-guest complex is formed while the protective group is immobilized.

21. A derivatized target compound of interest obtainable according to any one of claims 1-20.

22. A derivatized aminoglycoside antibiotic, preferably a neomycin or paromomycin derivative, characterized in that the aminoglycoside is derivatized only on the N.sup.6 (IV)-, N.sup.2 (IV)- or N.sup.2 (IV), NG (IV)-amine group(s).

23. An aminoglycoside derivative according to claim 22, having the general formula ##STR00016##

24. An aminoglycoside derivative according to claim 23, having the general formula ##STR00017##

25. An aminoglycoside derivative according to claim 22, selected from the group consisting of N.sup.6(IV) acetyl neomycin B (Formula 1), N.sup.6(IV) dimethylacetyl neomycin B (Formula 2), N.sup.6(IV) pent-3-inoyl neomycin B (Formula 3), N.sup.2(IV) {[(phenyl)amino]carbonyl}amino neomycin B (Formula 4), N.sup.2(IV) {[(4-methoxyphenyl)amino]carbonyl}amino neomycin B (Formula 5), N.sup.2(1V) {[(4-methoxyphenyl)amino]carbonyl}amino paromomycin (Formula 6) and N.sup.2(IV), N.sup.6 (IV)-bis{[(allylamino)carbonyl]amino} neomycin B (Formula 7), N.sup.6(IV) acetyl paromomycin, N.sup.6(IV)--sulfhydryl-propionyl neomycin B, N.sup.6-(IV)-azido neomycin B, N.sup.6-(IV), N.sup.2-(IV)-diazido neomycin B, N.sup.2(IV)-(propylamino)carbonyl neomycin B, N.sup.2(IV)-(isopropylamino)carbonyl neomycin B, N.sup.2(IV)-(tert-butylamino)carbonyl neomycin B, N.sup.2(IV), N.sup.6(IV)-bis-N-(propylamino)carbonyl neomycin B, N.sup.2(IV), N.sup.6(IV)-bis-N-(isopropylamino)carbonyl neomycin B, N.sup.2(TV), N.sup.6(IV)-bis-N-(tert-butylamino)carbonyl neomycin B, N.sup.6(II)- -sulfhydryl butanoyl neomycin B and pharmaceutically acceptable salts thereof.

26. A derivatized aminoglycoside antibiotic, preferably a kanamycin derivative, characterized in that the aminoglycoside is derivatized only on the N.sup.6 (II) amine group.

27. An aminoglycoside derivative according to claim 26, selected from the group consisting of N.sup.6(II)-acetyl kanamycin A or B, N.sup.6(II)-2-methylpropionyl kanamycin A or B, N.sup.6(II)-2-butynyl kanamycin A or B or N.sup.6(II)- -sulfhydryl butanoyl kanamycin A or B, and pharmaceutically acceptable salts thereof.

28. A pharmaceutical composition comprising an aminoglycoside antibiotic derivative according to any one of claims 22 to 27.

29. An aminoglycoside antibiotic derivative according to any one of claims 22 to 27 for use as a medicament.

30. An aminoglycoside antibiotic derivative according any one of claims 22 to 27 for use in a method of treatment of a bacterial infection, preferably an infection with Methicillin-resistant Staphylococcus aureus (MRSA) or vancomycin-resistant enterococci (VRE).

31. Use of compound according to any one of claims 22 to 27 as active biocide.

32. Use of an oligonucleotide or oligopeptide aptamer as a chemo-, regio- and/or stereoselective protective group.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0058] FIG. 1A shows neomycin B RNA aptainer complex; FIG. 1B shows a model of neomycin B tRNA interaction based on the basis of the crystallographic coordinates from Mikkelsen27; FIG. 1C)shows a sequence of RNA aptamers apt1 and apt2; FIG. 1D shows the structure of representative aminoglycoside antibiotics. The arrow indicates the free amine group of neomycin B for derivatization; The arrow indicates ring IV, which is directed outwards towards the solvents in 10 mM phosphate buffer. FIG. 1E shows a concept of chemo- and regioselective modification of a complex molecule applying APG technology. (1) non-covalent protection of target antibiotic with APG; (2) selective modification of unprotected functional group(s); (3) deprotection of the new derivative.

[0059] An ESI-Mass spectra of reaction mixture on neoB 1 with activated ester 4a in presence of RNA can be seen in the next group of drawings. FIG. 2A shows the spectra in the presence of RNA, and FIG. 2B shows the spectra without RNA. FIG. 2C shows 1H-NMR (500 MHz, D20) of monoacetylated neomycin B in presence of the aptamer apt1 and FIG. 2D shows it without apt1. FIGS. 2A and 2B show S=neomycin B, I-VI=degree of acetylation of neoB. FIG. 3 shows regioselective transformation of N6(IV) amine group of aminoglycosides.

[0060] FIG. 4A shows 11-1-NMR spectrum (500 MHz, D20) of neomycin B6 HFBA 1.

[0061] FIG. 4B shows HSQC spectrum (500 MHz, D20) of neomycin B6 HFBA 1.

[0062] FIG. 5A shows 41-NMR spectrum (500 MHz, 1)20) of N.sup.6(1V)-acetyl neomycin B5 HFBA 5a. FIG. 5B shows HSQC spectrum (500 MHz, D20) of N.sup.6(IV)-acetyl neomycin B5 HFBA 5a.

[0063] FIG. 6Ashows 11-NMR (500 MHz, D20) spectrum of N.sup.6(IV)-2,2-dimethylacetyl neomycin B5 HFBA G. FIG. 6 (Panel B) shows HSQC (500 MHz, D20) spectrum of N.sup.6(IV)-2,2-dimethylacetyl neomycin B5 HFBA 6.

[0064] FIG. 7A shows 11-NMR (500 MHz, D20) spectrum of N.sup.6(IV)-prop-3-inoyl neomycin B5 HFBA 7. FIG. 7B shows HSQC (500 MHz, D20) spectrum of N.sup.6(IV)-prop-3-inoyl neomycin B5 HFBA 7.

[0065] FIG. 8 shows HSQC (500 MHz, D20) spectrum of N.sup.6(IV)-acetyl paromomycin4 HFBA 8.

[0066] FIG. 9 shows Attached Proton Tests (APT) of monoacetylated neomycin B after transformation in presence of RNA (Panel A) and in absence of RNA (Panel B). APT of neomycin B (Panel C). It proves the regioselective transformation of the N.sup.6(IV) amine group of the neomycin B to 5a, while the spectrum in panel B shows the presence of two monoacetylated neomycin B derivatives, the N(IV).sup.6 acetyl neomycin B 5a and the N(II)6 acetyl neomycin B 5b.

[0067] FIG. 10 schematic representation of the use of phage display technology to select oligopeptide aptamers. Immobilization of a target compound (e.g. antibiotic) to a solid support is directed in such a manner that the zone which is accessible to the phage comprises the reactive group(s) to be protected against derivatization, whereas the reactive group(s) to be derivatized is/are not accessible to the phages. See also Example 2.

[0068] FIG. 11 shows schematic illustration of the regio selective modification of the neamine antibiotic Neomycin B using immobilized aptameric protective groups (APGs) on solid support (agarose): 1. Complexation of antibiotic with immobilized APG; 2. Acylation using N-hydroxysuccinimide ester; 3. Elution of modified antibiotic.

EXPERIMENTAL SECTION

Example 1

[0069] This Example demonstrates the power of the novel SPG strategy for the highly regioselective modification of aminoglycoside antibiotics to obtain new and biologically active saccharide derivatives within a single reaction step. It is proven for the first time that non-covalent protective groups can be generated with the help of a well established in vitro evolution process and do not require the tailored design and synthesis of ligands to protect individual target molecules for highly selective chemical transformations.

[0070] For the straightforward proof of concept that RNA can act as a SPG, a well characterized host-guest complex of an 23mer RNA aptamer (sequence: 5-GGA CUG GGC GAG AAG UUU AGU CC-3, apt1) and neoB was chosen. X-ray crystallography and nuclear magnetic resonance (NMR) measurements revealed that one of the six amine groups, i.e., the 6-amine group of ring IV, is not involved in complex formation and extends into the solvent (FIG. 1A).sup.29. Reaction of the N-acetoxy succinimide ester (10 equivalents) with the apt1 protected neoB resulted only in the formation of mono-and diacetylated neoB molecules according to mass spectrometric analysis (FIG. 2A).

[0071] In stark contrast, the unprotected transformation of neoB under the same conditions yielded di-, tri-, tetra-, penta and hexaacetylated products of the antibiotic (FIG. 2B). These experiments clearly indicated the ability of apt1 to inhibit the reactivity of several amine groups present within neoB. To obtain further insights into the regioselectivity of the transformations at the neoB scaffold reaction conditions were optimized to yield monoacylated neoB for the protected (FIG. 3 and table 1) and unprotected transformations. RNA-neoB complexes were reacted with 30 equivalents of the activated ester 4a while the unprotected neoB was incubated with only one equivalent of the succinimidyl ester. After purification by high performance liquid chromatography (HPLC) the monoacyl-neoB products were characterized and regioselectivities were determined by 11-1-NMR spectroscopy. It turned out that the RNA-protected neoB was acylated at the 6-amine group of ring IV with an extremely high regioselectivity of 95% to form 5a (FIG. 2C) while the unprotected neoB was converted to N.sup.6(IV)-acetyl neoB 5a and the N.sup.6(11)-acetyl neoB 5b in a ratio of 9:11 (FIG. 2D). These results were confirmed by Heteronuclear Single Quantum Coherence spectra (FISQC) and Attached Proton Test (APT) measurements. To test whether larger residues at the acyl group are tolerated by apt1, neoB was functionalized by employing the succinimidyl esters of isobutyric acid 4b and 4-pentynoic acid 4c (FIG. 3 and Tab. 1). Again, remarkable regioselectivities of 97% and 98% were achieved for 6 and 7, respectively, even exceeding the values of the acetylation reaction. It is important to mention that apt1 during preparation of 6 and 7 even tolerated the addition of the organic solvent dimethylformamide (DMF), which allowed easy solubilization of the hydrophobic activated esters 4b and 4c. Besides the high regioselectivities found for all the acylation reactions, the conversions for obtaining 5a, 6 and 7 were very high with values of 76%, 71% and 83%, respectively.

[0072] To demonstrate the generality of the concept regarding nucleotide ap tamers, an even shorter 21 mer aptamer (sequence: 5-CUG CAG UCC GAA AAG GGC CAG-3, apt2) was employed as SPG for neoB (tab. 1). This oligonucleotide was obtained in the same SELEX experiment as apt1.sup.10. Again, high regioselectivities were obtained for 5a, 6 and 7 (94%, 89% and 96%, respectively). The conversions for the reactions were slightly lower than for apt1, reaching 63%, 59% and 67%, respectively.

[0073] To show that the SPGs mentioned above arc also valuable for the generation of other drug derivatives with a similar pharmacophore, the related antibiotic paromomycin 2 was subjected to an acylation reaction employing apt1 (FIG. 1 and Table 1). It was found that 2 was exclusively transformed into the regioisomer N.sup.6(IV)-acetyl paromomycin 8.

[0074] After the successful synthesis of novel neomycin B derivatives the compounds 5a and 6 were investigated regarding their antimicrobial activities against E. coli ATCC 25922, which is a standard strain to evaluate the efficiency of antibiotics.sup.1. Two methods, the Kirby-Bauer Disk Test and the determination of the Minimal Inhibitory Concentration (MIC), were employed for that purpose (Table 2). It turned out that, despite of the removal of the positive charge of ring IV by acylation, the aminoglycoside derivatives 5a and 6 were still highly active. As shown in Table 2 the activity of 5a exhibiting the acetyl residue is slightly lower than neoB 1 with MIC-values of 6.3 and 3.1 M, respectively, while the derivative 6 modified with the more hydrophobic isobutyrate residue shows the same biological activity as paromomycin 2. These results confirm the finding that the 6-amine group of neomycin B ring TV is well suited for functionalization of the antibiotic and at the same time the high affinity of the neamine core 3 (ring I and II, FIG. 1D) to the prokaryotic ribosomal RNA is maintained.sup.1.

[0075] In conclusion, the effective use of short RNA sequences as non-covalent SPGs was demonstrated for the highly chemo- and regioselective derivatization of complex molecules bearing several functional groups with similar reactivity. It should be emphasized that the generation of such protective groups based on oligonucleotides relies on a well-established in vitro evolution process and hinders for a large variety of target molecules bearing different structural features can be evolved. In regard to the previously reported macrocyclic hosts, 5, the current limitations of SPG strategies, i.e., being only effective for molecules with a simple structure and the need for complicated design and synthesis of the host, were overcome. RNA SPGs allow the convenient functionalization of ring IV of the antibiotic neoB 1 with a regioselectivity of up to 98% and conversions of 83% in only one reaction step whereas conventional synthesis requires more than 20 steps including conventional covalent protection group chemistry' accompanied by much lower overall yields. Furthermore, the generality of the concept was demonstrated by employing RNAs with different sequence compositions and for aminoglycosides antibiotics with different functionalities at the pharmacophore, such as neomycin B 1 and paromomycin 2. These results suggest that SPGs based on oligonucleotides will become an indispensable and effective tool for the derivatization of natural products and drugs that can otherwise be only synthesized with demanding efforts and high costs.

TABLE-US-00001 TABLE 1 Regioselectivity of transformation dependent on size of activated ester antibiotic aptamer R.sub.2 Conv.{circumflex over ()}(%) r.s.*(%) 5a apt1 Me 76 95** 5a apt2 Me 63 94 6.sup. apt1 i-Pr 71 97** 6.sup. apt2 i-Pr 59 89 7.sup. apt1 3-butinyl 83 98** 7.sup. apt2 3-butinyl 67 96 8 apt1 Me 60 >99 All reactions were carried out using the general procedure (Supplementary Information) *regioselectivity (r.s.) of monoacylated neomycin B determined by .sup.1H-NMR, HSQC and APT. **regioselectivity average of three runs {circumflex over ()}maximal observed conversion of neomycin B to N.sup.6 acyl neomycin B as isomere mixture .sup.reaction were carried out in a mixture of 10 mM sodium phosphate buffer pH 6.8 and 6.7% DMF

TABLE-US-00002 TABLE 2 Antimicrobial Activity of Aminoglycosides against E. coli ATCC 25922 Amount Diameter.sup.a MIC.sup.b Antibiotic (nmol) (mm) (M) 1* 17.5 16.8 3.1 1{circumflex over ()} 17.5 16.1 3.1 2{circumflex over ()} 17.5 13.2 12.5 5a 17.5 13.9 6.3 6 17.5 11.5 12.5 *All compounds were used as HFBA salts, except for neomycin B sulphate 1* (Sigma Aldrich) {circumflex over ()}antibiotic x 6 HFBA, purified by HPLC using the same conditions of the neomycin B derivatives 5d and 6 (see Methods Summary). All compounds were used as HFBA salts, expect of neomycin B sulphate 1* (Sigma Aldrich) .sup.aThe zones of inhibition as determined by Kirby-Bauer disk method are given. For all compounds, the molar amount was kept constant at 17.5 mmol. .sup.bThe minimum inhibitory concentrations are given in M.

General Procedure for the Synthesis and Testing of Antibiotic Derivates 5a, 6, 7 and 8.

Materials

[0076] All chemicals and reagents were purchased from commercial suppliers and used without further purification, unless otherwise noted. Neomycin B trisulfate x hydrate (VETRANAL), paromomycin sulfate salt (98%), N,N-dimethylformamide (DMF, 99%), N-hydroxysuccinimide (NHS, 98%), trifluoroacetic anhydride (99%), dichloromethane (DCM, 99.5%), tetrahydrofurane (THF, 99.9%), pyridine (99%), 4-pentynoic acid (95%), acetic acid (99%), isobutyric acid (99%) and toluene (99.8%) were purchased from Sigma Aldrich and used as received. For HPLC purification heptafluorobutyric acid (IIFBA) (Fluka, puriss. p.a., for ion chromatography), acetone (Sigma-Aldrich, HPLC grade) were used. Ultrapure water (specific resistance >18.4 M cm) was obtained by Milli-Q water purification system (Sartorius). RNA aptamers (82-91% of purity) were purchased from BioSpring (Frankfurt am Main, Germany). All used N-hydroxysuccinimide ester (4a-c) were prepared according to standard literature procedures.sup.1,2. For the regioselective transformation Milli-Q water was treated with diethylpyrocarbonate (DEPC) and sterilized using an autoclave (121 C., 20 min).

General Procedure for Regioselective Transformation of Aminoglycosides.

[0077] The general procedure for regioselective transformation of aminoglycosides is schematically outlined in FIG. 3.

[0078] 816 L of a 6.1 mM RNA aptamer solution in 10 mM sodium phosphate buffer (pH 6.8) were heated to 85 C. for 10 min and afterwards stored for 15 min at room temperature. 684 L of a 4.8 mM solution of the antibiotic (3.28 mol) in 10 mM sodium phosphate buffer (pH 7.5) were added and the mixture was allowed to stand for 30 min at room temperature. 30 equiv. activated ester (98.4 mol) dissolved in 1.5 mL sodium phosphate buffer (pH 7.5) (for activated ester 4a) or in 106 l DMF (for activated esters 4b and 4c) were added and the reaction mixture was allowed to react for 24 hours at room temperature. After addition of 126 L of a 7 wt. % ethylamine water solution and further incubation for 30 min at room temperature the crude mixture was heated to 95 C. for 10 min. To the hot solution 3 mL of a 53 mM aqueous solution of didodecyldimethylammonium bromide (DDDMABr) were added to precipitate the RNA. After incubation for 15 min at room temperature and centrifugation for 30 min at 6 C. (16.1 u/s) the supernatant was freeze dried and dissolved in 400 L water. Each 30 L fraction was purified by HPLC using a Waters Spherisorb ODS-2C18 analytic column (water/acetone 6:5 containing 11.5 mM HFBA) and a flow rate of 1 ml/min at 40 C. to afford the antibiotic derivatives 5a, 6, 7 and 8.

Analytical Data

[0079] ##STR00007##

N.SUP.6.(VI)-acetyl neomycin B5 HFBA (5a)

[0080] The title compound was prepared according to the general procedure described above. Derivative 5a was obtained as a white solid. For the measurement of regioselectivity and the characterization of the compound .sup.1H-NMR, HSQC as well as APT spectra were recorded and electrospray ionization (ESI)-MS was employed. The yield was determined by HPLC: R.sub.t=6.57 min, conversion 76%, 27% yield. .sup.1H-NMR (D20, 500 MHz) 6 6.06 (d, .sup.3J=4 Hz, 1H, 1-H.sup.I), 5.44 (d, .sup.3J=2 Hz, 1H, 1-H.sup.II), 5.20 (d, .sup.3J=1.5 Hz, 1H, 1-H, 4.44 (t, .sup.3J=5.75 Hz, 1H, 3-H.sup.II), 4.39 (dd, .sup.2J=5 Hz, .sup.3J=2 Hz, 1H, 2-H.sup.II), 4.26 (t, .sup.3J=3 Hz, 1H, 3-H.sup.III, 4.24 (m, 1H, 4-H.sup.II), 4.09 (t, .sup.3J=6.75 Hz, 1H, 5-H.sup.II), 4.07 (m, 1H, 4-H), 4.01 (t, .sup.3J=10 Hz, 1H, 5-H.sup.I), 3.98-3.92 (m, 3H, 5-H.sup.II, 5-H, 3-H.sup.I), 3.76 (dd, 1H, .sup.2J=12.5 Hz, .sup.3J=5.5 Hz, 5-H), 3.72-3.68 (m, 2H, 4-H.sup.II, 6-H), 3.60 (dd, .sup.2J=14 Hz, .sup.3J=7.5 Hz, 1H, 6a-H.sup.III), 3.56 (m, 2H, 3-H, 2-H.sup.III), 3.53-3.41 (m, 4H, 6a-H.sup.I, 2-H.sup.I, 6b-H.sup.III, 4-H.sup.I), 3.38 (m, 1H, 1-H), 3.32 (dd, .sup.2J=14 Hz, .sup.3J=6 Hz, 1H, 6b-H.sup.I), 2.51 (dt, .sup.2J=12.5 Hz; .sup.3J=3.8 Hz, 1H, 2-H.sub.e), 2.04 (s, 3H, CH.sub.3), 1.89 (dd, .sup.3J=.sup.2J=12.7 Hz, 1H, 2-H.sub.a) ppm. APT (D.sub.2O, 500 MHz) 174.49 (Carbonyl-C), 110.00 (C-1.sup.II), 95.49 (C-1.sup.I), 95.51 (C-1.sup.III, 84.62 (C-5), 81.66 (C-4.sup.II), 75.39 (C-3.sup.II), 75.29 (C-4), 73.58 (C-2.sup.II), 72.45 (C-5.sup.III, 72.42 (C-6), 70.35 (C-4.sup.I), 69.22 (C-5.sup.I), 67.88 (C-3.sup.I), 67.56 (C-3.sup.III), 66.10 (C-4.sup.III, 60.00 (C-5.sup.II), 53.15 (C-2.sup.I), 50.90 (C-2.sup.III), 49.65 (C-1), 48.16 (C-3), 39.85 (C-6.sup.I), 39.33 (C-6.sup.111), 27.88 (C-2), 21.74 (CH.sub.3) ppm. MS (EI+) m/z: 657.32739 [M+H].sup.+.

##STR00008##

N.SUP.6.(VI)-isobutyl neomycin B5 HFBA (6)

[0081] The title compound was prepared according to the general procedure described above. Derivative 6 was obtained as a white solid. For the measurement of regioselectivity and the characterization of the compound .sup.1H-NMR, HSQC as well as APT spectra were recorded and ESI-MS was employed. The yield was determined by HPLC: R.sub.t=9.65 min. conversion 65%, 31% yield. .sup.1H-NMR (D.sub.2O, 500 MHz) 5.98 (s,1H, 1-H.sup.I), 5.38 (s, 1H.sup.II), 5.15 (s, 1H.sup.III), 4.39 (d, .sup.3J=6.0 Hz, 1H, 3-H.sup.II), 4.37 (m, 1H, 2-H.sup.II), 4.21 (m, 1H, 3H1.sup.III), 4.17 (m, 1H, 4-H.sup.II), 4.06-4.01 (m, 2H, 5-H.sup.III, 4-H), 3.96 (t, .sup.3J=10.3 Hz, 1H, 5-H.sup.I), 3.92-3.88 (m, 3H, 5-H.sup.II, 5-H, 3-H.sup.I), 3.70 (dd, 1H, .sup.2J=14 Hz, .sup.3J=5.8 Hz, 5-H.sup.II), 3.65-3.61 (m, 2H, 4-11.sup.III, 6-H), 3.54-3.50 (m, 3H, 6a-H.sup.III, 3- H, 2-H.sup.III), 3.46 (m, 1H, 4-H.sup.I), 3.44-3.38 (m, 3H, 6a-H.sup.I, 2-H.sup.I, 6b-H.sup.III), 3.56-3.33 (m, 1H, 1-H), 3.28 (dd, .sup.2J=13.5 Hz, .sup.3J=6 Hz, 1H, 6b-H.sup.I), 2.47 (m, 2H, CH(CH.sub.3).sub.2, 2-H.sub.e), 1.86 (dd, .sup.3J=12.2 Hz, 1H, 2-H.sub.a), 1.09 (s, .sup.3J=6.5 Hz, 6H, CH.sub.3) ppm. APT (D.sub.2O, 500 MHz) 176.68 (carbonyl-C), 110.31 (C-1.sup.II), 95.48 (C-1.sup.I, 95.18 (C-1.sup.III), 84.72 (C-5), 81.54 (C-4.sup.II), 75.23 (C-4), 74.90 (C-3.sup.II), 73.49 (C-2.sup.II), 72.47 (C-5.sup.III), 72.39 (C-6), 70.42 (C-4.sup.I), 69.23 (C-5.sup.I), 67.87 (C-3.sup.I), 67.48 (C-3.sup.III), 66.08 (C-4.sup.III), 60.04 (C-5.sup.II), 53.33 (C-2.sup.I), 50.87 (C-2.sup.III), 49.59 (C-1), 48.22 (C-3), 39.94 (C-6.sup.I), 39.03 (C-6.sup.III), 34.93 (CH(CH.sub.3).sub.2), 27.95 (CH(CH.sub.3).sub.2), 27.89 (C-2) ppm, MS (EI+) m/z: 685.36176 [M+H].sup.+, 707.34387 [M+Na].sup.+, 343.18356 [M+2H].sup.2+.

##STR00009##

N.SUP.6.(VI)-pent-4-inoyl neomycin B5 HFBA (7)

[0082] The title compound was prepared according to the general procedure described above. Derivative 7 was obtained as a white solid. For the measurement of regioselectivity and the characterization of the compound .sup.1H-NMR, HSQC as well as APT spectra were recorded and ESI-MS was employed. The yield was determined by HPLC: R.sub.t,=9.8 min, conversion 83%, 45% yield. .sup.1H-NMR (D.sub.2O, 500 MHz) 6.06 (d, .sup.3J=4 Hz, 1H, 1-H.sup.I), 5.43 (s, 1H, 1-H.sup.II), 5.19 (s, 1H, 1-H .sup.III), 4.47 (d, .sup.3J=5.75 Hz, 1H, 3-H.sup.II), 4.41 (m, 1H, 2-H.sup.II), 4.26 (m, 1H, 3-H .sup.III, 4.22 (m, 1H, 4-H.sup.II), 4.11-4.06 (m, 2H, 5-H.sup.III 4-H), 4.01 (t, .sup.3J=10 Hz, 1H, 5-H.sup.I), 3.97-3.94 (m, 3H, 5-H.sup.II, 5-H, 3-H.sup.I), 3.77 (dd, .sup.2J=13 Hz, .sup.3J=5 Hz, 1H, 5-H.sup.II), 3.75- 3.68 (m, 2H, 4-H.sup.III, 6-H), 3.61 (dd, .sup.2J=14 Hz, .sup.3J=7.5 Hz, 1H, 6a-H.sup.III), 3.56-3.53 (m, 2H, 3-H, 2-H.sup.III), 3.52-3.45 (m, 4H, 4-H.sup.I, 6a-H.sup.I, 2-H.sup.I, 6b-H.sup.III), 3.36 (m, 1H, 1-H), 3.33 (dd, .sup.2J=14 Hz, .sup.3J=6 Hz, 1H, 6b-H.sup.I), 2.56-2.43 (m, 6H, (CH.sub.2).sub.2, 2-H.sub.e, CCH), 1.90 (dd, .sup.3J=12.3 Hz, 1H, 2-H.sub.a) ppm. APT (D.sub.2O, 500 MHz) 175.10 (carbonyl-C), 110.11 (C-1.sup.II), 95.54 (C-1.sup.I, 95.33 (C-1.sup.III), 84.71 (C-5), 81.56 (C-4.sup.II), 75.17 (C-3.sup.II), 75.12 (C-4), 73.52 (C-2.sup.II), 72.72 (C-5.sup.III, 72.35 (C-6), 70.50 (C-4.sup.I), 83.47 (CCH), 70.35 (CCH), 69.24 (C-5.sup.I, 67.92 (C-3.sup.I, 67.51 (C-3.sup.III), 66.05 (C-4.sup.III), 60.05 (C-5.sup.II), 53.33 (C-2.sup.I), 50.88 (C-2.sup.III), 49.52 (C-1), 48.20 (C-3), 39.90 (C-6.sup.I), 39.34 (C-6.sup.III), 34.23 (COCH.sub.2CH.sub.2), 27.80 (C-2), 14.54 (COCH.sub.2CH.sub.2) ppm. MS (EI+) m/z: 695.34564 [MH].sup.+, 717.32770 [M+Na].sup.+, 348.17706 [M+2H].sup.2+.

##STR00010##

N.SUP.6.(VI)-acetyl paromomycin4 HFBA (8)

[0083] The title compound was prepared according to the general procedure described above. Derivative 8 was obtained as a white solid. For the measurement of regioselectivity and the characterization of the compound .sup.1H-NMR, HSQC as well as APT spectra were recorded and ESI-MS was employed. The yield was determined by HPLC: R.sub.t=4.78 min, conversion 60%, 22% yield. .sup.1H-NMR (D.sub.2O, 500 MHz) 5.81 (d, .sup.3J=3.5 Hz, 1H, 1-H.sup.I), 5.39 (s, 1H, 1-H.sup.III), 5.20 (s, 1H, 1-H.sup.III), 4.44 (t, .sup.3J=5.5 Hz, 1H, 3-H.sup.III), 4.36 (m, 1H, 1- H.sup.II), 4.25 (m, 1H, 3-H.sup.III), 4.22 (m, 1H, 4-H.sup.II), 4.10 (t, .sup.3J=6.5 Hz, 1H, 5-H.sup.III), 4.04 (t, .sup.3J=7.8 Hz, 1H, 4-H), 3.97-3.89 (m, 4H, 6-H.sup.I.sub.a, 3-H.sup.I, 5-H), 3.81-3.75 (m, 3H, 6-H.sup.I.sub.a, 5-H.sup.II.sub.b, 5-H.sup.I), 3.73-3.67 (m, 2H, 4-H.sup.III, 6-H), 3.62-3.57 (m, 2H, 6-H.sup.III.sub.a, 3-H), 3.55 (m, 1H, 2-H.sup.III), 3.51 (t, .sup.3J=9.3 Hz, 4-H.sup.I), 3.43 (m, 1H, 2-H.sup.II), 3.42 (dd, .sup.2J=15 Hz, .sup.3J=6 Hz, 1H, 6-H.sup.III.sub.b), 3.38-3.35 (m, 1H, 1-H), 2.51 (dt, .sup.2J=12, 5 Hz, .sup.3J=2.5 Hz, 1H, 2-H.sub.e), 2.04 (s, 3H, CH.sub.3), 1.86 (dd, .sup.3J=12.5 Hz, 1H, 2-H.sub.a) ppm. APT (D.sub.2O, 500 MHz) 174.93 (carbonyl-C), 109.78 (C-1.sup.II), 95.98 (C-1.sup.I), 95.56 (C-1.sup.III), 84.11 (C-5), 81.53 (C-4.sup.II), 77.23 (C-4), 75.53 (C-3.sup.II), 73.73 (C-5.sup.1), 73.44 (C-2.sup.II, 72.51 (C-5.sup.III), 72.16 (C-6), 69.08 (C-4.sup.I), 68.63 (C-3.sup.I), 67.56 (C-3.sup.III), 66.13 (C-4.sup.III), 60.11 (C-6I, C-5.sup.II), 53.161 (C-2.sup.I), 50.88 (C-2.sup.III), 49.48 (C-1), 48.63 (C-3), 39.38 (C-6.sup.III), 27.87 (C-2), 21.71 (CH3) ppm. MS (EI+) m/z: 685.31303 m/z [M+H].sup.+, 680.29454 [M+Na].sup.+, 329.66013 [M+2H].sup.2+.

Antimicrobial Tests

Materials

[0084] Mueller Hinton TT Broth powder (BDcat. no. 212322)

[0085] Agar (Rothcat. no. 5210.2)

[0086] 6 mm paper disks (BBLcat. no. 231039)

Kirby-Bauer Test

[0087] Antibiotic disks preparation. Paper disks (6 mm diameter, BBL Microbiology Systems) were wetted through with 20, 25, 30 and 35 L of a solution containing the antibiotic sample at a concentration of 0.5 nmol/L. The wet disks were dried in a desiccator overnight, and used the next day.

[0088] Culture preparation. A colony picked from a freshly made plate of the bacteria strain E. coli ATCC 25922 was used to inoculate 20 mL of Mueller-Hinton broth and the obtained culture was grown overnight at 37 C. and 250 RPM shaking.

[0089] Kirby-Bauer test procedure. A new culture was made adding 1 mL of overnight culture in 99 mL of fresh Mueller-Hinton broth. The culture was grown at 37 C. and 250 RPM shaking until it reached an OD600 of 0.132 (0.5 McFarland) and a series of Mueller-Ilinton-Agar plates preheated at 37 C. were inoculated spreading 200 L of that culture with sterile cotton. The plates were then dried for 30 minutes. Then on each plate 3 or 4 antibiotic paper disks were placed. The plates were incubated overnight at 37 C. and subsequently the diameter of the inhibition growth zone was measured.

Minimum Inhibitory Concentration (MIC) Test

[0090] All test solutions contained 40 nmol of antibiotic. After drying, the samples were resuspended in 100

[0091] L of Mueller-Hinton broth. Each solution of antibiotic in broth media was pipetted in the first lane of a 96 well plate (volume of well 500 L). The remaining lanes were filled with 50 L of E. coli ATCC 25922 culture with an OD600 of 0.264.

[0092] Series of 2-fold dilution were made removing from the first lane 50 L of the antibiotic solution and resuspending it with the culture contained in the next well. At the end, all wells were filled with 100 L of E. coli ATCC 25922 culture in Mueller-Hinton with an OD600 of 0.132 (0.5 McFarland) and the appropriate amount of antibiotic.

[0093] The 96-well plates were incubated overnight at 37 C. and 350 RPM shaking. The OD600 of all wells were measured using an E. coli ATCC 25922 culture with an OD600 of 0.132 as reference and the MIC value was determined by taking the lowest concentration where no bacterial growth was observed.

[0094] See also National Committee for Clinical Laboratory Standards, Performance Standards for antimicrobial susceptibility testing, 8th Informational Supplement (2002); John, D. T., et al. Antimicrobial Susceptibility testing: General Considerations, Manual of Clinical Microbiology 7th edition, 1469-1473 (1999); Laitha, M. K., Manual on antimicrobial susceptibility testing, 7-39 (2004).

Example 2

Selection of Aminoglycoside-Binding Oligopeptides using Phage Display

[0095] This example discloses the selection of short peptide sequences capable of binding to Neomycin B and protecting the molecule partially to allow chemical reactions at the functional groups that are not involved in the host-guest interaction. The strategy to evolve peptide sequences that are able to recognize and bind to Neomycin B relies on the well established Phage Display technique (Smith G P. Science 1985; 228:1315-1317; Kay B K, et al. Gene 1993; 128:59-65).

[0096] For that purpose, the target molecule (Neomycin B) is covalently linked to a solid surface. In this case a Nunc Amino Immobilizer 96-wells plate was chosen as surface to couple Neomycin B. The plate contains in its wells a modified surface able to react with the amino-groups of the target molecule. The density of grafted complexes is approx. 10.sup.14 cm.sup.2.

[0097] In this way, the whole surface of the Neomycin B can interact with the peptide that is exposed on the recombinant phage particle, except for the site where it is covalently linked to the surface it is masked and therefore is not able to interact with the peptide.

Step1Immobilization of Neomycin B

[0098] The plate used (Nunc Immobilizer Amino F96 clearNunc #436006) is optimized for peptide and protein coupling. Several conditions were tested for the immobilization. Antibiotic binding was assayed by an ELISA test using an anti-neomycin antibody. The optimized protocol for the immobilization of the Neomycin is described in the following.

[0099] The coupling reaction was carried out by filling a well with 3000 of Neomycin B (1 mg/ml in sodium-phosphate 0.1M, pH 9.6). The plate was then placed on a shaker at 250 RPM at room temperature, for 12 h. The well was then washed ten times using 3000 of TBST buffer (Tris-HCl 50mM +NaCl 0.15M pH 7.5+2% of Tween 20) and shaking 10 min at 250 RPM and room temperature for each washing step.

Step 2Phage Panning

[0100] Before starting the panning procedure the phages were incubated in an empty well to allow the plastic binder phages to be adsorbed on the plastic surface of the well. Therefore, 100 l of a phage suspension containing 10.sup.12 pfu/ml in sodium-phosphate 0.1M pH 7 were incubated in a blank well for 1 h at room temperature shacked at 320 RPM. This procedure was repeated 3 times by taking the supernatant and putting it into another blank well. The final supernatant was used to perform the phage panning.

[0101] Three rounds of phage panning were carried out. Each round consists of 3 phases: incubation, washing and elution.

[0102] Incubation: 100 l/well of phage suspension were pipetted in each Neomycin B coated well and the plate was shaken gently for 60 min to allow the host-guest interaction to take place. The unbound phages were discarded by pouring off and slapping the plate face-down onto a clean paper towel.

[0103] Washing: each well was washed by adding 300 l of TBST and shaking for 10 min at room temperature. The unbound phages were discarded by pouring off and slapping the plate face-down onto a clean paper towel. The washing procedure was repeated 5 times during the first round of selection then 7 times during the second round and 10 times during the third one.

[0104] Elution: Sodium Phosphate 0.1M at pH 7.0 was used as elution buffer with the addition of Neomycin B at a concentration of 1 M as competitor to avoid non specific elution of phage particles.

Step 3Sequencing

[0105] Because of the use of Neomycin B as competitor, the cells (E. coli ER2738) were previously transformed with pET28 that carries the Kanamycin resistance that allows cell growth on Neomycin B containing media. Phage particles eluted after the third round of selection were used to infect ER2738 cells (already containing pET28). Subsequently, the cells were plated on LB-agar plate. After overnight incubation at 37 C. several single colonies were picked and grown for 3 h in 3 ml of LB medium. Single stranded DNA was then purified from the phage particles from the culture's supernatant using the QIAprep Spin M13 Kit (QIAGEN #27704) and prepared for sequencing.

[0106] The following peptide sequences were derived from the analysis of the DNA sequences VNRSSDHWNLTT, DYDTLRTVAFTR, NGSLQRSFVISH, HVRIYVDTIEIR, GAMHLPWHMGTL and GAMHPPRHMGPL.

Example 3

Selection of Aminoglycoside-Binding Oligopeptides using In-Vivo Screening for Small Antibiotic Binders

[0107] An in-vivo screening for small antibiotic binders was performed by infecting a culture of E. coli ER2738 with a 12 amino acid random peptide-pIII phage library. The ability of the culture to survive under different antibiotic conditions was tested by measuring the ODGOO after overnight growth.

[0108] A fresh culture of ER2838 was prepared by inoculation of 20 ml of LB medium with 50 l glycerol stock solution of the strain. The culture (OD600=0.05) was infected with 100 l of phage suspension containing 10.sup.13 pfu/ml with an overall complexity of 10.sup.9 different sequences. After 1 h of growth at 37 C. shaking at 150-200 RPM the culture was aliquoted in 15 ml tubes resulting in a culture volume of 3 ml and the antibiotics at different concentrations were added.

[0109] The cultures were grown overnight at 37 C., shaking at 150-200 RPM. The OD600 of ten-fold diluted sample of each culture was estimated using a spectrophotometer. Not infected ER2738 and the same cells infected with a wild type M13 were used as negative control. The resistance against ampicillin, neomycin B and chloramphenicol was tested. It was observed that the cells infected with the library were more resistant in the presence of ampicillin and neomycin compared to the controls, but not in the case of chloramphenicol (data not shown). This demonstrates that the presence of the phage library influences the resistance of the cells at higher antibiotic concentration. The resistant cells were plated on LB-agar and the phage DNA was sequenced.

Example 4

Aptameric Protective Groups allow Diverse Modifications

[0110] This example demonstrates that the APG approach for regioselective transformation according to the present invention is compatible with diverse reagents in different reactions. Scheme 1 shows the chemo- and regioselective transformation of ring IV of neomycin as exemplary target compound 1. All reactions were performed in 10 mM phosphate buffer at room temperature for 24 h in presence of 1.5 equiv. of APG apt1. (1) Acylation: 15 equiv. of 7, pH 6.9; (2) Thiolation: 5 equiv. of 9, pH 6.9; (3) Azide introduction: 8 equiv. of 11, 0.36 mol % CuSO.sub.4, Na.sub.2CO.sub.3, NaOH, pH 8.0; (4) Urea bond formation: 30 equiv. of 14a-c, DMF, pH 6.9.

##STR00011## ##STR00012##

General Procedures

Synthesis of Sodium 4-(acetoxy)-2,3,5,6-tetrafluorobenzenesulfonate 7

[0111] 10.1 g of 2,3,5,6-tetrafluorophenol (61.4 mmol) was taken up in 22 mL fuming sulphuric acid (30% SO3) and stirred at ambient temperature for 18 h before pouring the mixture into 200 mL iced brine. The product was precipitated by adding 6 g of NaCl and stirred until no further precipitate was formed. This mixture was filtered through a sintered glass disc and the collected solids were taken up in 330 mL boiling acetonitril, filtered while hot, and allowed to cool slowly to ambient temperature. The colourless crystalline product was collected by filtration and dried in vacuum yielding 5.42 g (20.2 mmol, 33% yield) of 4-sulfo-tetrafluoro phenol sodium salt. 270 mg of this sodium salt (1.0 mmol) and 53.8 L of acetic acid (0.94 mmol) were dissolved in 30 mL acetone. After 230 mg 1,3-dicyclohexylcarbodiimide (1.1 mmol) were added the mixture was stirred at room temperature for 20 hour. The resulting precipitate was removed by filtration and the filtrate was concentrated under reduced pressure. The crude mixture was purified by column chromatography using a 4:1 acetone/chloroform mixture. 163 mg (0.56 mmol, 56% yield), white solid. Rt (acetone/chloroform 4:1)=0.55. 1H-NMR (D2O, 400 MHz): [ppm]=2.46 (s, 3H, CH3-CO). 13C-NMR (D2O, 50.43 MHz): [ppm]=170.07 (1C, CO); 147.01 (dq), 144.09 (ddd) (2C, 3-CAr, 5-CAr); 145.03 (dq), 142.10 (ddd) (2C, 2-CAr, 6-CAr), 131.14 (1C, C-SO2); 127.61 (1C, CCOCH3); 22.82 (1C, CH3-CO).

Synthesis of diazo-transfer reagent Imidazole-1-sulfonyl Azide Hydrochloride 11

[0112] Sulfuryl chloride (1 6 mL, 20 mmol) was added dropwise to an ice-cooled suspension of sodium azide (1.3 g, 20 mmol) in acetonitril (20 mL) and the mixture was stirred overnight at room temperature. Imidazole (2.6 g, 38 mmol) was added portion-wise to the ice-cooled mixture and the resulting slurry was stirred for additional 3 h at room temperature. The mixture was diluted with ethyl acetate (40 mL), washed with water (240 mL) and then with saturated aqueous sodium hydrogen carbonate (240 mL), dried over MgSO4 and filtered. The filtrate was cooled in an ice-batch and a 3M HCl methanolic solution (10 mL) was added drop wise to precipitate the product. Finally, the filer cake was washed with EtOAc (310 mL) to obtain 11. Yield: 1.9 g (9.1 mmol, 45% yield). 1H-NMR (D2O, 400 MHz) (p.p.m.) 9.53 (s, 1H, H-2), 8.07 (s, 1H, H-5), 7.67 (s, 1H, H-4). 13C-NMR (D2O, 400 MHz) (p.p.m.) 137.6, 122.6, 120.18. HRMS (EI+) (m/z): found 174.0078 [MC1]+, calc. 174.0080 [MC1]+.

(1) Acylation of Amino Group in C6 Position of Ring IV.

[0113] ##STR00013##

[0114] A volume of 900 L of a 5.54 mM RNA aptamer solution (4.98

[0115] mol) in 10 mM sodium phosphate buffer (pH 6.8) was heated to 85 C. for 10 min and was afterwards kept at room temperature for 15 min. 684 L of a 4.8 mM solution of neomycin B sulphate (3.28 mol) in 10 mM sodium phosphate buffer (pH 7.4) was added and the mixture was allowed to stand for 30 min at room temperature. Then, 15 equiv. activated ester, acetyl sulfo-NHS ester 4 or STP-ester 5 (49.2 mol) 500 L 10 mM sodium phosphate buffer (pH 7.4), or 5 equiv. 2-iminothiolane hydrochloride (16.4 mol) dissolved in 42 L 10 mM sodium phosphate buffer (pH 7.4) were added and the reaction mixture was allowed to react for 24 hours at room temperature. After addition of 180 L of a 7 wt. % ethylamine water solution and further incubation for 30 min at room temperature, 486 l of a 2 M sodium hydroxide solution were added and the crude mixture was heated to 90 C. for 30 min. After cooling to room temperature each 50 L fraction was purified by HPLC using a Waters Spherisorb ODS-2C.sub.18 analytic column (water/acetone 1:0.81 containing 16.9 mM HFBA) at a flow rate of 1 ml/min at 40 C. to afford the antibiotic derivatives 10 and 14. After evaporation of acetone and freeze-drying of collected fractions the product was taken up in 150 L of D.sub.2O for NMR-studies.

(2) Urea Bond Formation at 2C & 6C Position

[0116] ##STR00014##

[0117] 900 L of a 5.54 mM RNA aptamer solution (4.98 mol) in 10 mM sodium phosphate buffer (pH 6.8) was heated to 85 C. for 10 min and was afterwards kept at room temperature for 15 min. 684 L of a 4.8 mM solution of the aminoglycoside antibiotic (3.28 mol) in 10 mM sodium phosphate buffer (pH 7.4) was added and the mixture was allowed to stand for 30 min at room temperature. 15 equiv. (49.2 mol) aromatic isocyanate 7a-c or 30 equiv. (98.4 mol) of aliphatic isocyanates 9a-c dissolved in 108 L DMF were added and the reaction mixture was allowed to react for 24 hours at room temperature. After addition of 180 L of a 7 wt. % ethylamine water solution and further incubation for 30 min at room temperature, 280 l of a 2 M sodium hydroxide solution was added and the crude mixture was heated to 90 C. for 30 min. After cooling to room temperature each 50 L fraction was purified by HPLC using a Waters Spherisorb ODS-2C.sub.18 analytic column (water/acetone 1.0:0.81 containing 16.9 mM HFBA) at a flow rate of 1 ml/min at 40 C. to afford the antibiotic derivatives 15a-c, 16, 19a-c and 20a-b. After evaporation of acetone and freeze-drying the product was taken up in 150 L of D.sub.2O for NMR-studies.

(3) Azide Introduction at 2C and 6C Position

[0118] ##STR00015##

[0119] 900 L of a 5.54 mM RNA aptamer solution (4.98 mol) in 10 mM sodium phosphate buffer (pH 6.8) was heated to 85 C. for 10 min and was afterwards kept at room temperature for 15 min. 684 L of a 4.8 mM solution of neomycin B sulphate (3.28 mol in 10 mM sodium phosphate buffer (pII 7.4) was added and the mixture was allowed to stand for 30 min at room temperature. 540 L of an 4.8 mM aqueous solution of diazo-transfer reagent 8 (10 mg/mL), which was adjusted to pH 8 by approx. 25 L of adding 2 M NaOH solution, was added into the solution of the antibiotic 1-apt1 complex solution. After 59 L of an aqueous solution of sodium carbonate (10 mg/mL) and 50 L of an aqueous solution of copper sulfate (2 mg/mL) were added and the mixture was reacted for 24 hours at room temperature. After addition of 180 L of a 7 wt. % ethylamine water solution and further incubation for 30 min at room temperature, 375 l of a 2 M sodium hydroxide solution was added and the crude mixture was heated to 90 C. for 30 min. After cooling to room temperature each 50 L fraction was purified by HPLC using a Waters Spherisorb ODS-2C.sub.18 analytic column (water/acetone 1.0:0.81 containing 16.9 mM HFBA) at a flow rate of 1 ml/min at 40 C. to afford the antibiotic derivatives 17 and 18. After evaporation of acetone and freeze-drying of collected fractions the product was taken up in 150 L of D.sub.2O for NMR-studies.

REFERENCES

[0120] 1. Alper, P. B., Hendrix, M., Sears, P., Wong, C.-H. Probing the specificity of aminoglycoside ribosomal RNA interactions with designed synthetic analogs. J. Am. Chem. Soc. 120, 1956-1978 (1998). [0121] 2. Usui, T., Umezawa, S. Total synthesis of neomycin B, Carbohydrate Research 174, 133-143 (1988). [0122] 3. Hanessian, S. et at.6-Hydroxy to 6-amino tethered ring-to-ring macrocyclic aminoglycosides as probes for APH(3)-IIIa kinase, Bioorg. & Med. Chem. Lett. 17, 3221-3225 (2007). [0123] 4. Coquire, D., de la Lande, A., Parisel, O., Prang, T., Reinaud, O. Directional control and supramolecular protection allowing the chemo and regioselective Transformation of a Triamine. Chem. Eur. J. 15, 11912-11917 (2009). [0124] 5. Cafeo, G., Kohnke, F. H., Valenti, L., Cafeo, G. et al. Regioselective 0-alkylations and acylations of polyphenolic substrates using a calix141pyrrole derivative. Tetrahedron Letters 50, 2009, 4138-4140 (2009). [0125] 6. Sun, W., Du, L., Li, M. Aptamer-based carbohydrate recognition. Current Pharmaceutical Design 16, 2269-2278 (2010). [0126] 7. Beta, H. et al. Aptamers that recognize the lipid moiety of the antibiotic moenomycin A. Biol. Chem. 284, 1497-1500 (2003). [0127] 8. Jiang, L., Suri, A. K., Fiala, R., Patel, D. J. Saccharide-RNA recognition in an aminoglycoside antibiotic-RNA aptamer complex. Chemistry & Biology 4, 35-50 (1997). [0128] 9. Wang, K. Y., McCurdy, S., Shea, R. G., Swaminathan, S., Bolton, P. H. A DNA aptamer which bind to and inhibits thrombin exhibits a new structural motif for DNA. Biochemistry 32, 1899-1904 (1993). [0129] 10. Wallis, M. G., von Ahsen, U., Schroeder, R, Famulok, M. A novel RNA motif for neomycin recognition. Chemistry and Biology 2, 543-552 (1995). [0130] 11. Connell, G. J., Illangesekare, M., Yarus, M. Three small ribooligonucleotides with specific arginine sites , Biochemistry 32, 5497-5502 (1993). [0131] 12. Sassanfar, M., Szostak, J. W. An RNA motif that binds ATP, Nature 364, 550-553 (1993). [0132] 13. Huizenga, D. E., Szostak, J. W. Identification of N2-(1-Carboxyethyl)guanine (CEG) as a Guanine Advanced Glycosylation End Product, Biochemistry 34, 656-665 30 (1995). [0133] 14. Burke, D. H., Hoffmann, D. C. A novel acidophilic RNA motif that recognizes Coenzyme A, Biochemistry 37, 4653-4663 (1998). [0134] 15. Ellington, A. D., Szostak, J. W. Selection in citro of single-stranded DNA molecules that fold into specific ligand-binding structures, Nature 355, 850-852 (1992). [0135] 16. Li, Y. F., Geyer, C. R. Sen, D. Recognition of anionic porphyrins by DNA aptamers, Biochemistry 35, 6911-6922 (1996). [0136] 17. Wang, C. Y. E., Su, W. P. D., Kurtin, P. J. Subcutaneous panniculitic T-Cell Lymphoma, Int. J. Dermat. 35, 1-8 (1996). [0137] 18. Moazed, D., Noller, H. F. Interaction of antibiotics which functional sites in 16S ribosomal RNA. Nature 327, 389-394 (1987). [0138] 19. Purohi, P., Stern, S. Interaction of small RNA with antibiotics and RNA ligands to the 30S subunit. Nature 370, 659-662 (1994). [0139] 20. Zhou, J.,Wang, G., Zhang, Ye, X.-S. Modification of aminoglycoside antibiotics targeting RNA. Med. Res. Rev. 27, 279-316 (2007). [0140] 21. Samantaray, S., Marathe, U., Dasgupta, S., Nandicoori, V. K., Roy, R. P. Peptide-sugar ligation catalyzed by transpeptidase sortase: a facile approach to neoglycoconjugate synthesis. J. Am. Chem. Soc. 130, 2132-2133 (2009). [0141] 22. Chang, C.-W. T. et al. Surprising alteration of antibacterial activity of 5-modified neomycin against resistant bacteria. J. Med. Chem. 51, 7563-7573 (2008). [0142] 23. Kirk, S. R., Tor, Y. tRNAPhP binds aminoglycoside antibiotics. Bioorg. & Med. Chem. 7, 1979-1991 (1999). [0143] 24. Tok, J. B.-H., Fenker, J. Novel synthesis and RNA-binding properties of aminoglycoside dimers conjugated via a naphthalene diimide-based intercalator. Bioorg. & Med. Chem. Lett. 11, 2987-2991 (2001). [0144] 25. Bera, S., Zhanel, G. G., Schweizer, F. Evaluation of amphiphilic aminoglycoside-peptide triazole conjugates as antibacterial agents. Bioorg. & Med. Lett. 20, 3031-3035 (2010). [0145] 26. Francois, B. et al. Crystal structures of complexes between aminoglycosides and decoding A site oligonucleotides: role of the number of rings and positive charges in the specific binding leading to miscoding. Nucleic Acids 33, 5677-5690 (2005). [0146] 27. Mikkelsen, N. E., Johansson, K., Virtanen, A., Kirsebom, L. A. Aminoglycosides binding displaces a divalent metal ion in a tRNA-neomycin B complex. Nat. Struct. Biol. 8, 510-514 (2001). [0147] 28. Stampfl, S., Lempradl, A., Koehler, G. Schroeder, R. Monovalent ion dependence 15 of neomyin B binding to an RNA aptamer characterized by spectroscopic methods. Chem. Bio. Chem. 8, 1137-1145 (2007). [0148] 29. Jiang, L. et al. Saccharide-RNA recognition in a complex formed between neomycin B and a RNA aptamer. Structure 7, 817-827 (1999).

INCORPORATION OF SEQUENCE LISTING

[0149] Incorporated herein by reference in its entirety is the Sequence Listing for the application. The Sequence Listing is disclosed on a computer-readable ASCII text file titled, Sequence_Listing_294_435_PCT_US_ST25.txt, created on Dec. 1, 2015. The Sequence Listing text file is 2.56 KB/2,623 bytes in size.