Synthesis of Ring II/III Fragment of Kanamycin: A New Minimum Structural Motif for Aminoglycoside Recognition

A novel protocol has been established to prepare the kanamycin ring II/III fragment, which has been validated as a minimum structural motif for the development of new aminoglycosides on the basis of its bactericidal activity even against resistant strains. Furthermore, its ability to act as a AAC-(6′) and APH-(3′) binder, and as a poor substrate for the ravenous ANT-(4′), makes it an excellent candidate for the design of inhibitors of these aminoglycoside modifying enzymes.


Introduction
Since the discovery of streptomycin in 1944 [1], aminoglycosides have found widespread clinical use due to its broad antimicrobial spectrum and rapid bactericidal effects [2,3].Their mode of action involves binding to the 16S ribosomal subunit at the t-RNA acceptor A-site (aminoacyl site), where they interfere with the ability of the ribosome to correctly decode mRNA during protein synthesis [4][5][6][7].Unfortunately, irreversible ototoxic side effects and growing bacterial resistance have narrowed the significance of aminoglycosides as antibiotics in the clinical practice [8].
Kanamycins (1)(2)(3) are natural antibiotics belonging to a group of aminoglycosides containing a 4,6-disubstituted 2-deoxystreptamine (ring II) core and have been used against both Gram-(+) and Gram-(−) bacteria for more than fifty-years (Figure 1A) [9].The pseudo-disaccharide I/II fragments (4)(5)(6) of this family of antibiotics, with slightly different OH/NH 2 patterns in unit I, is common to most aminoglycosides and have been considered until now essential for specific complex formation, and consequently, for antibiotic activity (Figure 1B) [10].For this reason, most approximations to new aminoglycosides described to date involve the synthesis of derivatives maintaining the I/II core and eliminating or modifying ring III [11][12][13].
Unfortunately, there are many aminoglycoside-modifying enzymes transferring acetyl, phosphoryl and adenyl groups in a cofactor-dependent manner to virtually every amino or hydroxyl substituent of the I/II structural motif (Figure 1A) [14].As a result, semisynthetic congeners of the 4,6-disubstituted 2-deoxystreptamine class were developed to overcome the inactivating action of a subset of the aforementioned enzymes and their different isoforms prevalent in other pathogens [15][16][17][18].Despite the urgent need of new anti-infective therapies, it should be noted that no new aminoglycoside antibiotic was introduced since the early 1980s [19], up until this year, when promising semisynthetic propylamycin was reported by Crich and coworkers [20].
minimum structural motif (MSM) that provides basic affinity and, subsequently, hit-to-lead optimization of the resulting structure.Herein, we report on the synthesis of novel pseudodisaccharide 7 that shows interesting properties as a novel MSM, while still retaining a considerable part of the antibiotic activity against several strains, including some expressing the most clinically relevant aminoglycoside modifying enzymes (Figure 1A).

Synthesis of Pseudo-Disaccharide 7
The preparation of pseudo-disaccharide 7 was carried out according to the sequence described in Scheme 1.Our synthetic effort began with the preparation of tetraazidokanamycin A (8), an intermediate suitably protected with azide groups, according with the procedure described previously by our laboratory [21].As a next step, simultaneous regioselective protection of C-6′′ and C-4′′ hydroxyl groups was achieved by treatment of 8 with di-tert-butylsilyl bis(trifluoromethanesulfonate) in pyridine to afford compound 9 in 80% yield [22].The protection of C-6′′ OH was proven to be necessary for minimizing the formation of byproducts and for allowing an easier purification after the regioselective protection of ring I, the key step of our strategy.After undergoing many frustrations attempting the regioselective protection of positions 2′ and 3′ in ring I of kanamycin A [23], we envisioned the use of 2,3-butanodione-bis-dimethyl acetal as a plausible strategy [24].Indeed, this reaction afforded an equimolar mixture of 10a and 10b, by protection of C-3′ and C-4′ hydroxyl groups and C-2′ and C-3′, respectively, decreasing the total yield of the synthesis A general way for the discovery of new aminoglycosides can be thus summarized on keeping a minimum structural motif (MSM) that provides basic affinity and, subsequently, hit-to-lead optimization of the resulting structure.Herein, we report on the synthesis of novel pseudo-disaccharide 7 that shows interesting properties as a novel MSM, while still retaining a considerable part of the antibiotic activity against several strains, including some expressing the most clinically relevant aminoglycoside modifying enzymes (Figure 1A).

Synthesis of Pseudo-Disaccharide 7
The preparation of pseudo-disaccharide 7 was carried out according to the sequence described in Scheme 1.Our synthetic effort began with the preparation of tetraazidokanamycin A (8), an intermediate suitably protected with azide groups, according with the procedure described previously by our laboratory [21].As a next step, simultaneous regioselective protection of C-6" and C-4" hydroxyl groups was achieved by treatment of 8 with di-tert-butylsilyl bis(trifluoromethanesulfonate) in pyridine to afford compound 9 in 80% yield [22].The protection of C-6" OH was proven to be necessary for minimizing the formation of byproducts and for allowing an easier purification after the regioselective protection of ring I, the key step of our strategy.After undergoing many frustrations attempting the regioselective protection of positions 2 and 3 in ring I of kanamycin A [23], we envisioned the use of 2,3-butanodione-bis-dimethyl acetal as a plausible strategy [24].Indeed, this reaction afforded an equimolar mixture of 10a and 10b, by protection of C-3 and C-4 hydroxyl groups and C-2 and C-3 , respectively, decreasing the total yield of the synthesis considerably given that 10a does not undergo the required β-elimination after periodate oxidation of the remaining diol.Despite this setback, this is to date the best procedure found for the selective protection of 2' and 3' hydroxyl groups of kanamycin A (1).In a next step, Williamson benzylation of the rest of the hydroxyl groups in compound 10b afforded 11 in 65% yield.Finally, the regioselective deprotection of the O-2 ,3 -butanodione-bis-dimethyl acetal using TFA provided diol 12 in 85% yield, the key intermediate in the preparation of 7.
the remaining diol.Despite this setback, this is to date the best procedure found for the selective protection of 2' and 3' hydroxyl groups of kanamycin A (1).In a next step, Williamson benzylation of the rest of the hydroxyl groups in compound 10b afforded 11 in 65% yield.Finally, the regioselective deprotection of the O-2′,3′-butanodione-bis-dimethyl acetal using TFA provided diol 12 in 85% yield, the key intermediate in the preparation of 7.
Oxidation of 12 was found to proceed efficiently with a moderate excess of sodium periodate in THF [25].Treatment of the resulting dialdehyde intermediate in methanol solution with triethylamine effected the desired β-elimination to give a solid product that was assigned as the expected pseudo-disaccharide structure 13.Finally, cleavage of the silyl-acetal function in the presence of TBAF, removal of the azide groups by Staudinger reduction and subsequent catalytic hydrogenolysis gave the desired pseudo-disaccharide 7 (Supplementary Material, Figure S1).Oxidation of 12 was found to proceed efficiently with a moderate excess of sodium periodate in THF [25].Treatment of the resulting dialdehyde intermediate in methanol solution with triethylamine effected the desired β-elimination to give a solid product that was assigned as the expected pseudo-disaccharide structure 13.Finally, cleavage of the silyl-acetal function in the presence of TBAF, removal of the azide groups by Staudinger reduction and subsequent catalytic hydrogenolysis gave the desired pseudo-disaccharide 7 (Supplementary Material, Figure S1).

Antibiotic Activity and Resistance Enzyme Susceptibility of 7
To assess the effect that ring I cleavage exhibits on the antibacterial capacity, minimum inhibitory concentration (MIC) values of the parent kanamycin A (1) and the fragments neamine (4) and pseudo-disaccharide 7, were measured against a battery of antibiotic non-resistant and resistant strains (Table 1).According to the obtained data, pseudo-disaccharide 7 showed an expected drop in efficacy (MIC = 50-100 µg mL −1 ) in comparison with the natural kanamycin A 1 (MIC = 1.5-6 µg mL −1 ).Remarkably, derivative 7 still represents an improvement over neamine (4), lowering the MIC value from 100 µg mL −1 to 50 µg mL −1 .Interestingly, according to the MIC values measured for compound 7 (Table 1, entries 5-6), while a complete loss of activity for antibiotic 1 and for neamine (4) was observed, fragment 7 maintains some activity against aminoglycoside inactivation performed by APH-(3 ) and AAC-( 6).These results are in agreement with the relative enzymatic activity observed: for APH-( 3) and for AAC-( 6) the rate of phosphorylation/acetylation (V rel (7)/V rel (1)) is zero, indicating that compound 7 is not inactivated by these enzymes.However, in the case of ANT(4 ), adenylation was much less effective [(V rel (5)/V rel (1) = 0.11], whereby position 4" of original III ring of 1 is being slowly modified [26].This is in agreement with results previously described by our group, where we demonstrated that ANT-(4 ) exhibits a remarkably low sensitivity toward the drug global shape and represents a paradigmatic example of substrate promiscuity [27].
Finally, we evaluated the capacity of kanamycin A (1), neamine (4) and pseudo-disaccharide 7 to bind the aforementioned enzymes ANT-(4 ), APH-(3 ) and AAC-(6 ) employing thermal melting shift experiments (Table 2).The change in unfolding transitions temperature (∆Tm) in the presence and in the absence of the ligands provides an estimation of the ligand/protein complex stability.Surprisingly, compound 7 proved to be an appropriate ligand not only for ANT-( 4), but also for AAC- (6 ) and APH-(3 ) even through it is not a substrate of these latter enzymes, producing clear thermal stabilizations of all of them (∆Tm = 5-7 • C).In conclusion, and considering the kinetic parameters, the estimated ligands binding affinity and the MIC values, compound 7 maintains a bactericidal activity even in resistant strains unlike neamine (4), where AAC-( 6) and APH-(3 ) were shown unable to modify the substrate and ANT-(4 ) only barely, while in all three cases 7 is a ligand of the enzymes.These results strongly suggest that compound 7 should be carefully considered in the design of novel antibiotics with improved activity against resistant strains and inhibitors of these aminoglycoside modifying enzymes.

General Procedures
All reactions were carried out in oven-dried glassware under a positive pressure of argon unless otherwise noted.Neomycin B and Kanamycin A free bases were prepared from the corresponding monosulfate salts (purchased from Santa Cruz Biotechnology, inc.and Sigma-Aldrich, respectively) by use of Amberlite-IRA 400 (OH − ) strongly basic ion-exchange resin.Solvents were dried in a Pure Solv system model PS-400-3-MD.Reactions were monitored by analytical thin-layer chromatography (TLC) on EM silica gel 60 F254 plates (0.25 mm), visualized by ultraviolet light and/or by staining with ceric ammonium molybdate, H 2 SO 4 or ninhydrin.Column chromatography was performed on Silice 60 (230-400 µM) and on Amberlite CG-50 (NH4 + ) cation exchange resin. 1 H NMR spectra were recorded on a Varian Inova-400 (400 MHz) and Varian UNITY 500 (500 MHz) in CDCl 3 , CD 3 OD and D 2 O solutions at ambient temperature.Data were reported as follows: chemical shift on the δ scale (either using TMS or residual proton solvent as internal standard), multiplicity (br = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constant(s) in hertz, and integration. 13C NMR spectra were recorded on a Varian Inova-400 (100 MHz) and Varian UNITY 500 (125 MHz).
Mass spectra were recorded on an AGILENT 6520 Accurate-Mass QTOF LC/MS spectrometer using the electrospray modes (ES).

MIC Determination
The selected bacterial strains were grown in Mueller-Hinton broth (1 mL) until an optical density at 600 nm (OD 600 ) of 0.5 units.At this time, kanamycin A (1), neamine (4) and pseudo-disaccharide 7, as free-bases, were added from stock solutions (0.5-400 ∝g/mL) prepared at different concentrations.These cultures were incubated at 37 • C for 24 h, after which the OD 600 of each sample was recorded.Herein, we considered the MIC as the lowest concentration of aminoglycoside which produced an inhibition of the bacterial growth greater than 90%.For resistant strains with overexpressed AAC-(6 )-Ib, ANT(4 ) or APH-(3 ) enzymes, E. coli (BL21) with the corresponding plasmids (pET-AAC(6 )-Ib, pET-ANT-(4 ), and pET-APH-(3 )) were induced with IPTG before adding the aminoglycosides.

Thermal Shift Assay
Thermal melting shift experiments were conducted on an iQ5 Real Time Detection System (Bio-Rad, Foster City, CA, USA) using the fluorescent dye SYPRO Orange [29].In a typical experiment, a solution of SYPRO Orange (2.8 mM) and protein (ANT-( 4

Figure 1 .
Figure 1.(A) Structure of kanamycin A (1), kanamycin B (2) and kanamycin C (3). (B) Ring I/II fragments (4-6) of kanamycins.The functional relevance of the drug/RNA contacts established through ring I amines are indicated in each case.Besides, typical ranges for the MIC values of natural kanamycins (1-3) and of the fragments thereof (4-6) are also presented.(C) Ring II/III fragment studied in this paper (7).

Figure 1 .
Figure 1.(A) Structure of kanamycin A (1), kanamycin B (2) and kanamycin C (3). (B) Ring I/II fragments (4-6) of kanamycins.The functional relevance of the drug/RNA contacts established through ring I amines are indicated in each case.Besides, typical ranges for the MIC values of natural kanamycins (1-3) and of the fragments thereof (4-6) are also presented.(C) Ring II/III fragment studied in this paper (7).