Our strategy is based on the fundamental observation that the key structural feature of (nearly) all aminoglycosides is not a carbohydrate but a diaminocyclohexitol ring termed 2-deoxystreptamine [44]. It was hypothesized that 2-deoxystreptamine is a crucial scaffold to build aminoglycoside libraries and that the all-equatorial substitution pattern is highly favorable to position other pharmacophores in the proper orientation. Although a large number of synthetic routes to 2-deoxystreptamine have been developed over the years [44], including contributions from our own lab [45,46,47,48], we realized that the most straightforward and cheapest route to 2-deoxystreptamine commences from natural neomycin. Apart from that, we surmised that partial degradation of neomycin would leave the ribofuranoside as a suitable substituent at the 5-position of 2-deoxystreptamine, as in structure 2 (Scheme 1). Thus, N-Boc-protected neomycin 1 [49] was reacted with 12 equivalents of sodium metaperiodate resulting in the oxidative cleavage of vicinal diols on both 2,6-diaminosugars. The intermediate tetraaldehyde was not purified due to its instability, but upon base treatment underwent smooth β-elimination under the influence of an amine base, to give 5-O-ribosyl-2-deoxystreptamine 2. Use of different bases like n-butyl amine, ammonium hydroxide and triethylamine gave the same results in terms of yield and reaction time. Importantly, 2 was purified by direct recrystallization from dichloromethane leading to highly pure product 2 in a 48% average yield over the two steps. The resulting pseudodisaccharide 2 was once again subjected to sodium metaperiodate cleavage of the diol to give a dialdehyde, this time followed by reductive amination with propargylamine, to afford morpholine 3 substituted with an acetylene moiety. Unfortunately, the formation of open chain diamine side products could not be fully avoided, thus significantly suppressing the yield.

Reagents and conditions: a) NaIO₄, H₂O, MeOH, overnight (o.n.); b) Et₃N, MeOH, o.n. (45–52%). c) i. NaIO₄, MeOH, H₂O, 2 h, ii. propargylamine, NaBH₃CN, AcOH, MeOH, 1 h (41%).

As aminoglycoside antibiotics function by selective recognition and binding to a specific RNA sequence, the removal of two of the aminoglycopyranosides is detrimental for RNA affinity. Therefore, we opted to restore the binding affinity by conjugation with other RNA ligands. To this end, we surmised that aminopyridines and aminoquinolines [50] could nicely serve our purpose because these molecules are of low molecular weight and contain no aliphatic amine functionality, but nevertheless have been reported to bind with E. coli A-site RNA in micromolar range. We selected the two tightest binders from the series of aminopyridines, e.g., 2-(2-aminoethylamino)-4-methylpyridine and 2-(2-aminoethylamino)-5-methylpyridine, and the best aminoquinoline ligand 2-(2-aminoethylamino)-4-methylquinoline (Scheme 2). In order to be able to conjugate the arylamine ligands to our morpholine compound, we designed a route involving reductive amination via the primary alcohol of 3. Therefore, we first prepared derivatives of the arylamines by treating the commercially available chloropyridines and a chloroquinoline with 1,2-ethylenediamine at 150 °C for 18 hours as shown in the Scheme 2, to afford the desired 2-aminoethyl modified arylamines 5–7 in reasonable yield.

Reagents and conditions: a) 1,2-ethylenediamine, 150 °C, 20 h (5: 46%, 6: 51%, 7: 48%).

Having the primary amines at hand, the next step involved the diversification of morpholine 3 by conjugation with the aminoethyl derivatives 5–7. For this purpose the primary hydroxyl of the compound 3 appeared most appropriate since it is most accessible and leaves the chiral secondary alcohols of 2-deoxystreptamine intact. Since reductive amination is such a robust conjugation technology, we aimed to selectively oxidize the primary alcohol of 3. Because it is known that Swern oxidations can be selectively executed at O-TES protected primary alcohols [51], we treated compound 3 with TESOTf and triethylamine in dichloromethane to protect all hydroxyls with the TES group. Subsequently, the triply TES-protected derivative was subjected to Swern oxidation conditions (oxalyl chloride, DMSO, -78 °C°0 °C), in order to selectively oxidize the primary hydroxyl. However, an inseparable mixture of compounds was obtained (Table 1, entry 1).

Because Swern oxidation of TES ethers was not successful, we opted for a selective oxidation of the primary alcohol withoutprotective groups. However, attempts to oxidize the primary hydroxyl with TCCA/TEMPO [52] (entry 2) or IBX [53] (entry 3) gave no or less than 10% conversion, respectively. Finally, we succeeded (entry 4) in oxidizing the primary hydroxyl group with Dess-Martin periodinane [54] to give selectively the desired aldehyde 4b.However, also in this case the reaction did not go to completion, reaching a maximum of about 60% conversion. Further attempts to optimize the reaction conditions, e.g., variation in temperature, solvents or stoichiometry of the oxidant did not improve the outcome.

The resulting aldehyde 4b was subjected to the next reaction without purification on account of its instability. We therefore proceeded to couple crude aldehyde 4b with aminopyridine and aminoquinoline derivatives 5–7 (Scheme 3). Therefore, compounds 5–7 were condensed with aldehyde 4b employing standard reductive amination conditions, e.g., NaBH₃CN and AcOH in methanol. The relatively low yields of the resulting compounds 8a-c can be ascribed to incomplete oxidation of the compound 3 in the proceeding step, apart from the fact that accompanying dialkylation of amines was observed during the reductive amination. However, in this case the desired conjugates 9a-c could be readily purified by silica gel column chromatography. Both for this purpose and for follow-up chemistry, it was found most convenient to convert the resulting secondary amino functions of the initial products 8a-c into Boc-protected carbamates under the influence of Boc₂O and DMAP in CH₂Cl₂. Thus, heteroconjugates 9a-c of truncated neomycin with non-aminoglycoside type RNA ligands were successfully prepared.

Obviously, the obtained arylamine-morpholine-2-deoxystreptamine conjugates contain a functional handle at the morpholine ring in the form of the propargyl moiety. Based on the popular copper(I)-catalyzed azide acetylene cycloaddition (CuAAC) [55,56], it is clear that the propargyl moiety provides an excellent handle for the preparation of further conjugates. Therefore, we next utilized the copper-catalyzed (3+2) cycloaddition chemistry to make immers with a range of mono- and bisazido-functionalized linkers. This approach provided maximum synthetic flexibility and proved effective for the synthesis of various monomers and immers from the building blocks 9a-c. Thus, the synthetic heteroconjugates were treated with a monoazide (benzyl azide, 1 equiv.) or bisazides A-E (0.5 equiv.) in a mixture of water and acetonitrile in the presence of copper-wire [57] to make the respective monomers 9a-cE and dimers 9a-cA to 9a-cD in reasonable to good yields (Table 2). Finally, acidic deprotection of the Boc protective groups was performed in a 1:2 mixture of TFA and DCM to afford the respective monomers and the dimers in quantitative yields, giving a final set of 15 arylamine-aminoglycoside conjugates (3 monomers, 12 dimers). For biological evaluation, all of the compounds were purified by reversed phase HPLC, giving the pure conjugates in 5–20 mg quantitities.

Reagents and conditions: a) L, CH₃CN, H₂O, Cu wire, o.n. b) CH₂Cl₂, TFA, 3 h (quantitative).

Having prepared the desired conjugates, we were interested to investigate the antibacterial activity of these compounds. Determination of antibacterial activity is normally expressed in MIC (minimal inhibitory concentration) by growing bacteria in medium containing increasing amounts of the presumed antibiotic. Such an assay typically requires substantial amounts of compounds (30–70 mg), clearly exceeding the amount of compound that we had prepared. With small amounts of substrate, antibacterial activity can also be determined with Kirby-Bauer disc test, but that provides only a qualitative measure of activity. Alternatively, a plethora of RNA-binding assays have been developed [58,59,60,61,62,63,64,65,66,67,68,69], but a strong drawback of such assays is that they do not include cell-wall penetration or resistance mechanisms, obviously key in determining the overall activity of an antibiotic [70]. An alternative approach, recently reported by some of us [71], determines MICs on whole bacteria in a simple and rapid assay and moreover requires only minimal amounts (< 2 mg) of substrate. The technique relies on a fluorescence-based cell viability assay and involves a hemocyanin-based oxygen biosensor to monitor oxygen consumption of bacteria and subsequently bacterial cell growth (Figure 1).

Deoxygenated hemocyanin has no absorption in the visible spectrum but upon oxygenation a strong band centered at 340 nm and a weak band around 570 nm appear (Figure 1B). If a fluorescent label is attached to the N-terminus of hemocyanin, this change in absorption can be translated into a change of fluorescence intensity of an attached label (Cy3, Cyanine 3 fluorescent dye) through a Förster resonance energy transfer (FRET) mechanism (Figure 1A) [72]. The efficiency of this energy transfer depends on the distance and orientation between the donor and the acceptor and also on the overlap integral between the donor emission and acceptor absorption spectra (Figure 1C). In this way, when the protein is in its deoxygenated state (Cy3-Deoxy-Hemocyanin), all the energy absorbed by the label is emitted normally as fluorescence. Upon oxygenation of hemocyanin (Cy3-Oxy-Hemocyanin), energy is transferred from Cy3 by radiationless decay to the copper center of hemocyanin, resulting in a decrease in the fluorescence intensity. Thus, when a bacterial culture grows aerobically, oxygen in the medium is gradually consumed. This results in a drop of the oxygen concentration, eventually resulting in an anaerobic medium. When hemocyanin-based oxygen biosensor is present, this oxygen depletion results in an increase of the fluorescence intensity emitted by the Cy3 label attached to protein [71].Error! Bookmark not defined. Tests were carried out as describedError! Bookmark not defined. [71] with an initial cell concentration of 10⁵ cfu/mL.

We determined the MICs of our heteroconjugates with the fluorescence-based cell viability assay against several bacterial strains (E. coli, E. coli kan.^r e.g.,kanamycin-resistant E. coli)using kanamycin as a positive control. As can be seen from Figure 2, using compound 9cA and 9cB as an example, the MIC is determined by performing the bacterial growth in medium containing increasing amounts of our compounds. The MIC values of our synthetic analogues can be determined from Figure 2 and are depicted in Table 3.

Unfortunately, our set of dimers of heteroconjugates exhibited lower antibacterial activity than that of natural kanamycin when tested against E. coli. In fact, only compound 9cB demonstrated reasonable antibacterial activity. Moreover, and in contrast to expectation, it is clear that the dimers do not show improved activity compared to the monomeric substrates 9a-cE.

Reagents and conditions: a) R-N₃, CH₃CN, H₂O, Cu-wire, 16 h (70–80%). b) CH₂Cl₂, TFA, 3 h (quantitative); ^a azides were reduced with PMe₃ prior to Boc removal.

The lack of improved activity is in contrast to findings by other groups [34,35,36,37,38,39,40,41,42,43], which may be explained in our case by the relatively short linkers employed. It is not excluded that a short linker is precluding cooperative binding of the other end of the molecule to a second binding pocket. Whatever the reason, the fact that the MIC value of the monomeric compound 9cE was comparable to that of different dimers stimulated us to explore whether antibacterial activity of the more simple, monomeric heteroconjugates could be further raised by modification of the azide partner for cycloaddition to the acetylene. Thus, we selected a range of aromatic and aliphatic azides and coupled these to acetylene derivatives 9a-c with CuAAC to obtain a set of monomeric compounds 10a-g with different substituents (Scheme 5).Thus, compounds 10a–e were synthesized by simply treating 9c with different monoazides in a mixture of water and acetonitrile in the presence of copper wire. For the synthesis of amino-terminated compounds 10f and 10g, acetylene 9c was treated with an excess of 1,4-bis(azidomethyl)benzene or 1,5-diazidopentane, to give the respective monosubstituted triazole adducts, followed by Staudinger reduction of the unreacted azide group at the other chain terminus under the action of PMe₃. Again, all compounds 10a-g were purified to homogeneity by reversed-phased HPLC before evaluation of antibacterial activity. Next, we tested the antibacterial activity of this set of compounds employing the same fluorescence-based cell viability assay and the results are summarized in Table 3. As evident from MIC values, para-bromo substitution of the aromatic ring enhanced the antibacterial activity of compounds 10a and 10b. The rest of the monomeric compounds, with a methoxy- or methylamino-substituent on the aromatic ring 10c-10f,or with the aliphatic chain 10g showed a reduced antibacterial activity compared to that of unsubstituted parent compound 9cE. Finally we tested the MIC of the most active compounds against the kanamycin resistant strains of E. coli (Table 4, entry 2). We were happy to find that compounds 10a and 10b have distinct antibacterial activity against E. coli. More importantly, compound 10b displayed an improved activity against the kanamycin-resistant E. coli strain revealing that omission of the diaminoglycosides indeed fulfills our desired aim to reduce susceptibility to resistance mechanisms. The search for further synthetic analogues of aminoglycosides with such improved traits is currently ongoing in our laboratory.

TLC analyses are performed on silica gel-coated plates (Merck 60 F254) with the indicated solvent mixture. Compounds were detected with ammonium molybdate or potassium permanganate staining, or UV light. Flash column chromatography was carried out using ACROS silica gel (0.035–0.070 mm, ca 6 nm pore diameter). Solvents were distilled from appropriate drying agents prior to use. Unless stated otherwise, all chemicals were purchased and used as such. IR spectra were recorded on an ATI Mattson Genesis Series FTIR spectrometer; absorption reported in cm^-1. NMR spectra were recorded on a Bruker DMX 300 (75 MHz), and a Varian 400 (400 MHz) spectrometer. ¹H-NMR and ¹³C-NMR spectra are reported in ppm units on the δ scale. Coupling constants are reported as J-values in Hz. Semipreparative RP-HPLC was performed using a C₁₈ column (10 × 250 mm, 5 µm) with HPLC grade MilliQ containing 0.1% TFA (eluant A) and acetonitrile containing 0.1% TFA (eluant B) with a gradient of 5 → 50% of B in 30 min. Compounds were detected by UV absorption at 215 and 254 nm.

To a solution of neomycin B (2 g, 2.2 mmol) in a mixture of H₂O/1,4-dioxane (30 mL, 1:1 v/v) was added Na₂CO₃ and di-tert-butyl dicarbonate. The reaction mixture was stirred at room temperature for 6 h. Dioxane was removed by evaporation and the residue was partitioned between EtOAc (100 mL) and H₂O (100 mL). The aqueous layer was extracted with EtOAc (2 ×50 mL) and the combined organic layers werewashed with brine (100 mL), dried (Na₂SO₄) and concentrated in vacuo. Purification by column chromatography (MeOH/CH₂Cl₂, 1/9) afforded 1 (2.2 g, 82%) as an amorphous white solid. R_F 0.49 (MeOH/CH₂Cl₂, 1/9). HRMS (ESI) m/z calcd. for C₅₃H₉₄N₆O₂₅ (M+Na)⁺: 1237.6166, found: 1237.6141.

 To a solution of 1 (1 g, 0.82 mmol) in 25 mL of MeOH, was added 25 mL of a H₂O solution of NaIO₄ (2.12 g, 9.8 mmol). The reaction mixture was stirred at room temperature for 16 h. Methanol was removed by evaporation and the residue was partitioned between EtOAc (100 mL) and H₂O (50 mL). The aqueous layer was extracted with EtOAc (2 × 50 mL) and the combined organic layers werewashed with brine (100 mL), dried (Na₂SO₄) and concentrated in vacuo. The residue was dissolved in MeOH (30 mL), and Et₃N (2 mL) was added. The reaction mixture was stirred at room temperature for 16 h and then concentrated in vacuo. Purification by crystallization from CH₂Cl₂ (100 mL) afforded compound 2 (212 mg, 52%) as a white amorphous solid. R_F 0.12 (MeOH/CH₂Cl₂, 1/9); ¹H-NMR (MeOD, 400 MHz) δ 5.21 (s, 1H), 4.32 (dd, J =4.5, 7.7 Hz, 1H), 3.99 (d, J =4.5 Hz, 1H), 3.90 (dt, J =2.6, 7.7 Hz, 1H), 3.78 (dd, J =2.3, 12.1 Hz, 1H), 3.60 (dd, J =2.6, 12.1 Hz, 1H), 3.40–3.15 (m, 10H), 2.02 (dt, J =4.2, 7.9 Hz, 1H), 1.41 (s, 18H), 1.21 (m, 1H); ¹³C-NMR (MeOD, 75 MHz): δ 156.3, 107.8, 83.8, 82.3, 78.2, 74.7, 72.7, 68.6, 59.7, 50.6, 33.8,26.8; HRMS (ESI) m/z calcd. for C₂₁H₃₈N₂O₁₁ (M+Na)⁺: 517.2373, found: 517.2375.

To a solution of 2 (830 mg, 1.68 mmol) in 20 mL of MeOH, was added 20 mL of a H₂O solution of NaIO₄ (431 mg, 2.016 mmol). The reaction mixture was stirred at room temperature for 2 h. The methanol was removed by evaporation and the residue was partitioned between EtOAc (100 mL) and H₂O (50 mL). The aqueous layer was extracted with EtOAc (2 ×50 mL) and the combined organic layers werewashed with brine (100 mL), dried (Na₂SO₄) and concentrated in vacuo. The residue was dissolved in 15 mL of MeOH, and NaBH₃CN (422 mg, 6.72 mmol) was added. To the resulting mixture was added drop-wise a solution of propargylamine (138 μL, 2.52 mmol) and AcOH (403 μL, 6.72 mmol) in MeOH (1 mL). The reaction mixture was stirred at room temperature for 2 h. The reaction was then quenched with 0.5 mL of Et₃N and concentrated in vacuo. The residue was taken up in EtOAc (100 mL), washed with saturated NaHCO₃ (2 × 50 mL) and brine (50 mL). The organic layer was dried (Na₂SO₄), filtered and concentrated in vacuo.Purification by column chromatography (MeOH/CH₂Cl₂, 1/9) afforded 1 (358 mg, 41%) as an amorphous white solid. R_F 0.34 (MeOH/CH₂Cl₂, 1/9); ¹H-NMR (MeOD, 400 MHz) δ 4.85 (dd, J = 2.3, 8.6 Hz, 1H), 3.70 (m, 1H), 3.57 (dd, J = 1.7, 5.1Hz, 2H), 3.42–3.19 (m, 11H), 3.01 (d, J = 10.5 Hz, 1H), 2.70 (d, J = 11.1 Hz, 1H), 2.65 (t, J = 2.3 Hz, 1H), 2.13 (m, 2H), 2.02 (dt, J = 3.9, 13.0 Hz, 1H), 1.41 (s, 18H), 1.21 (dd, J = 12.5 Hz, 1H). ¹³C-NMR (MeOD, 75 MHz): δ 156.2, 99.7, 85.2, 78.1, 76.7, 74.1, 73.5, 72.5, 61.9, 54.1, 51.3, 50.6, 50.4, 45.1, 33.6, 26.8; HRMS (ESI) m/z calcd. for C₂₄H₄₁N₃O₉ (M+H)⁺: 516.2921, found: 516.2925.

A solution of 2-chloro-4-methylpyridine (0.876 mL, 10 mmol) in 1,2-diaminoethane (6 mL) was heated at 150°C for 18 h. The volatiles were removed in vacuo. Purification by column chromatography (10% MeOH/CH₂Cl₂ to 20% MeOH/CH₂Cl₂) afforded 5 (700 mg, 46%) as a yellow oil; ¹H-NMR (MeOD, 300 MHz) δ 7.76 (d, J = 4.8 Hz, 1H), 6.36 (m, 2H), 3.31 (t, J = 6.2 Hz, 2H), 2.79 (t, J = 6.2 Hz, 2H), 2.17 (s, 3H); ¹³C-NMR (MeOD, 75 MHz): δ 156.5, 145.0, 136.2, 121.1, 106.6, 40.1, 39.9, 15.5.HRMS (ESI) m/z calcd. for C₈H₁₃N₃ (M+H)⁺: 152.1187, found: 152.1199.

 A solution of 2-chloro-5-methylpyridine (0.434 mL, 5 mmol) in 1,2-diaminoethane (3 mL) was heated at 150°C for 18 h. The volatiles were removed in vacuo. Purification by column chromatography (10% MeOH/CH₂Cl₂ to 20% MeOH/CH₂Cl₂) afforded 6 (386 mg, 51%) as a yellow oil; ¹H-NMR (MeOD, 300 MHz) δ 7.72 (m, 1H), 7.27 (dd, J = 2.5, 8.5 Hz, 1H), 6.46 (d, J = 8.5 Hz, 1H), 3.30 (t, J = 6.2 Hz, 2H), 2.79 (t, J = 6.2 Hz, 2H), 2.13 (s, 3H). ¹³C-NMR (MeOD, 75 MHz): δ 156.6, 145.3, 138.0, 120.3, 107.7, 43.5, 40.1, 15.5; HRMS (ESI) m/z calcd. For C₈H₁₃N₃ (M+H)⁺: 152.1187, found: 152.1199.

 A solution of 2-chloro-4-methylquinoline (1.7 g, 10 mmol) in 1,2-diaminoethane (6 mL) was heated at 150°C for 18 h. The volatiles were removed in vacuo. Purification by column chromatography (10% MeOH/CH₂Cl₂ to 20% MeOH/CH₂Cl₂) afforded 7 (963 mg, 48%) as a yellow oil; ¹H-NMR (MeOD, 300 MHz) δ 7.72 (dd, J = 1.2, 8.1 Hz, 1H), 7.59 (dq, J = 0.5, 1.2, 8.4 Hz, 1H), 7.45 (ddd, J = 1.4, 6.9, 8.4 Hz, 1H), 7.17 (ddd, J = 1.2, 6.9, 8.1 Hz, 1H), 6.59 (d, J = 1.0 Hz, 1H), 3.50 (t, J = 6.2 Hz, 2H), 2.86 (t, J = 6.2 Hz, 2H), 2.48 (d, J = 1.0 Hz, 3H). ¹³C-NMR (MeOD, 75 MHz): δ 156.9, 147.0, 144.2, 128.4, 124.5, 123.0, 122.8, 120.9, 111.9, 42.6, 40.3, 16.9; HRMS (ESI) m/z calcd. for C₁₂H₁₅N₃ (M+H)⁺: 202.1344, found: 202.1335.

To a solution of 3 (65 mg, 0.126 mmol) in DMF (1 mL), was added Dess-Martin periodinane (64 mg, 0.151mmol). The reaction mixture was stirred overnight at room temperature. The reaction was quenched with a saturated solution of NaHCO₃ (20 mL) and extracted with EtOAc (3 ×25 mL). The combined organic layers were washed with H₂O (2 × 25 mL) and brine (25 mL), dried (Na₂SO₄) and concentrated in vacuo. The residue was dissolved in 2 mL of MeOH, and NaBH₃CN (12 mg, 0.189 mmol) was added. To the reaction mixture was added a solution of 5 (57 mg, 0.378 mmol) and AcOH (11 μL, 0.189 mmol) in MeOH (0.5 mL). The reaction mixture was stirred at room temperature for 4 h. The reaction was quenched with 0.1 mL of Et₃N and concentrated in vacuo. The residue was taken up in EtOAc (50 mL), washed with saturated NaHCO₃ (2 × 25 mL) and brine (25 mL). The organic layer was dried (Na₂SO₄), filtered and concentrated in vacuo. Purification by column chromatography (MeOH/CH₂Cl₂, 1/9) afforded 8a (32 mg, 39%) as a white solid. R_F 0.22 (MeOH/CH₂Cl₂, 1/9); ¹H- NMR (MeOD, 400 MHz) δ 7.89 (d, J =  5.4 Hz, 1H), 6.52 (d, J = 5.4 Hz, 1H), 6.47 (s, 1H), 5.03 (d, J = 7.5 Hz, 1H), 3.96 (m, 1H), 3.57 (q, J = 5.6, 10.3 Hz, 2H), 3.41–3.15 (m, 12H), 2.94 (d, J = 10.3 Hz, 1H), 2.70 (m, 2H), 2.29 (dd, J = 7.7, 11.0 Hz, 1H), 2.22 (s, 3H), 1.99 (m. 1H), 1.41 (d, J= 8.4 Hz, 18H), 1.24 (m, 1H); ¹³C-NMR (MeOD, 75 MHz): δ 156.3, 156.2, 149.1, 144.9, 126.9, 114.3, 109.2, 99.5, 83.2, 78.2, 76.4, 74.8, 73.7, 72.8, 69.1, 54.0, 51.5, 48.7, 45.0, 38.2, 33.8, 26.8, 19.2; HRMS (ESI) m/z calcd. for C₃₂H₅₂N₆O₈ (M+H)⁺: 649.3924, found: 649.3923.

 To a solution of 3 (80 mg, 0.155 mmol) in DMF (1 mL), was added Dess-Martin periodinane (79 mg, 0.186 mmol). The reaction mixture was stirred overnight at room temperature. The reaction was quenched with a saturated solution of NaHCO₃ (20 mL) and extracted with EtOAc (3 × 25 mL). The combined organic layers were washed with H₂O (2 × 25 mL) and brine (25 mL), dried (Na₂SO₄) and concentrated in vacuo. The residue was dissolved in 3 mL of MeOH, and NaBH₃CN (14 mg, 0.232 mmol) was added. To the resulting mixture was added a solution of 6 (70 mg, 0.465 mmol) and AcOH (14 μL, 0.232 mmol) in MeOH (0.5 mL). The reaction mixture was stirred at room temperature for 4 h. The reaction was quenched with 0.1 mL of Et₃N and concentrated in vacuo. The residue was taken up in EtOAc (50 mL), washed with saturated NaHCO₃ (2 × 25 mL) and brine (25 mL). The organic layer was dried (Na₂SO₄), filtered and concentrated in vacuo. Purification by column chromatography (MeOH/CH₂Cl₂, 1/9) afforded 8b (41 mg, 41%) as a white solid. R_F 0.22 (MeOH/CH₂Cl₂, 1/9); ¹H- NMR (MeOD, 400 MHz) δ 7.74 (s, 1Η),7.28 (dd, J = 2.3, 8.5 Hz, 1H), 6.5 (d, J = 8.5 Hz, 1H), 4.85 (dd, J = 2.3, 8.6 Hz, 1H), 3.80 (m, 1H), 3.42–3.19 (m, 11H), 2.99 (d, J = 10.3 Hz, 1H), 2.18–2.00 (m, 5H), 1.40 (d, J = 7.2 Hz, 18H), 1.26 (m, 1H); ¹³C-NMR (MeOD, 75 MHz): δ 156.6, 156.3, 156.1, 145.1, 138.2, 120.5, 108.1, 99.8, 85.2, 78.2, 76.6, 76.2, 74.2, 73.5, 72.6, 71.7, 54.1, 52.5, 50.5, 50.0, 45.0, 39.9, 33.8, 26.8, 15.4; HRMS (ESI) m/z calcd. for C₃₂H₅₂N₆O₈ (M+H)⁺: 649.3924, found: 649.3923.

 To a solution of 3 (100 mg, 0.194 mmol) in DMF (1 mL), was added Dess-Martin periodinane (99 mg, 0.232 mmol). The reaction mixture was stirred overnight at room temperature. The reaction was quenched with saturated solution of NaHCO₃ (20 mL) and extracted with EtOAc (3 × 25 mL). The combined organic layers were washed with H₂O (2 × 25 mL) and brine (25 mL), dried (Na₂SO₄) and concentrated in vacuo. The residue was dissolved in 4 mL of MeOH, and NaBH₃CN (18 mg, 0.291 mmol) was added. To the resulting mixture was added a solution of 7 (116 mg, 0.582 mmol) and AcOH (17.5 μL, 0.291 mmol) in MeOH (0.5 mL). The reaction mixture was stirred at room temperature for 4 h. The reaction was quenched with 0.1 mL of Et₃N and concentrated in vacuo. The residue was taken up in EtOAc (50 mL), washed with saturated NaHCO₃ (2 × 25 mL) and brine (25 mL). The organic layer was dried (Na₂SO₄), filtered and concentrated in vacuo. Purification by column chromatography (MeOH/CH₂Cl₂, 1/9) afforded 8c (52 mg, 38%) as a white solid. R_F 0.21 (MeOH/CH₂Cl₂, 1/9); ¹H-NMR (MeOD, 400 MHz) δ 7.84 (d, J = 8.1 Hz, 1H), 7.62 (d, J = 3.4 Hz, 1H), 7.30 (q, J = 4.1, 8.1 Hz, 1H), 6.72 (d, J = 1.0 Hz, 1H), 5.06 (d, J = 6.3 Hz, 1H), 3.97 (m, 1H), 3.75 (t, J = 5.0 Hz, 2H), 3.44–3.18 (m, 13H), 2.95 (d, J = 11.0 Hz, 1H), 2.72–2.65 (m, 2H), 2.57 (d, J = 1.0 Hz, 3H), 2.30 (dd, J = 7.7, 11.0 Hz, 1H), 2.17 (dd, J = 9.3, 11.1 Hz, 1H), 2.03 (dt, J = 3.8, 12.5 Hz, 1H), 1.42 (d, J = 9.1 Hz, 18H), 1.32 (m, 1H); ¹³C-NMR (MeOD, 75 MHz): δ 156.7, 145.9, 145.2, 129.4, 123.7, 123.3, 123.1, 122.0, 112.0, 99.6, 83.5, 78.2, 76.4, 74.7, 73.7, 72.8, 69.2, 54.0, 51.6, 49.1, 48.5, 45.0, 38.4, 26.9, 17.0; HRMS (ESI) m/z calcd. for C₃₆H₅₄N₆O₈ (M+H)⁺: 699.4081, found: 699.4062.

To a solution of 8a-c (0.15 mmol) in CH₂Cl₂ (2 mL) was added di-tert-butyl dicarbonate (0.18 mmol, 1.2 equiv.) and DMAP (5 mg). The reaction mixture was stirred at room temperature for 5 h. The volatiles were removed in vacuo. Purification by column chromatography (MeOH/CH₂Cl₂, 1/9) gave the desired product.

5-O-(N-propyn-1-yl-2-(methylamino-N-(Tert-Butoxycarbonyl)ethylamino-N -4-methylpyridin-2-yl)-

morpholino)-2-deoxystreptamine 9a:Yield 87%, white solid. R_F 0.43 (MeOH/CH₂Cl₂, 1/9); HRMS (ESI) m/z calcd. for C₃₇H₆₀N₆O₁₀ (M+H)⁺: 749.4449, found: 749.4434.

5-O-(N-propyn-1-yl-2-(methylamino-N-(Tert-Butoxycarbonyl)ethylamino-N -5-methylpyridin-2-yl)-orpholino)-2-deoxystreptamine 9b: Yield 90%, white solid. R_F 0.43 (MeOH/CH₂Cl₂, 1/9); HRMS (ESI) m/z calcd. for C₃₇H₆₀N₆O₁₀ (M+H)⁺: 749.4449, found: 749.4434.

5-O-(N-propyn-1-yl-2-(methylamino-N-(Tert-Butoxycarbonyl)ethylamino-N -4-methylquinolin-2-yl)-morpholino)-2-deoxystreptamine 9c: Yield 84%, white solid. R_F 0.46 (MeOH/CH₂Cl₂, 1/9); HRMS (ESI) m/z calcd. for C₄₁H₆₂N₆O₁₀ (M+H)⁺: 799.4605, found: 799.4587.

To a solution of a commercially obtained dibromide (2.5 mmol) in 10 mL of DMF was added NaN₃ (6.0 mmol). The reaction mixture was stirred at 60 °C for 5 h. The reaction was then quenched with H₂O and extracted with Et₂O. The organic layers were washed with H₂O and brine, dried (Na₂SO₄) and concentrated in vacuo. Purification by column chromatography (EtOAc/heptane, 1/20) gave the pure product.

1,4-bis(Azidomethyl)benzene(A): Yield 86%, oil. R_F 0.34 (EtOAc/heptane, 1/8); ¹H-NMR (CDCl₃, 300 MHz) δ 7.34 (s, 4H), 4.35 (s, 4H); ¹³C-NMR (CDCl₃, 75 MHz): δ 135.0, 128.1, 53.9.

1,2-bis(Azidomethyl)benzene (B): Yield 82%, oil. R_F 0.36 (EtOAc/heptane, 1/8); ¹H-NMR (CDCl₃, 300 MHz) δ 7.38 (s, 4H), 4.43 (s, 4H); ¹³C-NMR (CDCl₃, 75 MHz): δ 133.4, 129.6, 128.5, 51.7.

1,5-Diazidopentane (C): Yield 91%, liquid. R_F 0.49 (EtOAc/heptane, 1/8); ¹H-NMR (CDCl₃, 300 MHz) δ3.25 (t, J = 6.7 Hz, 4H), 1.60 (m, 4H), 1.42 (m, 2H); ¹³C-NMR (CDCl₃, 75 MHz) δ 50.7, 27.9, 23.4.

1,4-Diazidobutane (D): Yield 86%, liquid. R_F 0.51 (EtOAc/heptane, 1/8); ¹H-NMR (CDCl₃, 300 MHz) δ 3.27(t, J = 5.7 Hz, 4H), 1.61 (m, 4H); ¹³C-NMR (CDCl₃, 75 MHz) δ 50.4, 25.6.

To a solution of 1-bromo-4-(bromomethyl)benzene (200 mg, 0.80 mmol) in 1 mL of DMF was added NaN₃ (52 mg, 0.80 mmol). The reaction mixture was stirred at 60 °Cfor 5 h. The reaction was then quenched with H₂O and extracted with Et₂O. The organic layers were washed with H₂O and brine, dried (Na₂SO₄)and concentrated in vacuo. Purification by column chromatography (EtOAc/heptane, 1/20) gave 1-bromo-4-(azidomethyl)benzene (156 mg, 92%) as a colorless liquid. R_F 0.51 (EtOAc/heptane, 1/8); ¹H-NMR (CDCl₃, 400 MHz) δ 7.47 (d, J = 8.4 Hz, 2H), 7.15 (d, J = 8.4 Hz, 2H), 4.24 (s, 2H); ¹³C-NMR (CDCl₃, 75 MHz) δ 133.9, 131.5, 129.3, 121.8, 53.6.

To a solution of NaN₃ (1.5 g, 24.3mmol) in a mixture of H₂O/CH₂Cl₂ (7.5 mL, 1:1 v/v) at 0 °C, was added Tf₂O (2.01 mL, 12.15mmol). The reaction mixture was stirred at room temperature for 2 h. After quenching with aqueous NaHCO₃, the layers were separated and the aqueous layer was extracted with CH₂Cl₂ (5.75 mL). The organic layers were combined to afford 9.5 mL of TfN₃ solution. Then, to a solution of para-bromoaniline (700mg, 4.06mmol) and CuSO₄ (5 mg) in H₂O (9.5 mL) was added the TfN₃ solution, MeOH (31 mL) and Et₃N (1.6 mL). The reaction mixture was stirred overnight at room temperature. Then solid NaHCO₃ (1.0 g) was added carefully and the organic solvents were evaporated. The aqueous residue was extracted with EtOAc (3 × 100 mL), and the organic layers were combined, dried (Na₂SO₄), and concentrated in vacuo to give a yellow liquid. Purification by column chromatography (EtOAc/heptane, 1/20) afforded 1-azido-4-bromobenzene (630mg, 78%) as a colorless liquid. R_F 0.61 (EtOAc/heptane, 1/8); ¹H-NMR (CDCl₃, 400 MHz) δ 7.44 (d, J = 8.9 Hz, 2H), 6.89 (d, J = 8.9 Hz, 2H); ¹³C-NMR (CDCl₃, 75 MHz) δ 138.7, 132.3, 120.1, 117.2.

To a solution of 1-(chloromethyl)-4-methoxybenzene (400 μL, 2.9 mmol) in 2 mL of DMF was added NaN₃ (188 mg, 2.9 mmol). The reaction mixture was stirred at 60 °C for 5 h. The reaction was then quenched with H₂O and extracted with Et₂O. The organic layers were washed with H₂O and brine, dried (Na₂SO₄) and concentrated in vacuo. Purification by column chromatography (EtOAc/heptane, 1/20) gave 1-(azidomethyl)-4-methoxybenzene (410 mg, 87%) as a colorless liquid. R_F 0.36 (EtOAc/heptane, 1/8); ¹H-NMR (CDCl₃, 400 MHz) δ 7.23(d, J = 8.8 Hz, 2H), 6.90 (d, J = 8.8 Hz, 2H), 4.24 (s, 2H), 3.78 (s, 3H); ¹³C-NMR (CDCl₃, 75 MHz) δ 159.2, 129.2, 126.9, 113.7, 54.7, 53.9.

To a solution of NaN₃ (1.5 g, 24.3mmol) in a mixture of H₂O/CH₂Cl₂ (7.5 mL, 1:1 v/v) at 0 °C, was added Tf₂O (2.01 mL, 12.15mmol). The reaction mixture was stirred at room temperature for 2 h. After quenching with aqueous NaHCO₃, the layers were separated and the aqueous layer was extracted with CH₂Cl₂ (5.75 mL). The organic layers were combined to afford 9.5 mL of TfN₃ solution. Then, to a solution of para-methoxyaniline (500mg, 4.06mmol) and CuSO₄ (5 mg) in H₂O (9.5 mL) was added the TfN₃ solution, MeOH (31 mL) and Et₃N (1.6 mL). The reaction mixture was stirred overnight at room temperature. Then solid NaHCO₃ (1.0 g) was added carefully and the organic solvents were evaporated. The aqueous residue was extracted with EtOAc (3 × 100 mL), and the organic layers were combined, dried (Na₂SO₄), and concentrated in vacuo to give yellow liqued. Purification by column chromatography (EtOAc/heptane, 1/20) afforded 1-azido-4-methoxybenzene (490mg, 81%) as a colorless liquid. R_F 0.42 (EtOAc/heptane, 1/8); ¹H-NMR (CDCl₃, 400 MHz) δ 6.94 (m, 2H), 6.87 (m, 2H), 3.78 (s, 3H); ¹³C-NMR (CDCl₃, 75 MHz) δ156.5, 131.8, 119.5, 114.6, 55.0.

To a solution of 1-(chloromethyl)-2-methoxybenzene (400 μL, 2.9 mmol) in 2 mL of DMF was added NaN₃ (188 mg, 2.9 mmol). The reaction mixture was stirred at 60 °C for 5 h. The reaction was then quenched with H₂O and extracted with Et₂O. The organic layers were washed with H₂O and brine, dried (Na₂SO₄) and concentrated in vacuo. Purification by column chromatography (EtOAc/heptane, 1/20) gave 1-(azidomethyl)-2-methoxybenzene (406 mg, 86%) as a colorless liquid. R_F 0.44 (EtOAc/heptane, 1/8). ¹H-NMR (CDCl₃, 400 MHz) δ 7.29(m, 2H), 6.94 (m, 2H), 4.35 (s, 2H), 3.84 (s, 3H).¹³C-NMR (CDCl₃, 75 MHz) δ 157.2, 129.6, 129.3, 123.4, 120.1, 110.1, 54.8, 49.7.

Dimer9aC: Yield 61%, white solid. ¹H-NMR (MeOD, 400 MHz): δ 7.95 (s, 2H), 7.78 (d, J = 6.4 Hz, 2H), 6.91 (s, 2H), 6.81 (dd, J = 6.6, 1.4 Hz, 2H), 5.09 (d, J = 7.7 Hz, 2H), 4.36 (t, J = 7.0 Hz, 4H), 4.10 (m, 2H), 3.89 (s, 2H), 3.77 (t, J = 6.0 Hz, 4H), 3.65 (t, J = 9.2 Hz, 2H), 3.48 (m, 6H), 3.35 (m, 4H), 3.21 (m, 6H), 2.39 (s, 6H), 2.36 (dt, J = 8.2, 3.8 Hz, 2H), 1.91 (m, 4H), 1.72 (q, J = 12.4 Hz, 2H), 1.27 (m, 2H). HRMS (ESI) m/zcalcd for C₄₉H₈₂N₁₈O₈ (M+H)⁺: 1051.6641, found: 1051.6577; RP-HPLC: t_R = 16.33 min.

Dimer9aD: Yield 60%, white solid. HRMS (ESI) m/z calcd. for C₄₈H₈₀N₁₈O₈ (M+H)⁺: 1037.6484, found: 1037.6448; RP-HPLC: t_R = 16.28 min.

Dimer 9aE: Yield 87%, white solid. HRMS (ESI) m/z calcd. for C₂₉H₄₃N₉O₄ (M+H)⁺: 582.3516, found: 582.3516; RP-HPLC: t_R = 15.12 min.

Dimer 9bA: Yield 73%, white solid. HRMS (ESI) m/z calcd. for C₅₂H₈₀N₁₈O₈ (M+H)⁺: 1085.6412, found: 1085.6419; RP-HPLC: t_R = 16.72 min.

Dimer 9bB: Yield 65%, white solid. HRMS (ESI) m/z calcd. for C₅₂H₈₀N₁₈O₈ (M+H)⁺: 1085.6484, found: 1085.6442; RP-HPLC: t_R = 16.44 min.

Dimer 9bC: Yield 66%, white solid. HRMS (ESI) m/z calcd. for C₄₉H₈₂N₁₈O₈ (M+H)⁺: 1051.6641, found: 1051.6641; RP-HPLC: t_R = 17.11 min.

Dimer 9bD: Yield 65%, white solid. HRMS (ESI) m/z calcd. for C₄₈H₈₀N₁₈O₈ (M+H)⁺: 1037.6484, found: 1037.6460; RP-HPLC: t_R = 16.81 min.

Dimer 9bE: Yield 92%, white solid. HRMS (ESI) m/z calcd. for C₂₉H₄₃N₉O₄ (M+H)⁺: 582.3516, found: 582.3516; RP-HPLC: t_R = 16.19 min.

Dimer9cA: Yield 60%, white solid. HRMS (ESI) m/z calcd. for C₆₀H₈₄N₁₈O₈ (M+H)⁺: 1185.6797, found: 1185.6878; RP-HPLC: t_R = 22.72 min.

Dimer9cB: Yield 58%, white solid; HRMS (ESI) m/z calcd. for C₆₀H₈₄N₁₈O₈ (M+H)⁺: 1185.6797, found: 1185.6781; RP-HPLC: t_R = 24.13 min.

Dimer 9cC: Yield 69%, white solid. ¹H-NMR (MeOD, 400 MHz): δ 8.09 (s, 2H), 7.96 (d, J = 8.1 Hz, 2H), 7.82 (s, 2H), 7.71 (t, J = 7.2 Hz, 2H), 7.49 (t, J = 8.1 Hz, 2H), 6.99 (s, 2H), 5.24 (d, J = 7.4 Hz, 2H), 4.44 (s, 2H), 4.34 (t, J = 7.0 Hz, 4H), 3.95 (s, 4H), 3.65 (t, J = 9.1 Hz, 4H), 3.38–3.60 (m, 8H), 3.27 (t, J = 6.6 Hz, 4H), 3.16 (m, 4H), 2.95 (s, 2H), 2.63 (s, 4H), 2.32 (dt, J = 12.2, 4.1 Hz, 2H), 1.87 (m, 4H), 1.69 (q, J = 12.2 Hz, 2H), 1.26 (m, 2H); HRMS (ESI) m/z calcd. for C₅₇H₈₆N₁₈O₈ (M+H)⁺: 1151.6954, found: 1151.6945; RP-HPLC: t_R = 21.61 min.

Dimer9cD: Yield 66%, white solid; HRMS (ESI) m/z calcd. for C₅₆H₈₄N₁₈O₈ (M+H)⁺: 1137.6797, found: 1137.6793; RP-HPLC: t_R = 23.12 min.

Dimer 9cE: Yield 90%, white solid; HRMS (ESI) m/z calcd. for C₃₃H₄₅N₉O₄ (M+H)⁺: 632.3672, found: 632.3669; RP-HPLC: t_R = 19.87 min.

Compound 10a:Yield 79%, white solid; ¹H-NMR (MeOD, 400 MHz): δ 8.04 (s, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.77 (t, J = 7.7 Hz, 1H), 7.55 (t, J = 7.7 Hz, 1H), 7.50 (d, J = 8.2 Hz, 2H), 7.24 (d, J = 8.2 Hz, 2H), 5.55 (s, 2H), 5.16 (d, J = 7.2 Hz, 1H), 4.17 (s, 3H), 3.98 (t, J = 6.0 Hz, 2H), 3.65 (t, J = 9.0 Hz, 1H), 3.56 (m, 1H), 3.48 (t, J = 9.7 Hz, 4H), 3.37 (dd, J = 12.8, 2.3 Hz, 1H), 3.20 (m, 4H), 2.69 (s, 3H), 2.60 (m, 2H), 2.36 (dt, J = 8.1, 3.7 Hz, 1H), 1.71 (q, J = 12.4 Hz, 1H); HRMS (ESI) m/z calcd. for C₃₃H₄₄N₉O₄Br (M+H)⁺: 710.2777, found: 710.2761; RP-HPLC: t_R = 20.43 min.

Compound 10b: Yield 71%, white solid; HRMS (ESI) m/z calcd. for C₃₂H₄₂N₉O₄Br (M+H)⁺: 696.2621, found: 696.2664; RP-HPLC: t_R = 21.18 min.

Compound 10c: Yield 77%, white solid; ¹H-NMR (MeOD, 400 MHz): δ 8.03 (s, 1H), 8.01 (d, J = 8.1 Hz, 1H), 7.76 (t, J = 7.7 Hz, 1H), 7.54 (t, J = 7.7 Hz, 1H), 7.28 (d, J = 8.7 Hz, 2H), 6.88 (d, J = 8.7 Hz, 2H), 5.49 (s, 2H), 5.23 (d, J = 7.0 Hz, 1H), 4.36 (s, 2H), 4.28 (t, J = 9.5 Hz, 1H), 3.99 (t, J = 5.5 Hz, 2H), 3.74 (s, 3H), 3.66 (t, J = 9.1 Hz, 1H), 3.63 (d, J = 11.5 Hz, 1H), 3.57 (dd, J = 12.8, 6.4 Hz, 1H), 3.36–3.55 (m, 4H), 3.31 (s, 3H), 2.84 (m, 2H), 2.68 (s, 2H), 2.37 (dt, J = 12.0, 4.0 Hz, 1H), 1.73 (q, J = 12.5 Hz, 1H); HRMS (ESI) m/z calcd. for C₃₄H₄₇N₉O₅ (M+H)⁺: 662.3778, found: 662.3758; RP-HPLC: t_R = 21.34 min.

Compound 10d: Yield 72%, white solid; HRMS (ESI) m/z calcd. for C₃₃H₄₅N9O₅ (M+H)⁺: 648.3621, found: 648.3600; RP-HPLC: t_R = 20.63 min.

Compound 10e: Yield 81%, white solid; ¹H-NMR (MeOD, 400 MHz): δ 8.02 (d, J = 8.2 Hz, 1H), 7.97 (s, 1H), 7.77 (dd, J = 8.2, 7.2 Hz, 1H), 7.55 (dd, J = 8.2, 7.2 Hz, 1H), 7.33 (m, 1H), 7.23 (dd, J = 7.4, 1.6 Hz, 1H), 7.00 (d, J = 8.3 Hz, 1H), 6.92 (dt, J = 7.5, 1.0 Hz, 1H), 5.55 (s, 2H), 5.19 (d, J = 8.3 Hz, 1H), 4.24 (s, 3H), 3.99 (t, J = 5.9 Hz, 2H), 3.83 (s, 3H), 3.68 (t, J = 9.2 Hz, 1H), 3.35–3.60 (m, 5H), 2.84 (m, 3H), 2.69 (s, 4H), 2.36 (dt, J = 12.1, 4.0 Hz, 1H), 1.71 (q, J =12.6 Hz, 1H); HRMS (ESI) m/zcalcd for C₃₄H₄₇N₉O₅ (M+H)⁺: 662.3778, found: 662.3769; RP-HPLC: t_R = 21.15 min.

Compound 10f: Yield 70%, white solid; HRMS (ESI) m/z calcd. for C₃₄H₄₈N₁₀O₄ (M+H)⁺: 661.3938, found: 661.3914; RP-HPLC: t_R = 21.32 min.

Compound 10g: Yield 68%, white solid; HRMS (ESI) m/z calcd. for C₃₁H₅₀N₁₀O₄ (M+H)⁺: 627.4094, found: 627.4084; RP-HPLC: t_R = 21.12 min.

A typical experiment using a 96-well U-bottom plate was performed as follows. Serial dilutions of a freshly grown cell culture were made in LB to achieve 10⁹ to 10² colony-forming units (cfu, confirmed by colony plate counting). Wells were prepared in triplicate by filling each well with 180 μl of the cell dilution plus 20 μL of Cy3-labeled hemocyanin solution (0.5–1 μM end-concentration). To avoid evaporation and rapid oxygen diffusion, 50 μL of mineral oil was added on top of each well. For antibiotic resistance tests, the initial number of cells was 10⁷ cfu/well. Kanamycin was added to a final concentration of 0.05 to 5000 μg/mL. Controls were performed by adding Cy3 or Cy3-labeled BSA instead of Cy3-labeled hemocyanin to the cell culture as well as by using unlabeled hemocyanin. The fluorescence of Cy3-labeled hemocyanin in LB without cells was also monitored as a control in the absence of oxygen consumption.