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Molecules 2018, 23(4), 899; doi:10.3390/molecules23040899

Article
Differential Effects of Linkers on the Activity of Amphiphilic Tobramycin Antifungals
Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0596, USA
*
Author to whom correspondence should be addressed.
Received: 2 April 2018 / Accepted: 9 April 2018 / Published: 13 April 2018

Abstract

:
As the threat associated with fungal infections continues to rise and the availability of antifungal drugs remains a concern, it becomes obvious that the need to bolster the antifungal armamentarium is urgent. Building from our previous findings of tobramycin (TOB) derivatives with antifungal activity, we further investigate the effects of various linkers on the biological activity of these aminoglycosides. Herein, we analyze how thioether, sulfone, triazole, amide, and ether functionalities affect the antifungal activity of alkylated TOB derivatives against 22 Candida, Cryptococcus, and Aspergillus species. We also evaluate their impact on the hemolysis of murine erythrocytes and the cytotoxicity against mammalian cell lines. While the triazole linker appears to confer optimal activity overall, all of the linkers incorporated into the TOB derivatives resulted in compounds that are very effective against the Cryptococcus neoformans species, with MIC values ranging from 0.48 to 3.9 μg/mL.
Keywords:
aminoglycosides; Aspergillus; Candida; Cryptococcus; cytotoxicity; hemolysis

1. Introduction

Aminoglycosides (AGs) represent a group of structurally diverse amino-modified sugars that have long been used for their antibacterial efficacy. Their broad-spectrum of activity against a plethora of pathogenic bacteria has, indeed, made them a valuable class of antibiotics [1]. However, as is the case with most antibiotics nowadays, AGs are suffering from the resurgence of bacterial resistance [2]. To circumvent this problem, several research groups have devoted efforts to the development of novel AGs with improved antibacterial activity [3]. Amphiphilic AGs in particular, which are AG-derived cationic amphiphiles, emerged as potent antimicrobial agents with a new mechanism of action [4]. They resulted from the incorporation of various hydrophobic groups to the polycationic and hydrophilic amikacin [5], tobramycin (TOB) [6,7,8,9,10,11], neamine [12,13,14], neomycin B [15,16,17,18,19], paromomycin [20,21], and kanamycins A (KANA) and B (KANB) [22,23].
A growing interest in the identification of new targets of AGs [24] has recently led researchers to the investigation of AGs’ action against fungi [23,25,26,27,28,29,30]. It is worth mentioning that the social and economic burden associated with fungal infections is considerable, both in medicine and agriculture. While the frequency of invasive fungal infections continues to rise, the number of antifungal agents available remains limited, calling for the development of additional effective antifungal drugs. Indeed, only three classes of antifungals are currently in clinical use [31]. These include the polyenes (for example, amphotericin B (AmB)), the azoles (for example, fluconazole, voriconazole (VOR), itraconazole, posaconazole), and the echinocandins (for example, caspofungin (CAS), micafungin, anidulafungin). These drugs exert their antifungal activity by either extracting ergosterol from the fungal plasma membrane [32], blocking the production of ergosterol through the inhibition of lanosterol 14α-demethylase [33], or inhibiting the biosynthesis of the fungal cell membrane component (1,3)-β-d-glucan [34], respectively. Unfortunately, resistance to these antifungal drugs has already been observed [35]. However, new strategies to combat fungal infections are being investigated, notably the development of derivatives of currently approved antifungals with improved activity and biological safety profiles [36,37,38,39,40], the combination therapy of current antifungal drugs working synergistically with other drugs [41], and the development of new classes of antifungal agents [42].
We have recently demonstrated that incorporating a linear alkyl chain (for example, the dodecyl group (C12) or tetradecyl group (C14)) at the 6″-position of TOB or KANB through a thioether linkage results in AGs with antifungal activity against C. albicans [23,43]. Although we found the TOB (C14) derivative to display stronger antifungal activity than TOB (C12), we elected to generate TOB (C12) analogues in this study because, as we previously demonstrated by investigating the hemolytic activity of KANB (C12) and (C14), KANB (C12) displayed lower hemolytic activity than KANB (C14). Herein, with the goal of improving on the activity of TOB (C12), we investigated the effects of different groups (for example, thioether, sulfone, triazole, amide, and ether) as linkers for the alkyl chain to the AG scaffold on the efficacy of TOB (C12) derivatives against 22 fungal strains. We investigated the hemolytic activity and cytotoxicity of the five most promising antifungals. We also performed time-kill studies and membrane permeabilization assays for the most active compound generated.

2. Results and Discussion

2.1. Chemistry

The synthesis started from the commercially available AG TOB. Modification at the 6″-position of TOB requires the selective conversion of the 6″-hydroxyl group into a good leaving group, which could be accomplished by Boc protection of the amino groups of TOB, followed by a reaction with 2,4,6-triisopropylbenzenesulfonyl chloride (TIPBSCl) in pyridine (Scheme 1). This gave the central intermediate 1 [44], which upon treatment with 1-dodecanethiol and Cs2CO3, afforded another intermediate compound 2 [8,10]. Treatment of compound 2 with trifluoroacetic acid (TFA) efficiently removed all the Boc protecting groups to give the target compound 3 [10], representing the TOB derivative with the thioether linkage. Furthermore, S-oxidation of compound 2 with m-CPBA followed by TFA treatment yielded the target sulfone derivative 4 [8].
Compound 1 was also subjected to a SN2 nucleophilic displacement reaction in the presence of tetrabutylammonium azide (TBAA) to give the azido intermediate 5 [11]. Using copper sulfate and sodium l-ascorbate in DMF under microwave conditions, a click reaction between compound 5 and commercially available alkynes (1-dodecyne and 1-tetradecyne) afforded the intermediates 6a and 6b [11], respectively, whose TFA treatment gave the target triazole derivatives 7a and 7b [11], respectively. The design for compound 7a (C10-triazole), which is two-carbon shorter than compound 7b, stems from the need to investigate whether or not the nitrogen atoms N2 and N3 of the triazole ring contribute to the overall length of the alkyl side chain. Compound 5 was also subjected to Staudinger reduction that converted the 6″-azido group into an amine, followed by amide coupling with lauric acid to yield compound 8, which was then converted to the target amide derivative 9.
The synthesis of the TOB derivative with the ether linkage started with the conversion of TOB into the known perazide 10 [45] with triflyl azide, which was then reacted with pivaloyl chloride to afford the 6″-O-pivalate 11 (Scheme 2). Benzyl protection of the remaining hydroxyl groups afforded compound 12, which was then subjected to base hydrolysis of the pivaloyl group to give compound 13, with a free 6″-hydroxyl group. Alkylation of compound 13 in the presence of sodium hydride and 1-bromododecane then afforded compound 14. Staudinger reduction of azide to amine, hydrogenolysis, and Boc protection of the free amino groups gave compound 15, which was converted to the target 6″-O-dodecyl-TOB derivative 16 through TFA deprotection.

2.2. In Vitro Antifungal Activity

The minimum inhibitory concentration (MIC) values of TOB and its derivatives 3, 4, 7a, 7b, 9, and 16 were determined (Table 1, Table 2 and Table 3). We focused our study on the Candida (albicans and non-albicans), Cryptococcus, and Aspergillus species, which are medically significant fungal pathogens [46]. We also included the FDA-approved antifungal drug CAS as a control; it is used clinically in the treatment of fungal infections caused by Candida and Aspergillus species.
Our initial study targeted the C. albicans species since, in addition to offering a point of comparison with our previously published data on compound 3 [43], they are responsible for the largest number of Candida-related fungal infections in humans [47]. They account for many invasive fungal infections observed in intensive care units [48]. These included the following seven strains: C. albicans ATCC 10231 (strain A), C. albicans ATCC 64124 (strain B), C. albicans ATCC MYA-2876 (strain C), C. albicans ATCC 90819 (strain D), C. albicans ATCC MYA-2310 (strain E), C. albicans ATCC MYA-1237 (strain F), and C. albicans ATCC MYA-1003 (strain G). While the derivatives appeared to be less effective than CAS, all but compound 16 were better than the parent TOB against all C. albicans strains tested (Table 1). Indeed, TOB was inactive against all strains tested with MIC values >62.5 μg/mL. On the other hand, compound 7b, with the triazole linkage, was the most active derivative with MIC values ranging from 3.9 to 15.6 μg/mL, which corresponds to a 4- to >16-fold improvement compared to TOB. The thioether derivative 3 and the sulfone 4 were the next most active derivatives. The MIC values for compound 3 corresponded within 2-fold to those previously published [43]. The MIC values of compounds 3 and 4 (7.8–31.3 μg/mL) were also not much different from each other, always within a 2-fold dilution, indicating that metabolic S-oxidation might not have a noticeable effect on potency. This is in agreement with our previous observation of KANB with a C12 chain in a thioether linkage at the 6″-position and its oxidized counterpart as antifungals [23]. Compound 16 (C12-ether) was the least effective (MIC values ≥62.5 μg/mL) followed by the C10-triazole 7a (MIC values ranging from 31.3 to 62.5 μg/mL) and the C11-amide 9 (MIC values ranging from 15.6 to 62.5 μg/mL).
These promising results encouraged us to expand our study to nine non-albicans Candida strains, including C. glabrata ATCC 2001 (strain H), C. krusei ATCC 6258 (strain I), C. parapsilosis ATCC 22019 (strain J), three C. glabrata clinical isolates (CG13), and three C. parapsilosis clinical isolates (CP13) (Table 2). As observed with the C. albicans yeast strains, the compounds 3, 4, 7a, 7b, 9, and 16 were more active than TOB, but less active than CAS. Once again, compound 7b, with the triazole linkage, displayed the lowest MIC values overall (1.95–7.8 μg/mL), representing an 8- to >32-fold improvement compared to TOB (MIC values >62.5 μg/mL). The thioether 3 and the sulfone 4 also exhibited good activity (MIC values ranging from 3.9 to 15.6 μg/mL), followed by the amide 9 (MIC values ranging from 7.8 to 31.3 μg/mL), the C10-triazole 7a (MIC values ranging from 15.6 to 62.5 μg/mL), and the C12-ether 16 (MIC values ranging from 31.3 to 62.5 μg/mL).
We had previously observed that Cryptococcus neoformans MYA-895 was exceptionally sensitive to compound 3, with a low MIC value of 1.95 μg/mL [43]. We thus decided to assess the potency of compounds 3, 4, 7a, 7b, 9, and 16 against C. neoformans clinical isolates CN13 (Table 2). Interestingly, all synthesized TOB derivatives displayed excellent activity, with MIC values ranging from 0.48 to 3.9 μg/mL, while TOB and CAS showed low to no activity. This represents a 16- to >128-fold improvement compared to TOB (MIC values >62.5 μg/mL). C. neoformans is responsible for cryptococcosis, which remains a significant cause of mortality and morbidity in immunocompromised individuals [49].
Finally, based on the very promising and encouraging results obtained against the C. neoformans clinical isolates, we decided to assess the antifungal activity of the derivatives generated against other types of fungal strains. We opted for the filamentous fungi Aspergillus flavus ATCC MYA-3631 (strain K), Aspergillus nidulans ATCC 38163 (strain L), and Aspergillus terreus ATCC MYA-3633 (strain M) (Table 3). In these cases, all the TOB derivatives, with the exception of compound 16, were better than both TOB and CAS, with a trend similar to that observed with the C. albicans and non-albicans Candida strains. Furthermore, while A. terreus ATCC MYA-3633 (strain M) was not sensitive to the TOB derivatives, compounds 3, 4, 7a, 7b, and 9 showed moderate activity against A. flavus ATCC MYA-3631 (strain K) (MIC values ranging from 7.8 to 31.3 μg/mL) and good activity against A. nidulans ATCC 38163 (strain L) (MIC values ranging from 1.95 to 3.9 μg/mL). Compound 16 only showed good activity against A. nidulans ATCC 38163 (strain L) with an MIC value of 3.9 μg/mL.
In light of these results, it appears that the linkers play a major role in the activity of alkylated TOB derivatives, with triazole > “better than” thioether/sulfone > amide > ether. This may stem from the distinct ability of these linkers to form interactions with the polar lipid head groups of fungi. Indeed, with its three nitrogen atoms, the triazole ring may form more hydrogen bonds than the sulfone which has two oxygens, the amide with a nitrogen and an oxygen, and the ether with only an oxygen atom. It is worth mentioning that, while compound 7b (C12-triazole) was the most effective at inhibiting the growth of yeasts and filamentous fungi, compound 7a (C10-triazole) was the least effective of all six TOB derivatives synthesized (except compound 16). This is no surprise as it is in accordance with the chain length-dependent antifungal activity observed with other amphiphilic AGs [23,43]. Indeed, amphiphilic AGs bear a hydrophilic core structure, which is rich in polar hydroxyl and amino groups that are positively charged under physiological conditions, and a lipophilic alkyl chain capable of interacting with the lipid-rich fungal cell membrane. As the length of the alkyl chain increases, the ability of amphiphilic AGs to puncture the lipid bilayers also increases, which may result in an enhanced cell membrane perturbation, a mechanism well-known for this type of molecules [8,27]. This also shows that the additional atoms N2 and N3 of the triazole ring do not contribute to the overall length of the alkyl chain.

2.3. Hemolysis

As potential antifungal agents, it is necessary to assess the ability of the synthesized compounds to selectively target fungal membranes. Since compound 16 was in general as inactive as the parent AG TOB, we decided to focus our efforts on the remaining TOB derivatives. We performed a hemolytic assay of the active TOB derivatives 3, 4, 7a, 7b, and 9 using murine red blood cells (mRBCs) (Figure 1 and Table S1). Although these amphiphilic TOB derivatives appear to affect mRBCs more than the parent TOB, they all displayed relatively lower hemolytic activity compared to AmB, which is an FDA-approved antifungal prescription medicine. Overall, the following trend was observed: 7a (C10-triazole) < “less hemolytic than” 9 (C11-amide) < 4 (C12-sulfone) < 3 (C12-thioether) < 7b (C12-triazole). Once again, we noticed a chain length-dependent effect on hemolysis, as previously observed with KANB-derived cationic amphiphiles [23]. It also appeared that the sulfone and amide linkers may impart less hemolysis than the thioether and triazole ones. Oxidation of the thioether derivative 3 to its corresponding sulfone 4 also seemed to lessen the hemolytic activity, suggesting that metabolic S-oxidation may not be detrimental in this case. While the sulfone derivative 4 and the amide 9 lysed ~15–25% and ~18–32% of mRBCs, respectively, at their MIC values against all seven C. albicans strains, the thioether derivative 3 and the triazole 7b lysed ~19–46% and ~13–41% of mRBCs, respectively. At their MIC values against non-albicans Candida strains, the sulfone derivative 4 and the amide 9 lysed ~6–11% and ~18–26% of mRBCs, respectively, while the thioether derivative 3 and the triazole 7b lysed ~19–46% and ~14–25% of mRBCs, respectively. While more hemolysis was observed at their MIC values against the Aspergillus strains (~4–32% for compounds 4 and 9, and ~12–74% for compounds 3 and 7b), the TOB derivatives 3, 4, 7a, 7b, and 9 caused little to no hemolysis when tested at their MIC values against C. neoformans, only lysing up to 13% of mRBCs at 3.9 μg/mL, which represents 2- to 8-fold their MIC values against the three clinical isolates tested. The generally low hemolytic activity observed strengthens the potential application of the newly synthesized TOB derivatives as antifungal agents against the C. neoformans species.

2.4. Cytotoxicity

To further evaluate the potential safety of the TOB derivatives 3, 4, 7a, 7b, and 9, we assessed the cytotoxicity of these compounds against two mammalian cell lines, BEAS-2B and A549 (Figure 2). Like the parent AG TOB, these derivatives displayed little to no toxicity against both cell lines. Indeed, compounds 4 (sulfone), 7a (C10-triazole), 7b (C12-triazole), and 9 (amide) all had IC50 values >62.5 μg/mL, which were 1- to 16-fold higher than their antifungal MIC values against the C. albicans species. Only compound 3 (thioether) had an IC50 value in the range of 31.3–62.5 μg/mL in BEAS-2B cells. This observed selectivity of the TOB derivatives in targeting fungal cells in the presence of mammalian cells may stem from the difference in lipid composition of the cell membranes in fungi and humans [50].

2.5. Time-Kill Studies

In light of the results presented so far, we selected the most promising compound 7b for further investigations. We evaluated the antifungal potency of 7b (C12-triazole) by determining its time-kill course against a representative C. albicans strain, ATCC 10231 (strain A); a representative non-albicans Candida strain, C. parapsilosis ATCC 22019 (strain J); and a representative non-Candida yeast, C. neoformans (strain CN1) over a 24-h period. We also included the parent AG TOB and the triazole antifungal agent VOR as controls in our study (Figure 3). While TOB did not affect the growth of any of the tested fungal strains, its triazole derivative 7b was able to reduce the C. albicans ATCC 10231 (strain A) CFU by ≥2 log10 after 9 h of treatment at 3.9 μg/mL (1× MIC) and after 6 h of treatment at 15.6 μg/mL (4× MIC) (Figure 3a). This suggests a dose-dependent effect as previously observed with other antifungal amphiphilic AGs [23,43]. Furthermore, compound 7b showed a similar growth inhibitory effect to that of VOR, since the latter also reduced the CFU of C. albicans ATCC 10231 (strain A) by ≥2 log10 after 9 h of treatment at its 1× MIC value of 1.0 μg/mL and maintained a fungistatic activity up to the 24-h period. Against C. parapsilosis ATCC 22019 (strain J), compound 7b displayed a fungistatic effect at 1.95 μg/mL (1× MIC) only for the first 3 h before the fungal cells followed a trend similar to the growth control (Figure 3b). A decrease in CFU by ≥2 log10 was only observed after 24 h of treatment. Meanwhile, at 7.8 μg/mL (4× MIC), compound 7b completely killed C. parapsilosis ATCC 22019 (strain J) after 24 h. At 0.49 μg/mL (1× MIC), compound 7b rapidly reduced the CFU of C. neoformans (strain CN1) by ≥2 log10 after 3 h of treatment (Figure 3c). In addition, complete fungal cell death was observed as early as 6 h after the treatment at 0.49 μg/mL (1× MIC) and 3 h after the treatment at 1.95 μg/mL (4× MIC), while TOB and VOR showed little to no growth inhibitory effect against C. neoformans (strain CN1). These results confirm that the TOB derivative 7b (C12-triazole) exhibits better fungal growth inhibition than the parent AG TOB. Furthermore, compound 7b displayed a similar fungistatic effect to that of the FDA-approved triazole antifungal agent VOR against C. albicans ATCC 10231 (strain A) and C. parapsilosis ATCC 22019 (strain J), and an enhanced fungicidal effect against C. neoformans (strain CN1). The promise of 7b as a fungicidal agent against Cryptococcus sp. is highly encouraging.

2.6. Antifungal Mechanism of Action

We have previously shown that amphiphilic TOB derivatives, notably compound 3, exert their antifungal activity by perturbing the fungal cell membrane [43]. We then decided to evaluate the ability of the most potent compound 7b to affect the fungal membrane integrity. Using propidium iodide (PI), a dye that fluoresces upon binding nucleic acids in membrane-compromised cells [51], we observed that C. albicans ATCC 10231 (strain A) cells that were treated with compound 7b at concentrations equivalent to 2× MIC or 4× MIC allowed a larger influx of PI dye compared to the untreated cells or those treated with the parent TOB, which only showed negligible staining (Figure 4). Yeast killed by heat were used as the positive control. These results show that compound 7b also affects the fungal membrane permeability and is more effective than TOB in causing PI permeation into the azole-resistant C. albicans ATCC 10231 (strain A) cells.

3. Conclusions

In summary, we have synthesized six TOB derivatives with various linkers, including thioether, sulfone, triazole, amide, and ether. While the C12-triazole derivative 7b was the most potent, the C12-ether derivative 16 was the least effective at inhibiting the growth of several fungal strains. We also noticed a chain length-dependent effect on the antifungal activity. Furthermore, the active TOB derivatives displayed low hemolytic activity and low mammalian cell toxicity. Finally, the compounds generated were very active against C. neoformans clinical isolates, leaving the door open for a future (outside of the scope of this study) investigative avenue for the development of novel therapies for cryptococcosis.

4. Materials and Methods

4.1. General Information

Tobramycin (TOB) was purchased from AK Scientific (Union City, CA, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. Compounds 1 [44], 5 [11], and 10 [45] were prepared as previously described. All microwave irradiation experiments were performed in a sealed glass microwave reaction vial using a Biotage® Initiator™ microwave synthesizer. Experiments were performed in temperature-control mode where the temperature was controlled using the built-in calibrated IR sensor. Chemical reactions were monitored by TLC (Merck, Kenilworth, NJ, USA, Silica gel 60 F254). Visualization was achieved using one of the following methods: H2SO4 stain (5% in MeOH), ceric molybdate stain (5 g (NH4)2Ce(NO3)6, 120 g (NH4)6Mo7O24·4H2O, 80 mL H2SO4, and 720 mL H2O), or p-anisaldehyde stain (54 mL EtOH, 3 mL H2SO4, and 3 mL p-anisaldehyde). Compounds were purified by SiO2 flash chromatography (Dynamic Adsorbents Inc., Norcross, GA, USA, Flash silica gel 32–62u). 1H and 13C-NMR spectra were recorded on a Varian 400 MHz spectrometer. High-resolution mass sprectra were recorded on an AB SCIEX Triple TOF 5600 System. All compounds tested for activity are ≥95% pure according to NMR spectra. TOB derivatives 3, 4, 7a, 7b, 9, and 16 were dissolved in sterile MQ-H2O at a final concentration of 10 mg/mL and stored at −20 °C.
The antifungal agent caspofungin (CAS) was purchased from Sigma-Aldrich (St. Louis, MO, USA), dissolved in DMSO at a final concentration of 5 mg/mL, and stored at −20 °C. Candida albicans ATCC 10231 (strain A), C. albicans ATCC 64124 (strain B), and C. albicans ATCC MYA-2876 (strain C) were kindly provided by Dr. Jon Y. Takemoto (Utah State University, Logan, UT, USA). C. albicans ATCC MYA-90819 (strain D), C. albicans ATCC MYA-2310 (strain E), C. albicans ATCC MYA-1237 (strain F), C. albicans ATCC MYA-1003 (strain G), Candida glabrata ATCC 2001 (strain H), Candida krusei ATCC 6258 (strain I), Candida parapsilosis ATCC 22019 (strain J), Aspergillus flavus ATCC MYA-3631 (strain K), and Aspergillus terreus ATCC MYA-3633 (strain M) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Aspergillus nidulans ATCC 38163 (strain N) was received from Dr. Jon S. Thorson (University of Kentucky, Lexington, KY, USA). All clinical fungal isolates, C. glabrata (strain CG13), C. parapsilosis (strain CP13), and Cryptococcus neoformans (strain CN13) were obtained from Dr. Nathan P. Wiederhold (University of Texas Health Science Center, San Antonio, TX, USA). Filamentous fungi and yeasts were cultivated at 35 °C in RPMI 1640 medium (with l-glutamine, without sodium biocarbonate, Sigma-Aldrich, St. Louis, MO, USA) buffered to a pH of 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) buffer (Sigma-Aldrich).
The human bronchus normal cell line BEAS-2B (ATCC CRL-9609) and the human lung carcinoma cell line A549 (ATCC CRL-185) were kind gifts from the laboratories of Dr. David K. Orren (University of Kentucky, Lexington, KY, USA) and Dr. Markos Leggas (University of Kentucky, Lexington, KY, USA). The mammalian cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (from ATCC) with 10% fetal bovine serum (FBS) (from ATCC) and 1% Pen/Strep (from ATCC). The cell lines were incubated at 37 °C and 5% CO2 and passaged by trypsinization with 0.05% trypsin and 0.53 mM EDTA (from ATCC). The cell confluency was determined by using a Nikon Eclipse TS100 microscope (Minato, Tokyo, Japan).

4.2. Synthesis of Compounds 216

O-3-Deoxy-3-[[(1,1-dimethylethoxy)carbonyl]amino]-6-S-dodecyl-6-thio-α-d-glucopyranosyl-(1→6)-O-[2,3,6-trideoxy-2,6-bis[[(1,1-dimethylethoxy)carbonyl]amino]-α-d-ribo-hexopyranosyl-(1→4)]-2-deoxy-N1,N3-bis[(1,1-dimethylethoxy)carbonyl]-d-streptamine (2). A solution of compound 1 (0.30 g, 0.24 mmol), Cs2CO3 (0.12 g, 0.36 mmol), and 1-dodecanethiol (0.29 mL, 1.22 mmol) in anhydrous DMF (3 mL) was stirred at room temperature overnight. Progress of the reaction was monitored by TLC (Hexanes:EtOAc/2:3, Rf 0.56). Purification by flash column chromatography (SiO2, pure Hexanes to Hexanes:EtOAc/2:3) gave the known compound 2 [8] (0.18 g, 64%) as a white solid: 1H-NMR (400 MHz, CD3OD, which matches the Reference [8], Figure S1) δ 5.07 (br s, 1H, H-1′), 5.01 (br d, J = 2.8 Hz, 1H, H-1″), 4.05 (ddd, J1 = 9.2 Hz, J2 = 7.6 Hz, J3 = 2.8 Hz, 1H, H-5″), 3.70–3.30 (m, 13H, H-1, H-3, H-4, H-5, H-6, H-2′, H-4′, H-5′, H-6′ (2H), H-2″, H-3″, H-4″), 2.97 (dd, J1 = 14.4 Hz, J2 = 2.8 Hz, 1H, H-6″), 2.61 (m, 1H, H-6″), 2.57 (t, J = 7.2 Hz, 2H, SCH2(CH2)10CH3), 2.14 (m, 1H, H-2eq), 1.99 (m, 1H, H-3′eq), 1.62 (app. q, J1 = J2 = J3 = 12.0 Hz, 1H, H-3′ax), 1.59–1.22 (m, 66H, H-2ax, 5× CO2(CH3)3, SCH2(CH2)10CH3), 0.88 (t, J = 7.2 Hz, 3H, SCH2(CH2)10CH3).
O-3-Amino-3-deoxy-6-S-dodecyl-6-thio-α-d-glucopyranosyl-(1→6)-O-[2,6-diamino-2,3,6-trideoxy-α-d-ribo-hexopyranosyl-(1→4)]-2-deoxy-d-streptamine (3). Compound 2 (26 mg, 0.023 mmol) was treated at room temperature with neat TFA (1 mL) for 3 min. The TFA was removed under reduced pressure, the residue was dissolved in a minimal volume of H2O and freeze-dried to afford the known compound 3 [8] (17 mg, 61%) as a white powder: 1H-NMR (400 MHz, D2O, which matches Reference [8], Figure S2) δ 5.60 (d, J = 3.6 Hz, 1H, H-1′), 4.95 (d, J = 4.0 Hz, 1H, H-1″), 3.90–3.77 (m, 4H, H-4, H-5′, H-2″, H-5″), 3.75 (app. t, J1 = J2 = 9.2 Hz, 1H, H-5), 3.65 (app. t, J1 = J2 = 9.6 Hz, 1H, H-6), 3.61–3.38 (m, 5H, H-1, H-3, H-2′, H-4′, H-4″), 3.32 (app. t, J1 = J2 = 10.8 Hz, 1H, H-3″), 3.29 (dd, J1 = 13.6 Hz, J2 = 3.6 Hz, 1H, H-6′), 3.13 (dd, J1 = 14.0 Hz, J2 = 7.2 Hz, 1H, H-6′), 2.91 (dd, J1 = 14.0 Hz, J2 = 2.0 Hz, 1H, H-6″), 2.63 (dd, J1 = 14.0 Hz, J2 = 8.0 Hz, 1H, H-6″), 2.48 (t, J = 7.6 Hz, 2H, SCH2(CH2)10CH3), 2.42 (app. dt, J1 = 12.4 Hz, J2 = J3 = 4.4 Hz, 1H, H-2eq), 2.17 (app. dt, J1 = 12.4 Hz, J2 = J3 = 4.4 Hz, 1H, H-3′eq), 1.88 (app. q, J1 = J2 = J3 = 12.0 Hz, 1H, H-3′ax), 1.80 (app. q, J1 = J2 = J3 = 12.4 Hz, 1H, H-2ax), 1.45 (p, J = 7.2 Hz, 2H, SCH2CH2(CH2)9CH3), 1.22–1.06 (m, 18H, SCH2CH2(CH2)9CH3), 0.72 (t, J = 7.2 Hz, 3H, SCH2(CH2)10CH3).
O-3-Amino-3,6-dideoxy-6-(dodecylsulfonyl)-α-d-glucopyranosyl-(1→6)-O-[2,6-diamino-2,3,6-trideoxy-α-d-ribo-hexopyranosyl-(1→4)]-2-deoxy-d-streptamine (4). m-Chloroperbenzoic acid (m-CPBA, 30 mg, 0.17 mmol) was added to a solution of compound 2 (56 mg, 0.049 mmol) in CHCl3 (3 mL) and the resulting mixture was stirred at room temperature for 16 h. The reaction mixture was then diluted with CHCl3 (10 mL) and washed with 1M KOH (3 × 2 mL). The organic layers were dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The crude product obtained was treated at room temperature with neat TFA (0.5 mL) for 3 min. The TFA was removed under reduced pressure, the residue was dissolved in a minimal volume of H2O and freeze-dried to afford the known compound 4 [8] (40 mg, 66%) as a white foam: 1H-NMR (400 MHz, D2O, which matches the Reference [8], Figure S3) δ 5.54 (d, J = 3.6 Hz, 1H, H-1′), 5.00 (d, J = 3.6 Hz, 1H, H-1″), 4.30 (app. t, J1 = J2 = 9.6 Hz, 1H, H-5″), 3.87–3.76 (m, 4H, H-4, H-5, H-5′, H-2″), 3.66 (app. t, J1 = J2 = 8.8 Hz, 1H, H-6), 3.61–3.37 (m, 8H, H-1, H-3, H-2′, H-4′, H-3″, H-4″, H-6″ (2H)), 3.28 (dd, J1 = 13.6 Hz, J2 = 3.6 Hz, 1H, H-6′), 3.19 (t, J = 7.6 Hz, 2H, SO2CH2CH2CH2(CH2)8CH3), 3.11 (dd, J1 = 13.6, J2 = 7.2 Hz, 1H, H-6′), 2.42 (dt, J1 = 12.4 Hz, J2 = J3 = 4.4 Hz, 1H, H-2eq), 2.16 (dt, J1 = 12.4 Hz, J2 = J3 = 4.4 Hz, 1H, H-3′eq), 1.86 (app. q, J1 = J2 = J3 = 12.8 Hz, 1H, H-3′ax), 1.78 (app. q, J1 = J2 = J3 = 12.8 Hz, 1H, H-2ax), 1.66 (p, J = 7.6 Hz, 2H, SO2CH2CH2CH2(CH2)8CH3), 1.30 (p, J = 7.6 Hz, 2H, SO2CH2CH2CH2(CH2)8CH3), 1.13 (m, 16H, SO2CH2CH2CH2(CH2)8CH3), 0.71 (t, J = 6.8 Hz, 3H, SO2CH2CH2CH2(CH2)8CH3).
O-3,6-Dideoxy-3-[[(1,1-dimethylethoxy)carbonyl]amino]-6-(4-decyl-1H-1,2,3-triazol-1-yl)-α-d-glucopyranosyl-(1→6)-O-[2,3,6-trideoxy-2,6-bis[[(1,1-dimethylethoxy)carbonyl]amino]-α-d-ribo-hexopyranosyl-(1→4)]-2-deoxy-N1,N3-bis[(1,1-dimethylethoxy)carbonyl]-d-streptamine (6a). To a microwave reaction vessel, compound 5 (60 mg, 0.060 mmol), sodium l-ascorbate (6 mg, 0.030 mmol), CuSO4·5H2O (6 mg, 0.024 mmol), 1-dodecyne (52 μL, 0.24 mmol), and DMF (1 mL) were added. The reaction vessel was then sealed and irradiated by microwaves for 4 min at 80 °C, twice. Progress of the reaction was monitored by TLC (Hexanes:EtOAc/3:7, Rf 0.49). Upon completion, the reaction mixture was filtered through celite®, and eluted with EtOAc. The filtrate was washed with H2O (3×) and brine (3×), dried over anhydrous MgSO4, filtered, and concentrated to give a crude product. Purification by column chromatography (SiO2, pure Hexanes to Hexanes:EtOAc/2:3) gave compound 6a (40 mg, 57%) as a white solid: 1H-NMR (400 MHz, CD3OD, Figure S4) δ 7.73 (s, 1H, triazole ring), 5.09 (d, J = 2.8 Hz, 1H, H-1″), 5.04 (br s, 1H, H-1′), 4.67 (br d, J = 11.2 Hz, 1H, H-6″), 4.55 (t, J = 12.0 Hz, 1H, H-6″), 4.39 (m, 1H, H-5″), 3.72 (t, J = 10.0 Hz, 1H, H-3″), 3.61–3.28 (m, 11H, H-1, H-3, H-4, H-5, H-6, H-2′, H-4′, H-5′, H-6′ (2H), H-2″), 2.96 (t, J = 10.0 Hz, 1H, H-4″), 2.66 (t, J = 7.6 Hz, 2H, CHN3CCH2CH2(CH2)7CH3), 2.01 (m, 2H, H-2eq, H-3′eq), 1.69–1.57 (m, 3H, H-3′ax, CHN3CCH2CH2(CH2)7CH3), 1.44–1.27 (m, 60H, H-2ax, 5× CO2(CH3)3, CHN3CCH2CH2(CH2)7CH3), 0.88 (t, J = 7.2 Hz, 3H, CHN3CCH2CH2(CH2)7CH3); 13C-NMR (100 MHz, CD3OD, Figure S5) δ 158.0, 157.8, 156.4, 156.3 (2 carbons), 147.6, 123.2, 98.4, 97.3, 81.6, 80.3, 79.3, 79.0 (2 carbons), 78.9, 78.7, 75.1, 72.1, 70.4, 70.2, 69.0, 65.0, 55.6, 50.3, 50.2, 49.8, 40.6, 34.6 (2 carbons), 32.9, 31.6, 29.32, 29.30, 29.2, 29.1, 29.0, 28.9, 27.4 (15 carbons), 24.9, 22.3, 13.0; HR-MS (Figure S6) calcd for C55H99N8O18 [M + H]+ 1159.7072; found 1159.7096.
O-3,6-Dideoxy-3-[[(1,1-dimethylethoxy)carbonyl]amino]-6-(4-dodecyl-1H-1,2,3-triazol-1-yl)-α-d-glucopyranosyl-(1→6)-O-[2,3,6-trideoxy-2,6-bis[[(1,1-dimethylethoxy)carbonyl]amino]-α-d-ribo-hexopyranosyl-(1→4)]-2-deoxy-N1,N3-bis[(1,1-dimethylethoxy)carbonyl]-d-streptamine (6b). To a microwave reaction vessel, compound 5 (60 mg, 0.060 mmol), sodium l-ascorbate (6 mg, 0.030 mmol), CuSO4·5H2O (6 mg, 0.024 mmol), 1-tetradecyne (66 μL, 0.24 mmol), and DMF (1 mL) were added. The reaction vessel was then sealed and irradiated by microwaves for 4 min at 80 °C, twice. Progress of the reaction was monitored by TLC (Hexanes:EtOAc/3:7, Rf 0.58). Upon completion, the reaction mixture was filtered through celite®, and eluted with EtOAc. The filtrate was washed with H2O (3×) and brine (3×), dried over anhydrous MgSO4, filtered, and concentrated to give a crude product. Purification by column chromatography (SiO2, pure Hexanes to Hexanes:EtOAc/2:3) gave the known compound 6b [11] (45 mg, 63%) as a white solid: 1H-NMR (400 MHz, CD3OD, which matches the Reference [11], Figure S7) δ 7.73 (s, 1H, triazole ring), 5.09 (br s, 1H, H-1″), 5.03 (br s, 1H, H-1′), 4.66 (br d, J = 10.4 Hz, 1H, H-6″), 4.55 (t, J = 14.4 Hz, 1H, H-6″), 4.40 (m, 1H, H-5″), 3.72 (t, J = 10.8 Hz, 1H, H-3″), 3.61–3.24 (m, 11H, H-1, H-3, H-4, H-5, H-6, H-2′, H-4′, H-5′, H-6′ (2H), H-2″), 2.96 (t, J = 10.8 Hz, 1H, H-4″), 2.66 (t, J = 7.2 Hz, 2H, CHN3CCH2CH2(CH2)9CH3), 2.01 (m, 2H, H-2eq, H-3′eq), 1.69–1.57 (m, 3H, H-3′ax, CHN3CCH2CH2(CH2)9CH3), 1.44–1.27 (m, 64H, H-2ax, 5× CO2(CH3)3, CHN3CCH2CH2(CH2)9CH3), 0.88 (t, J = 7.6 Hz, 3H, CHN3CCH2CH2(CH2)9CH3).
O-3-Amino-3,6-dideoxy-6-(4-decyl-1H-1,2,3-triazol-1-yl)-α-d-glucopyranosyl-(1→6)-O-[2,6-diamino-2,3,6-trideoxy-α-d-ribo-hexopyranosyl-(1→4)]-2-deoxy-d-streptamine (7a). Compound 6a (30 mg, 0.026 mmol) was treated at room temperature with neat TFA (1 mL) for 3 min. The TFA was removed under reduced pressure, the residue was dissolved in a minimal volume of H2O, extracted with CH2Cl2 (3×), and freeze-dried to afford compound 7a (30 mg, 94%) as a white powder: 1H-NMR (400 MHz, D2O, Figure S8) δ 7.62 (s, 1H, triazole ring), 5.64 (d, J = 3.6 Hz, 1H, H-1′), 4.88 (d, J = 3.6 Hz, 1H, H-1″), 4.58 (m, 1H, H-6″), 4.44 (dd, J1 = 14.8 Hz, J2 = 7.2 Hz, 1H, H-6″), 4.18 (m, 1H, H-5″), 3.81–3.68 (m, 3H, H-4, H-5′, H-2″), 3.64–3.50 (m, 4H, H-5, H-6, H-2′, H-4′), 3.44–3.30 (m, 4H, H-1, H-3, H-3″, H-4″), 3.27 (dd, J1 = 13.6 Hz, J2 = 3.6 Hz, 1H, H-6′), 3.07 (dd, J1 = 14.0 Hz, J2 = 7.6 Hz, 1H, H-6′), 2.51 (t, J = 7.2 Hz, 2H, CHN3CCH2CH2(CH2)7CH3), 2.36 (dt, J1 = 12.8 Hz, J2 = J3 = 3.6 Hz, 1H, H-2eq), 2.16 (dt, J1 = 12.4 Hz, J2 = J3 = 4.4 Hz, 1H, H-3′eq), 1.87 (app. q, J1 = J2 = J3 = 12.0 Hz, 1H, H-3′ax), 1.74 (app. q, J1 = J2 = J3 = 12.8 Hz, 1H, H-2ax), 1.45 (p, J = 6.4 Hz, 2H, CHN3CCH2CH2(CH2)7CH3) 1.20–1.00 (m, 14H, CHN3CCH2CH2(CH2)7CH3), 0.67 (t, J = 6.8 Hz, 3H, CHN3CCH2CH2(CH2)7CH3); 13C-NMR (100 MHz, D2O, Figure S9) δ 148.6, 125.0, 100.8, 93.8, 83.9, 76.6, 74.2, 70.8, 69.9, 67.7, 66.5, 64.4, 54.6, 50.2, 49.5, 48.2, 47.7, 39.8, 31.1, 29.3, 28.54, 28.47, 28.4, 28.3, 28.2, 27.9, 27.6, 24.3, 21.9, 13.3; HR-MS (Figure S10) calcd for C30H59N8O8 [M + H]+ 659.4450; found 659.4457.
O-3-Amino-3,6-dideoxy-6-(4-dodecyl-1H-1,2,3-triazol-1-yl)-α-d-glucopyranosyl-(1→6)-O-[2,6-diamino-2,3,6-trideoxy-α-d-ribo-hexopyranosyl-(1→4)]-2-deoxy-d-streptamine (7b). Compound 6b (35 mg, 0.029 mmol) was treated at room temperature with neat TFA (1 mL) for 3 min. The TFA was removed under reduced pressure, the residue was dissolved in a minimal volume of H2O, extracted with CH2Cl2 (3×), and freeze-dried to afford the known compound 7b [11] (38 mg, quant.) as a white powder: 1H-NMR (400 MHz, D2O, which matches Reference [11], Figure S11) δ 7.62 (s, 1H, triazole ring), 5.64 (d, J = 3.6 Hz, 1H, H-1′), 4.88 (d, J = 3.6 Hz, 1H, H-1″), 4.63 (m, 1H, H-6″), 4.44 (dd, J1 = 14.8 Hz, J2 = 7.2 Hz, 1H, H-6″), 4.18 (m, 1H, H-5″), 3.80–3.69 (m, 3H, H-4, H-5′, H-2″), 3.64–3.50 (m, 4H, H-5, H-6, H-2′, H-4′), 3.42–3.30 (m, 4H, H-1, H-3, H-3″, H-4″), 3.27 (dd, J1 = 13.6 Hz, J2 = 2.8 Hz, 1H, H-6′), 3.07 (dd, J1 = 13.6 Hz, J2 = 7.6 Hz, 1H, H-6′), 2.51 (t, J = 7.2 Hz, 2H, CHN3CCH2CH2(CH2)9CH3), 2.36 (dt, J1 = 12.8 Hz, J2 = J3 = 4.4 Hz, 1H, H-2eq), 2.16 (dt, J1 = 12.4 Hz, J2 = J3 = 4.4 Hz, 1H, H-3′eq), 1.87 (app. q, J1 = J2 = J3 = 12.0 Hz, 1H, H-3′ax), 1.74 (app. q, J1 = J2 = J3 = 12.4 Hz, 1H, H-2ax), 1.45 (p, J = 6.4 Hz, 2H, CHN3CCH2CH2(CH2)9CH3) 1.20–1.00 (m, 18H, CHN3CCH2CH2(CH2)9CH3), 0.67 (t, J = 6.8 Hz, 3H, CHN3CCH2CH2(CH2)9CH3).
O-3,6-Dideoxy-3-[[(1,1-dimethylethoxy)carbonyl]amino]-6-[(1-oxododecyl)amino]-α-d-glucopyranosyl-(1→6)-O-[2,3,6-trideoxy-2,6-bis[[(1,1-dimethylethoxy)carbonyl]amino]-α-d-ribo-hexopyranosyl-(1→4)]-2-deoxy-N1,N3-bis[(1,1-dimethylethoxy)carbonyl]-d-streptamine (8). A solution of compound 7 (80 mg, 0.081 mmol) in THF (2 mL) and H2O (0.1 mL) was treated with 1 M PMe3 in THF (0.11 mL, 0.11 mmol) and the resulting mixture was stirred at 50 °C for 1 h. Progress of the reaction was monitored by TLC (CH2Cl2:MeOH/9:1 + NH4OH (0.7%), Rf 0.25). Upon completion, the solvents were removed and the crude material obtained was filtered through silica gel, eluting with Hexanes:EtOAc/1:3 and CH2Cl2:MeOH/9:1 + NH4OH (0.7%). The filtrate collected from the latter eluting solvent was concentrated, redissolved in anhydrous DMF, and added to a pre-stirred, ice-cooled solution of lauric acid (32 mg, 0.16 mmol), EDC·HCl (39 mg, 0.20 mmol), HOBt (31 mg, 0.20 mmol), DIPEA (0.11 mL, 0.64 mmol), and anhydrous DMF (2 mL). The resulting mixture was stirred overnight till room temperature. Progress of the reaction was monitored by TLC (Hexanes:EtOAc/2:3, Rf 0.24). Upon completion, the reaction mixture was diluted with H2O and extracted with EtOAc (3×). The combined organic layers were washed with H2O (3×) and brine (3×), dried over anhydrous MgSO4, filtered, and concentrated to give a crude product. Purification by flash column chromatography (SiO2, pure Hexanes to Hexanes:EtOAc/2:3) gave compound 8 (51 mg, 55%) as a white solid: 1H-NMR (400 MHz, CD3OD, Figure S12) δ 5.11 (br s, 1H, H-1′), 5.01 (d, J = 3.2 Hz, 1H, H-1″), 4.01 (m, 1H, H-5″), 3.66–3.28 (m, 14H, H-1, H-3, H-4, H-5, H-6, H-2′, H-4′, H-5′, H-6′ (2H), H-2″, H-3″, H-6″ (2H)), 3.14 (t, J = 9.6 Hz, 1H, H-4″), 2.19 (t, J = 7.2 Hz, 2H, NHCOCH2(CH2)9CH3), 2.10–1.90 (m, 2H, H-2eq, H-3′eq), 1.70–1.20 (m, 65H, H-2ax, H-3′ax, 5× CO2(CH3)3, NHCOCH2(CH2)9CH3), 0.88 (t, J = 7.2 Hz, 3H, NHCOCH2(CH2)9CH3); 13C-NMR (100 MHz, CD3OD, Figure S13) δ 175.6, 157.9, 157.8, 156.6, 156.33, 156.25, 98.4, 98.0, 81.8, 81.1, 79.3, 79.1, 78.9 (2 carbons), 78.7, 75.5, 72.0, 71.2, 70.7, 69.3, 65.1, 55.6, 50.3, 49.7, 49.5, 40.6, 40.1, 35.7, 34.4, 32.9, 31.7, 29.4, 29.34, 29.26, 29.1, 29.0, 28.4, 27.4 (15 carbons), 25.6, 22.3, 13.0; HR-MS (Figure S14) calcd for C55H101N6O19 [M + H]+ 1149.7116; found 1149.7128.
O-3-Amino-3,6-dideoxy-6-[(1-oxododecyl)amino]-α-d-glucopyranosyl-(1→6)-O-[2,6-diamino-2,3,6-trideoxy-α-d-ribo-hexopyranosyl-(1→4)]-2-deoxy-d-streptamine (9). Compound 8 (41 mg, 0.036 mmol) was treated at room temperature with neat TFA (1 mL) for 3 min. The TFA was removed under reduced pressure, the residue was dissolved in a minimal volume of H2O, extracted with CH2Cl2 (3×), and freeze-dried to afford compound 9 (40 mg, 93%) as a white powder: 1H-NMR (400 MHz, D2O, Figure S15) δ 5.63 (d, J = 3.6 Hz, 1H, H-1′), 4.88 (d, J = 4.0 Hz, 1H, H-1″), 3.84–3.71 (m, 4H, H-4, H-5′, H-2″, H-5″), 3.66 (t, J = 9.6 Hz, 1H, H-5), 3.61–3.23 (m, 10H, H-1, H-3, H-6, H-2′, H-4′, H-6′, H-3″, H-4″, H-6″ (2H)), 3.09 (dd, J1 = 13.6 Hz, J2 = 6.8 Hz, 1H, H-6′), 2.37 (dt, J1 = 12.4 Hz, J2 = J3 = 4.4 Hz, 1H, H-2eq), 2.13 (m, 1H, H-3′eq), 2.09 (t, J = 8.0 Hz, 2H, NHCOCH2CH2(CH2)8CH3), 1.85 (app. q, J1 = J2 = J3 = 12.0 Hz, 1H, H-3′ax), 1.76 (app. q, J1 = J2 = J3 = 12.8 Hz, 1H, H-2ax), 1.40 (p, J = 6.0 Hz, 2H, NHCOCH2CH2(CH2)8CH3), 1.09 (m, 16H, NHCOCH2CH2(CH2)8CH3), 0.67 (t, J = 6.8 Hz, 3H, NHCOCH2CH2(CH2)8CH3); 13C-NMR (100 MHz, D2O, Figure S16) δ 178.1, 100.8, 94.0, 83.5, 77.2, 74.1, 70.9, 70.2, 68.0, 66.4, 64.3, 54.5, 49.6, 48.2, 47.6, 39.7, 38.8, 35.7, 31.1, 29.2, 28.6 (2 carbons), 28.5, 28.4, 28.24, 28.16, 27.7, 25.3, 21.9, 13.3; HR-MS (Figure S17) calcd for C30H60N6O9 [M + H]+ 649.4495; found 649.4494.
O-3-Azido-3-deoxy-α-d-glucopyranosyl-(1→6)-O-[2,6-diazido-2,3,6-trideoxy-α-d-ribo-hexopyranosyl-(1→4)]-1,3-diazido-1,2,3-trideoxy-d-myo-inositol (10). The synthesis started with the preparation of triflic azide (TfN3). Note: extreme caution should be exercised while handling azides! A solution of NaN3 (8.34 g, 128.3 mmol) in H2O (25 mL) was cooled down to 0 °C in an ice-H2O bath and vigorously stirred. CH2Cl2 (25 mL) was added, followed by Tf2O (10.8 mL, 64.2 mmol), and the resulting mixture was stirred for 2 h at 0 °C. 30 min before completion of the TfN3 reaction, tobramycin (2.0 g, 4.28 mmol) and ZnCl2 (29 mg, 0.21 mmol) were dissolved in H2O (65 mL). Et3N (8.9 mL, 64.2 mmol) was added, followed by dropwise addition of MeOH (215 mL). The resulting mixture was stirred at room temperature. Meanwhile, saturated aqueous NaHCO3 was carefully added to the stirred solution of TfN3 until the reaction mixture stopped fizzing. The TfN3 mixture was then extracted with CH2Cl2 (2 × 20 mL), and the combined CH2Cl2 solutions of TfN3 were washed again with saturated NaHCO3, and filtered directly into the tobramycin solution. The resulting mixture was vigorously stirred for 3 h. The reaction was quenched by addition of solid NaHCO3, filtered through a bed of celite®, and the filtrate was dried in vacuo. Purification by flash column chromatography (SiO2, pure CH2Cl2 to CH2Cl2:MeOH/19:1, Rf 0.29 in CH2Cl2:MeOH/19:1) gave the known compound 10 [45] (2.4 g, 92%) as a white solid: 1H-NMR (400 MHz, CD3OD, which matches Reference [45], Figure S18) δ 5.58 (d, J = 3.2 Hz, 1H, H-1′), 5.23 (d, J = 3.6 Hz, 1H, H-1″), 4.08 (ddd, J1 = 9.2 Hz, J2 = 5.6 Hz, J3 = 2.4 Hz, 1H), 4.02 (ddd, J1 = 9.6 Hz, J2 = 4.4 Hz, J3 = 2.4 Hz, 1H), 3.76 (dd, J1 = 12.0 Hz, J2 = 2.4 Hz, 1H), 3.68–3.29 (m, 10H), 3.21–3.15 (m, 3H), 2.37 (dt, J1 = 12.4 Hz, J2 = J3 = 4.4 Hz, 1H, H-2eq), 2.13 (dt, J1 = 11.2 Hz, J2 = J3 = 4.4 Hz, 1H, H-3′eq), 1.99 (app. q, J1 = J2 = J3 = 11.2 Hz, 1H, H-3′ax), 1.55 (app. q, J1 = J2 = J3 = 12.0 Hz, 1H, H-2ax).
O-3-Azido-3-deoxy-6-O-pivaloyl-α-d-glucopyranosyl-(1→6)-O-[2,6-diazido-2,3,6-trideoxy-α-d-ribo-hexopyranosyl-(1→4)]-1,3-diazido-1,2,3-trideoxy-d-myo-inositol (11). A solution of compound 10 (500 mg, 0.84 mmol) in pyridine (3 mL) at 0 °C was treated with trimethylacetyl chloride (0.12 mL, 1.01 mmol) and the resulting mixture was stirred at 0 °C for 4 h. Progress of the reaction was monitored by TLC (Hexanes:EtOAc/2:3, Rf 0.38). Upon completion, the reaction was quenched with H2O and extracted with EtOAc (2×). The combined organic layers were washed with H2O and brine, dried over anhydrous MgSO4, filtered, and concentrated to give a crude product. Purification by flash column chromatography (SiO2, Hexanes:EtOAc/3:2) gave compound 11 (360 mg, 63%) as a colorless liquid: 1H-NMR (400 MHz, CD3OD, Figure S19) δ 5.60 (d, J = 3.6 Hz, 1H, H-1′), 5.26 (d, J = 4.0 Hz, 1H, H-1″), 4.35 (dd, J1 = 11.6 Hz, J2 = 2.0 Hz, 1H), 4.29 (ddd, J1 = 10.0 Hz, J2 = 4.4 Hz, J3 = 2.0 Hz, 1H), 4.12 (dd, J1 = 12.0 Hz, J2 = 4.8 Hz, 1H), 4.08 (ddd, J1 = 10.0 Hz, J2 = 5.6 Hz, J3 = 2.0 Hz, 1H), 3.69–3.62 (m, 3H), 3.61 (dd, J1 = 11.2 Hz, J2 = 4.0 Hz, 1H), 3.55 (ddd, J1 = 11.2 Hz, J2 = 4.8 Hz, J3 = 2.0 Hz, 1H), 3.51 (dd, J1 = 8.0 Hz, J2 = 2.0 Hz, 1H), 3.50 (m, 1H), 3.48 (dd, J1 = 13.6 Hz, J2 = 2.8 Hz, 1H), 3.40 (dd, J1 = 10.4 Hz, J2 = 3.6 Hz, 1H), 3.39–3.31 (m, 2H), 3.20 (dt, J1 = 12.8 Hz, J2 = J3 = 4.0 Hz, 1H), 2.38 (dt, J1 = 12.8 Hz, J2 = J3 = 4.0 Hz, 1H, H-2eq), 2.14 (dt, J1 = 11.2 Hz, J2 = J3 = 4.8 Hz, 1H, H-3′eq), 1.99 (app. q, J1 = J2 = J3 = 12.4 Hz, 1H, H-3′ax), 1.54 (app. q, J1 = J2 = J3 = 12.4 Hz, 1H, H-2ax), 1.18 (s, 9H); 13C-NMR (100 MHz, CD3OD, Figure S20) δ 178.6, 97.9, 97.0, 80.2, 79.2, 74.9, 72.7, 70.8, 70.0, 68.7, 66.7, 65.1, 62.9, 60.5, 59.3, 56.4, 51.1, 38.6, 32.0, 30.8, 26.2 (3 carbons); HR-MS (Figure S21) calcd for C23H35N15O10Na [M + Na]+ 704.2589; found 704.2589.
O-3-Azido-3-deoxy-2,4-bis-O-(phenylmethyl)-6-O-pivaloyl-α-d-glucopyranosyl-(1→6)-O-[2,6-diazido-2,3,6-trideoxy-4-O-(phenylmethyl)-α-d-ribo-hexopyranosyl-(1→4)]-1,3-diazido-1,2,3-trideoxy-5-O-(phenylmethyl)-d-myo-inositol (12). A solution of compound 11 (355 mg, 0.52 mmol) in DMF (3 mL) at 0 °C was treated with 60% sodium hydride (125 mg, 3.13 mmol). The reaction mixture was stirred at 0 °C for 15 min followed by the addition of benzyl bromide (0.37 mL, 3.13 mmol) and TBAI (96 mg, 0.26 mmol). The reaction mixture was allowed to warm to room temperature and stirred overnight. Progress of the reaction was monitored by TLC (Hexanes:EtOAc/5:1, Rf 0.44). The reaction mixture was quenched with H2O and extracted with EtOAc (2×). The combined organic layers were washed with H2O and brine, dried over anhydrous MgSO4, filtered, and concentrated to give a crude product. Purification by flash column chromatography (SiO2, pure Hexanes to Hexanes:EtOAc/4:1) gave compound 12 (410 mg, 76%) as a white foam: 1H-NMR (400 MHz, CDCl3, Figure S22) δ 7.43–7.25 (m, 13H, aromatic), 7.20 (dd, J1 = 7.6 Hz, J2 = 4.0 Hz, 2H, aromatic), 7.10–7.06 (m, 3H, aromatic), 7.04 (dd, J1 = 7.6 Hz, J2 = 3.6 Hz, 2H, aromatic), 5.64 (d, J = 3.6 Hz, 1H, H-1′), 5.43 (d, J = 4.0 Hz, 1H, H-1″), 4.96 (d, J = 12.0 Hz, 1H), 4.92 (d, J = 12.0 Hz, 1H), 4.81 (d, J = 11.6 Hz, 1H), 4.73 (d, J = 11.6 Hz, 1H), 4.65 (d, J = 10.8 Hz, 1H), 4.63 (d, J = 11.2 Hz, 1H), 4.44 (d, J = 11.2 Hz, 1H), 4.28 (d, J = 11.2 Hz, 1H), 4.19 (ddd, J1 = 9.6 Hz, J2 = 4.4 Hz, J3 = 2.0 Hz, 1H), 4.03 (dd, J1 = 12.0 Hz, J2 = 2.4 Hz, 1H), 3.92 (dd, J1 = 10.0 Hz, J2 = 2.4 Hz, 1H), 3.80 (app. t, J1 = J2 = 10.0 Hz, 1H), 3.72–3.50 (m, 6H), 3.48–3.38 (m, 3H), 3.31 (dd, J1 = 10.8 Hz, J2 = 4.0 Hz, 1H), 3.10 (app. t, J1 = J2 = 9.6 Hz, 1H), 2.99 (dt, J1 = 13.2 Hz, J2 = J3 = 4.0 Hz, 1H), 2.39 (dt, J1 = 13.6 Hz, J2 = J3 = 4.4 Hz, 1H, H-2eq), 2.31 (dt, J1 = 11.6 Hz, J2 = J3 = 4.4 Hz, 1H, H-3′eq), 1.99 (app. q, J1 = J2 = J3 = 12.4 Hz, 1H, H-3′ax), 1.62 (app. q, J1 = J2 = J3 = 12.8 Hz, 1H, H-2ax), 1.16 (s, 9H); 13C-NMR (100 MHz, CDCl3, Figure S23) δ 177.9, 137.44, 137.40, 137.2, 137.1, 128.54 (2 carbons), 128.52 (2 carbons), 128.3 (4 carbons), 128.12, 128.09 (2 carbons), 128.0, 127.9 (2 carbons), 127.8 (2 carbons), 127.7, 127.3, 125.9 (2 carbons), 96.3, 95.1, 83.3, 77.8, 77.2, 77.1, 76.4, 75.0, 74.7, 73.1, 71.8, 71.0, 70.8, 68.7, 65.1, 62.3, 60.2, 59.4, 56.2, 51.2, 38.8, 31.9, 27.8, 27.2 (3 carbons); HR-MS (Figure S24) calcd for C51H59N15O10Na [M + Na]+ 1064.4467; found 1064.4483.
O-3-Azido-3-deoxy-2,4-bis-O-(phenylmethyl)-α-d-glucopyranosyl-(1→6)-O-[2,6-diazido-2,3,6-trideoxy-4-O-(phenylmethyl)-α-d-ribo-hexopyranosyl-(1→4)]-1,3-diazido-1,2,3-trideoxy-5-O-(phenylmethyl)-d-myo-inositol (13). A solution of compound 12 (395 mg, 0.38 mmol) was dissolved in MeOH (5 mL), and sodium metal (3.3 mg, 0.14 mmol) was added. The resulting mixture was stirred at 60 °C for 6 h and progress of the reaction was monitored by TLC (Hexanes:EtOAc/4:1, Rf 0.20). The reaction was quenched with H2O and MeOH was removed. The resulting mixture was extracted with EtOAc (2×). The combined organic layers were washed with H2O and brine, dried over anhydrous MgSO4, filtered, and concentrated to give a crude product. Purification by flash column chromatography (SiO2, Hexanes:EtOAc/4:1) gave compound 13 (294 mg, 81%) as a white solid: 1H-NMR (400 MHz, CDCl3, Figure S25) δ 7.43–7.26 (m, 13H, aromatic), 7.20 (dd, J1 = 6.8 Hz, J2 = 2.8 Hz, 2H, aromatic), 7.13–7.08 (m, 5H, aromatic), 5.58 (d, J = 4.0 Hz, 1H, H-1′), 5.42 (d, J = 3.6 Hz, 1H, H-1″), 4.94 (d, J = 11.6 Hz, 1H), 4.90 (d, J = 11.6 Hz, 1H), 4.80 (d, J = 12.0 Hz, 1H), 4.74 (d, J = 11.6 Hz, 1H), 4.68 (d, J = 11.6 Hz, 1H), 4.63 (d, J = 11.2 Hz, 1H), 4.44 (d, J = 11.2 Hz, 1H), 4.41 (d, J = 11.2 Hz, 1H), 4.19 (ddd, J1 = 9.6 Hz, J2 = 4.8 Hz, J3 = 2.4 Hz, 1H), 3.81 (app. t, J1 = J2 = 10.0 Hz, 1H), 3.74–3.66 (m, 2H), 3.62 (app. t, J1 = J2 = 9.2 Hz, 1H), 3.58–3.50 (m, 3H), 3.48–3.37 (m, 4H), 3.31 (dd, J1 = 10.8 Hz, J2 = 3.6 Hz, 1H), 3.22 (app. t, J1 = J2 = 10.0 Hz, 1H), 3.17 (m, 1H), 2.99 (dt, J1 = 12.8 Hz, J2 = J3 = 4.0 Hz, 1H), 2.38 (dt, J1 = 12.8 Hz, J2 = J3 = 4.8 Hz, 1H, H-2eq), 2.32 (dt, J1 = 11.2 Hz, J2 = J3 = 4.4 Hz, 1H, H-3′eq), 1.99 (app. q, J1 = J2 = J3 = 12.4 Hz, 1H, H-3′ax), 1.63 (app. q, J1 = J2 = J3 = 12.8 Hz, 1H, H-2ax); 13C-NMR (100 MHz, CD3OD, Figure S26) δ 138.1, 138.0, 137.8, 137.6, 128.1 (2 carbons), 128.0 (2 carbons), 127.9 (2 carbons), 127.82 (2 carbons), 127.78 (2 carbons), 127.62, 127.55 (2 carbons), 127.5 (2 carbons), 127.4, 127.1, 126.7, 125.9 (2 carbons), 95.9, 95.0, 83.1, 78.0, 77.3, 77.0, 75.7, 74.3, 74.1, 72.6, 72.2, 71.2, 70.7, 70.4, 65.3, 60.2, 59.8, 59.7, 56.1, 51.0, 31.4, 27.3; HR-MS (Figure S27) calcd for C46H51N15O9Na [M + Na]+ 980.3892; found 980.3896.
O-3-Azido-3-deoxy-6-O-dodecyl-2,4-bis-O-(phenylmethyl)-α-d-glucopyranosyl-(1→6)-O-[2,6-diazido-2,3,6-trideoxy-4-O-(phenylmethyl)-α-d-ribo-hexopyranosyl-(1→4)]-1,3-diazido-1,2,3-trideoxy-5-O-(phenylmethyl)-d-myo-inositol (14). A solution of compound 13 (80 mg, 0.08 mmol) in THF (1.5 mL) at 0 °C was treated with 60% sodium hydride (20 mg, 0.84 mmol). The reaction mixture was stirred at 0 °C for 15 min followed by the addition of 1-bromododecane (0.20 mL, 0.84 mmol), and a catalytic amount of TBAI (3 mg). The reaction mixture was allowed to warm to room temperature and stirred for 12 h. Progress of the reaction was monitored by TLC (Hexanes:EtOAc/4:1, Rf 0.65). The reaction was quenched with H2O and extracted with EtOAc (2×). The combined organic layers were washed with H2O and brine, dried over anhydrous MgSO4, filtered, and concentrated to give a crude product. Purification by flash column chromatography (SiO2, Hexanes:EtOAc/4:1) gave compound 14 (77 mg, 82%) as a white solid: 1H-NMR (400 MHz, CD3OD, Figure S28) δ 7.43 (d, J = 7.6 Hz, 2H, aromatic), 7.35 (t, J = 7.6 Hz, 2H, aromatic), 7.31–7.23 (m, 11H), 7.12–7.09 (m, 5H, aromatic), 5.68 (d, J = 3.6 Hz, 1H, H-1′), 5.44 (d, J = 3.6 Hz, 1H, H-1″), 4.97 (d, J = 11.6 Hz, 1H), 4.92 (d, J = 11.6 Hz, 1H), 4.81 (d, J = 11.6 Hz, 1H), 4.71 (d, J = 11.6 Hz, 1H), 4.64 (d, J = 12.0 Hz, 1H), 4.63 (d, J = 11.2 Hz, 1H), 4.46 (d, J = 11.6 Hz, 1H), 4.40 (d, J = 10.8 Hz, 1H), 4.19 (m, 1H), 3.85 (m, 1H), 3.79–3.57 (m, 7H), 3.52–3.44 (m, 2H), 3.39 (dd, J1 = 10.4 Hz, J2 = 3.2 Hz, 1H), 3.36 (dd, J1 = 13.6 Hz, J2 = 5.6 Hz, 1H), 3.24 (t, J = 10.0 Hz, 2H, OCH2CH2CH2(CH2)8CH3), 3.18–3.05 (m, 3H), 2.38 (dt, J1 = 12.0 Hz, J2 = J3 = 4.4 Hz, 2H, H-2eq, H-3′eq), 1.95 (app. q, J1 = J2 = J3 = 11.6 Hz, 1H, H-3′ax), 1.85 (p, J = 3.2 Hz, 2H, OCH2CH2CH2(CH2)8CH3), 1.57 (app. q, J1 = J2 = J3 = 12.4 Hz, 1H, H-2ax), 1.43 (p, J = 6.8 Hz, 2H, OCH2CH2CH2(CH2)8CH3), 1.25 (m, 16H, OCH2CH2CH2(CH2)8CH3), 0.87 (t, J = 6.0 Hz, 3H, OCH2CH2CH2(CH2)8CH3); 13C-NMR (100 MHz, CDCl3, Figure S29) δ 138.0, 137.4, 137.3 (2 carbons), 128.50 (2 carbons), 128.48 (2 carbons), 128.18 (2 carbons), 128.16 (2 carbons), 128.15 (2 carbons), 128.05, 128.02, 127.8 (2 carbons), 127.7 (2 carbons), 127.5, 127.1, 126.2 (2 carbons), 96.3, 95.7, 83.2, 77.6, 77.1, 75.9, 74.9, 74.5, 73.0, 71.8, 71.6, 70.9, 70.8, 70.1, 68.3, 68.0, 65.3, 60.2, 59.4, 56.1, 51.2, 31.9, 29.69, 29.65, 29.62 (2 carbons), 29.57, 29.4, 29.3, 27.8, 26.1, 25.6, 22.7, 14.1; HR-MS (Figure S30) calcd for C58H75N15O19Na [M + Na]+ 1148.5770; found 1148.5786.
O-3-Deoxy-3-[[(1,1-dimethylethoxy)carbonyl]amino]-6-O-dodecyl-α-d-glucopyranosyl-(1→6)-O-[2,3,6-trideoxy-2,6-bis[[(1,1-dimethylethoxy)carbonyl]amino]-α-d-ribo-hexopyranosyl-(1→4)]-2-deoxy-N1,N3-bis[(1,1-dimethylethoxy)carbonyl]-d-streptamine (15). A solution of compound 14 (95 mg, 0.084 mmol) in THF (2 mL) and 0.1 M NaOH (0.5 mL) was treated with 1 M PMe3 in THF (0.59 mL, 0.59 mmol) and the resulting mixture was stirred at 50 °C for 2 h. Upon completion, the solvents were removed and the crude material obtained was dissolved in 5 mL of degassed AcOH:H2O/1:3. A catalytic amount of Pd(OH)2/C was added and the resulting mixture was stirred at room temperature overnight under H2 atmosphere. The following day, the mixture was filtered through celite®, and the residue was washed with H2O. The filtrate was freeze-dried overnight. The residue obtained was dissolved in THF (5 mL) and treated with Boc2O (110 mg, 0.51 mmol) and Et3N (0.14 mL, 1.01 mmol). The resulting mixture was stirred at 60 °C for 6 h. Progress of the reaction was monitored by TLC (Hexanes:EtOAc/2:3, Rf 0.34). The solvents were then removed and the crude material obtained was purified by column chromatography (SiO2, pure Hexanes to Hexanes:EtOAc/3:7) to give compound 15 (31 mg, 32% over 3 steps) as a white solid: 1H-NMR (400 MHz, CD3OD, Figure S31) δ 5.10 (br s, 1H, H-1′), 5.02 (br d, J = 3.2 Hz, 1H, H-1″), 4.01 (m, 1H, H-5″), 3.70–3.30 (m, 17H, H-1, H-3, H-4, H-5, H-6, H-2′, H-4′, H-5′, H-6′ (2H), H-2″, H-3″, H-4″, H-6″ (2H), OCH2CH2(CH2)9CH3), 2.09 (m, 1H, H-2eq), 1.99 (m, 1H, H-3′eq), 1.62 (app. q, J1 = J2 = J3 = 12.0 Hz, 1H, H-3′ax), 1.55 (p, J = 6.8 Hz, 2H, OCH2CH2(CH2)9CH3), 1.60–1.22 (m, 64H, H-2ax, 5× CO2(CH3)3, OCH2CH2(CH2)9CH3), 0.88 (t, J = 7.2 Hz, 3H, OCH2CH2(CH2)9CH3); 13C-NMR (100 MHz, CD3OD, Figure S32) δ 158.1, 157.9, 156.5, 156.3 (2 carbons), 98.2, 97.8, 82.6, 80.9, 79.3, 78.9 (2 carbons), 78.7, 75.5, 72.3, 72.1, 71.6, 70.6, 69.6, 68.6, 65.1, 55.9, 50.2, 49.7, 49.6, 40.6, 34.2, 32.9, 31.7, 29.4 (2 carbons), 29.35, 29.25 (2 carbons), 29.23, 29.1 (2 carbons), 27.4 (15 carbons), 25.8, 22.3, 13.0; HR-MS (Figure S33) calcd for C55H101N5O19Na [M + Na]+ 1158.6988; found 1158.7010.
O-3-Amino-3-deoxy-6-O-dodecyl-α-d-glucopyranosyl-(1→6)-O-[2,6-diamino-2,3,6-trideoxy-α-d-ribo-hexopyranosyl-(1→4)]-2-deoxy-d-streptamine (16). Compound 15 (22 mg, 0.019 mmol) was treated at room temperature with neat TFA (1 mL) for 3 min. The TFA was removed under reduced pressure, the residue was dissolved in a minimal volume of H2O, extracted with CH2Cl2 (3×), and freeze-dried to afford compound 16 (22 mg, 96%) as a white foam: 1H-NMR (400 MHz, D2O, Figure S34) δ 5.61 (d, J = 3.2 Hz, 1H, H-1′), 4.90 (d, J = 4.0 Hz, 1H, H-1″), 3.85–3.70 (m, 4H, H-4, H-5′, H-2″, H-5″), 3.67 (app. t, J1 = J2 = 9.2 Hz, 1H, H-5), 3.62–3.28 (m, 11H, H-1, H-3, H-6, H-2′, H-4′, H-6′ (2H), H-3″, H-4″, OCH2(CH2)10CH3), 3.25 (dd, J1 = 14.0 Hz, J2 = 2.8 Hz, 1H, H-6″), 3.08 (dd, J1 = 13.2 Hz, J2 = 6.8 Hz, 1H, H-6″), 2.37 (app. dt, J1 = 12.4 Hz, J2 = J3 = 3.6 Hz, 1H, H-2eq), 2.12 (app. dt, J1 = 12.4 Hz, J2 = J3 = 4.0 Hz, 1H, H-3′eq), 1.85 (app. q, J1 = J2 = J3 = 12.0 Hz, 1H, H-3′ax), 1.76 (app. q, J1 = J2 = J3 = 12.8 Hz, 1H, H-2ax), 1.39 (p, J = 6.8 Hz, 2H, OCH2CH2(CH2)9CH3), 1.20–1.00 (m, 18H, OCH2CH2(CH2)9CH3), 0.67 (t, J = 7.2 Hz, 3H, OCH2(CH2)10CH3); 13C-NMR (100 MHz, D2O, Figure S35) δ 100.8, 93.9, 83.7, 77.0, 74.0, 71.89, 71.86, 70.3, 67.92, 67.86, 65.2, 64.3, 54.7, 49.5, 48.2, 47.6, 39.7, 31.1, 29.2, 28.7 (2 carbons), 28.62, 28.57, 28.5, 28.4, 28.3, 27.7, 25.0, 21.9, 13.3; HR-MS (Figure S36) calcd for C30H62N5O9 [M + H]+ 636.4542; found 636.4546.

4.3. Antifungal Susceptibility Testing

The MIC values of compounds 3, 4, 7a, 7b, 9, and 16, as well as the parent compound TOB and the antifungal CAS against yeasts (strains AG (Table 1), and strains HJ, CG13, CP13, and CN13 (Table 2)) were evaluated in 96-well microtiter plates as described in the CLSI document M27-A3 [52] with minor modifications. The final concentrations of compounds 3, 4, 7a, 7b, 9, and 16, as well as that of TOB tested in this study ranged from 0.24–62.5 μg/mL. CAS was used as a positive control and the final concentrations tested for CAS ranged from 0.03–31.3 μg/mL. Briefly, overnight yeast cultures were grown in yeast peptone dextrose (YPD) broth and the cell density was adjusted to an OD600 of 0.12 (~1 × 106 CFU/mL) by using a spectrophotometer. Yeast cell suspensions were further diluted to achieve 1–5 × 103 CFU/mL in RPMI 1640 medium, and 100 μL of these yeast cells was added to 96-well microtiter plates containing RPMI 1640 medium and titrated compounds. Each test was performed in triplicate. The plates were incubated at 35 °C for 48 h. The MIC values for compounds 3, 4, 7a, 7b, 9, and 16, CAS, and TOB were defined as the lowest drug concentration that prevented visible growth (also known as MIC-0) when compared to the growth control. These data are presented in Table 1 (for strains AG) and Table 2 (for strains HJ, CG13, CP13, and CN13).
Similarly, the MIC values of compounds 3, 4, 7a, 7b, 9, and 16, as well as that of all control drugs against filamentous fungi (strains KM (Table 3)) were determined as previously described in CLSI document M38-A2 [53]. The spores were harvested from sporulating cultures growing on potato dextrose agar (PDA) by filtration through sterile glass wool and enumerated by using a hemocytometer (Hausser Scientific, Horsham, PA, USA) to obtain the desired inoculum size. Two-fold serial dilutions of compounds 3, 4, 7a, 7b, 9, and 16, as well as CAS and TOB were made in sterile 96-well microtiter plates to obtain the final concentration range of 0.24–62.5 μg/mL for TOB and TOB derivatives as well as 0.03–31.3 μg/mL for CAS in the RPMI 1640 medium. The spore suspensions were added to the wells to afford a final concentration of 5 × 105 spores/mL. The plates were incubated at 35 °C for 72 h. The MIC values of all compounds, including compounds 3, 4, 7a, 7b, 9, and 16 as well as CAS and TOB against filamentous fungi were based on the complete inhibition of growth (optically clear well) when compared to the growth control (MIC-0). Each test was performed in triplicate. These data are also presented in Table 3 (strains KM).

4.4. Hemolytic Activity Assays

The hemolytic activity of compounds 3, 4, 7a, 7b, and 9 as well as the parent compound TOB was determined by using previously described methods with minor modifications [23]. Murine whole blood (1 mL) was suspended in 4 mL of PBS and centrifuged at 1000 rpm for 10 min at room temperature to obtain murine red blood cells (mRBCs). The mRBCs were washed four times in PBS and resuspended in the same buffer to a final concentration of 107 erythrocytes/mL. Two-fold serial dilutions of compounds 3, 4, 7a, 7b, and 9 were prepared using 100 μL of PBS buffer in Eppendorf tubes followed by the addition of 100 μL of mRBC suspension that made the final concentration of compounds and mRBCs to be 0.48–62.5 μg/mL and 5 × 106 erythrocytes/mL, respectively. The tubes were incubated at 37 °C for 1 h. The tubes with PBS buffer (200 μL) and Triton X-100® (1% v/v, 2 μL) served as the negative (blank) and positive controls, respectively. The percentage of hemolysis was calculated using the following equation: % hemolysis = [(absorbance of sample) − (absorbance of blank)] × 100/(absorbance of positive control). These data are presented in Figure 1 and Table S1.

4.5. In Vitro Cytotoxicity Assays

Mammalian cytotoxicity assays were performed as previously described with minor modifications [29]. The normal human bronchial epithelial cells BEAS-2B and the human lung carcinoma epithelial cells A549 were grown in DMEM containing 10% fetal bovine serum (FBS) and 1% antibiotics. The confluent cells were then trypsinized with 0.05% trypsin and 0.53 mM EDTA, and resuspended in fresh DMEM medium. The cells were transferred into 96-well microtiter plates at a density of 3000 cells/well and were grown for 24 h. The following day, the medium was replaced by a fresh culture medium containing serially diluted compounds 3, 4, 7a, 7b, and 9, as well as the parent drug TOB at final concentrations of 0.48–62.5 μg/mL or sterile ddH2O (negative control). The cells were incubated for an additional 24 h at 37 °C with 5% CO2 in a humidified incubator. To evaluate cell survival, each well was treated with 10 μL (25 μg/mL) of resazurin sodium salt (Sigma-Aldrich) for 4–6 h. Metabolically active cells can convert resazurin (blue) to the highly fluorescent dye resorufin (pink), which was detected at λ560 excitation and λ590 emission wavelengths by using a SpectraMax M5 plate reader. Triton X-100® (1% v/v) gave the complete loss of cell viability and was used as the positive control. The percentage survival rates were calculated by using the following formula: % cell survival = [(fluorescence of sample) − (fluorescence of blank)] × 100/[(fluorescence of negative control) − (fluorescence of blank)]. These data are presented in Figure 2.

4.6. Time-Kill Assays

Time-kill assays were used to assess the inhibitory efficiency of 7b against three yeast strains, C. albicans ATCC 10231 (strain A), C. parapsilosis ATCC 22019 (strain J), and C. neoformans clinical isolate CN1. The protocol for time-kill assays followed methods previously described [29,54] with minor modifications. Yeast cultures were grown overnight in YPD broth at 35 °C with shaking (200 rpm). A working stock of fungal cells was made by diluting cultures in RPMI 1640 medium to an OD600 of 0.125, approximately 1 × 106 CFU/mL. From the working stock, 100 μL of cells was added to 4.9 mL of RPMI 1640 medium in sterile culture tubes, making the starting fungal cell concentration approximately 105 CFU/mL. Compounds were then added to the fungal cells. The treatment conditions included sterile control, growth control, TOB (parent; negative control), voriconazole (VOR) at 1× MIC (positive control), 7b at 1× and 4× MIC. The treated fungal cultures were incubated at 35 °C with shaking (200 rpm) for 24 h. The samples were taken from the different treatments at regular time points (0, 3, 6, 9, 12, and 24 h.) and plated in duplicates. For each time point, cultures were vortexed, 100 μL of culture was aspirated, and 10-fold serial dilutions were made in sterile ddH2O. From the appropriate dilutions, 100 μL of fungal suspension was spread on PDA plates and incubated at 35 °C for 48 h before the colony counts were determined. At 24 h, 50 μL of 1 mM resazurin was added to the treatments and incubated at 35 °C with rotation for 2 h in the dark for visual inspection. Experiments were performed in duplicate. These data are presented in Figure 3.

4.7. Membrane Permeabilization Assay Using Propidium Iodide Staining

A fresh culture of C. albicans ATCC 10231 (strain A) was prepared in 5 mL of YPD broth in a Falcon tube and was grown overnight at 35 °C at 200 rpm. An overnight culture (40 μL) was transferred to RPMI 1640 medium (1 mL) containing no drug (negative control) or compound 7b at concentrations of 3.9 μg/mL (2× MIC) and 7.8 μg/mL (4× MIC). TOB (31.3 μg/mL) was used as a negative control. The cell suspensions were then treated for 2 h at 35 °C with continuous agitation (200 rpm). To prepare a positive control sample, C. albicans ATCC 10231 (strain A) cells were killed by heat shock at 95 °C for 5 min as described by Ocampo and Barrientos [55]. The cells were then centrifuged and resuspended in 500 μL of PBS buffer (pH 7.2 adjusted at room temperature). Subsequently, the cells were treated with propidium iodide (9 μM, final concentration) and incubated for 20 min at room temperature in the dark. Glass slides prepared with 10 μL of each mixture were observed in bright field and fluorescence modes (using Texas red filter set with λ535 excitation and λ617 emission wavelengths) using a Zeiss Axiovert 200 M fluorescence microscope. The data were obtained from at least two independent experiments. The images were also post-processed, utilizing automatic contrast and brightness setting in Microsoft PowerPoint 2013 to eliminate background noise. These images are presented in Figure 4.

Supplementary Materials

The supplementary materials include 1H and 13C-NMR as well as mass spectra (Figures S1–S36) for the molecules synthesized. All compounds tested for activity are ≥95% pure according to NMR spectra. Table S1 with the % hemolysis ± SDEV displayed in Figure 1 is also provided. These materials are available free of charge via the Internet.

Acknowledgments

This work was supported by NIH grant AI090048 (to S.G.-T.).

Author Contributions

M.Y.F., N.T.C. and S.G.-T. conceived and designed the study; M.Y.F. and N.T.C. performed the syntheses; S.K.S., E.K.D. and K.D.G. performed the biological studies; M.Y.F. and S.G.-T. analyzed the data and wrote the paper.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

AcOHacetic acid
AGaminoglycoside
AmBamphotericin B
Boc2Odi-tert-butyl dicarbonate
CAScaspofungin
CFUcolony forming unit
DIPEAN,N-diisopropylamine
DMFN,N-dimethylformamide
EDC·HClN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride
FDAUS food and drug administration
HOBthydroxybenzotriazole
KANAkanamycin A
KANBkanamycin B
m-CPBAmeta-chloroperbenzoic acid
mRBCmurine red blood cell
MICminimum inhibitory concentration
PivClpivaloyl chloride
TBAAtetrabutylammonium azide
TFAtrifluoroacetic acid
Tf2Otrifluoromethane sulfonic anhydride
THFtetrahydrofuran
TIPBSCl2,4,6-triisopropylbenzenesulfonyl chloride
TOBtobramycin
VORvoriconazole

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  • Sample Availability: Samples of the compounds 3, 4, 7a, 7b, 9, and 16 are available from the authors.
Scheme 1. The synthesis of tobramycin (TOB) derivatives 3, 4, 7a, 7b, and 9. Reagents: (a) TIPBSCl, pyridine, 59%; (b) C12H25SH, Cs2CO3, N,N-dimethylformamide (DMF), 79%; (c) Trifluoroacetic acid (TFA), 61%; (d) m-CPBA/CHCl3 then TFA, 66%; (e) Tetrabutylammonium azide (TBAA), DMF, 75 °C, 65%; (f) R-CCH, CuSO4·5H2O, sodium l-ascorbate, DMF, microwave, 57–63%; (g) TFA, 94%-quantitative; (h) PMe3, THF/H2O, 50 °C then C11H23CO2H, EDC·HCl, HOBt, DIPEA, DMF, 55%; (i) TFA, 93%.
Scheme 1. The synthesis of tobramycin (TOB) derivatives 3, 4, 7a, 7b, and 9. Reagents: (a) TIPBSCl, pyridine, 59%; (b) C12H25SH, Cs2CO3, N,N-dimethylformamide (DMF), 79%; (c) Trifluoroacetic acid (TFA), 61%; (d) m-CPBA/CHCl3 then TFA, 66%; (e) Tetrabutylammonium azide (TBAA), DMF, 75 °C, 65%; (f) R-CCH, CuSO4·5H2O, sodium l-ascorbate, DMF, microwave, 57–63%; (g) TFA, 94%-quantitative; (h) PMe3, THF/H2O, 50 °C then C11H23CO2H, EDC·HCl, HOBt, DIPEA, DMF, 55%; (i) TFA, 93%.
Molecules 23 00899 sch001
Scheme 2. The synthesis of TOB derivative 16. Reagents: (a) NaN3, Tf2O, H2O, CH2Cl2 then ZnCl2, Et3N, H2O, MeOH, 92%; (b) PivCl, pyridine, 0 °C, 63%; (c) NaH, BnBr, TBAI, DMF, 76%; (d) Na, MeOH, 60 °C, 81%; (e) NaH, C12H25Br, TBAI, DMF, 82%; (f) PMe3, NaOH, THF; (g) Pd(OH)2/C, H2, AcOH, H2O; (h) Boc2O, Et3N, THF, 60 °C, 32% over 3 steps; (i) TFA, 96%.
Scheme 2. The synthesis of TOB derivative 16. Reagents: (a) NaN3, Tf2O, H2O, CH2Cl2 then ZnCl2, Et3N, H2O, MeOH, 92%; (b) PivCl, pyridine, 0 °C, 63%; (c) NaH, BnBr, TBAI, DMF, 76%; (d) Na, MeOH, 60 °C, 81%; (e) NaH, C12H25Br, TBAI, DMF, 82%; (f) PMe3, NaOH, THF; (g) Pd(OH)2/C, H2, AcOH, H2O; (h) Boc2O, Et3N, THF, 60 °C, 32% over 3 steps; (i) TFA, 96%.
Molecules 23 00899 sch002
Figure 1. The hemolytic activity of tobramycin (TOB); its derivatives 3, 4, 7a, 7b, and 9; as well as AmB on mRBCs. Note: the exact values used to generate this graph are presented in Table S1.
Figure 1. The hemolytic activity of tobramycin (TOB); its derivatives 3, 4, 7a, 7b, and 9; as well as AmB on mRBCs. Note: the exact values used to generate this graph are presented in Table S1.
Molecules 23 00899 g001
Figure 2. The mammalian cell cytotoxicity of TOB and its derivatives 3, 4, 7a, 7b, and 9 against (A) the BEAS-2B cell line and (B) the A549 cell line.
Figure 2. The mammalian cell cytotoxicity of TOB and its derivatives 3, 4, 7a, 7b, and 9 against (A) the BEAS-2B cell line and (B) the A549 cell line.
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Figure 3. The representative time-kill studies of TOB derivative 7b against (A) C. albicans ATCC 10231 (strain A), (B) C. parapsilosis ATCC 22019 (strain J), and (C) Cryptococcus neoformans clinical isolate CN1. In all panels, the cultures were exposed to a no drug control (black circle), TOB at 15.6 μg/mL (white circle), VOR at 1.0 or 0.25 μg/mL (black inverted triangle), and 7b at 1× MIC (white triangle) and 4× MIC (black square). Each test tube represents the corresponding sample treated with resazurin, which was added for the visualization of fungal growth. Note: Blue = no fungal growth; orange-pink = fungal growth.
Figure 3. The representative time-kill studies of TOB derivative 7b against (A) C. albicans ATCC 10231 (strain A), (B) C. parapsilosis ATCC 22019 (strain J), and (C) Cryptococcus neoformans clinical isolate CN1. In all panels, the cultures were exposed to a no drug control (black circle), TOB at 15.6 μg/mL (white circle), VOR at 1.0 or 0.25 μg/mL (black inverted triangle), and 7b at 1× MIC (white triangle) and 4× MIC (black square). Each test tube represents the corresponding sample treated with resazurin, which was added for the visualization of fungal growth. Note: Blue = no fungal growth; orange-pink = fungal growth.
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Figure 4. The representative dose-dependent membrane permeabilization effects of TOB and its derivative 7b on C. albicans ATCC 10231 (strain A). From top to bottom: propidium iodine (PI) dye uptake by yeast cells without drug, with TOB (31.3 μg/mL), killed by heat, with 7b (2× MIC), and with 7b (4× MIC).
Figure 4. The representative dose-dependent membrane permeabilization effects of TOB and its derivative 7b on C. albicans ATCC 10231 (strain A). From top to bottom: propidium iodine (PI) dye uptake by yeast cells without drug, with TOB (31.3 μg/mL), killed by heat, with 7b (2× MIC), and with 7b (4× MIC).
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Table 1. The minimum inhibitory concentration (MIC) values a (in μg/mL) determined for tobramycin (TOB); its derivatives 3, 4, 7a, 7b, 9, and 16; as well as CAS, against a panel of seven C. albicans strains.
Table 1. The minimum inhibitory concentration (MIC) values a (in μg/mL) determined for tobramycin (TOB); its derivatives 3, 4, 7a, 7b, 9, and 16; as well as CAS, against a panel of seven C. albicans strains.
Yeast Strains
Cpd #ABCDEFG
TOB>62.5>62.5>62.5>62.5>62.5>62.5>62.5
315.615.615.615.615.67.815.6
415.631.315.615.615.615.615.6
7a31.331.362.562.631.362.562.5
7b3.93.915.615.67.87.87.8
931.315.631.362.531.331.331.3
1662.562.5>62.562.562.562.562.5
CAS0.9750.240.060.12 0.120.240.48
a MIC-0 values are reported for all compounds tested. Yeast strains: A = Candida albicans ATCC 10231, B = C. albicans ATCC 64124, C = C. albicans ATCC MYA-2876(S), D = C. albicans ATCC 90819(R), E = C. albicans ATCC MYA-2310(S), F = C. albicans ATCC MYA-1237(R), G = C. albicans ATCC MYA-1003(R). NOTE: Here, the (S) and (R) indicate that ATCC reports these strains to be susceptible (S) or resistant (R) to itraconazole and fluconazole.
Table 2. The MIC values a (in μg/mL) determined for TOB; its derivatives 3, 4, 7a, 7b, 9, and 16; as well as CAS, against a panel of nine non-albicans Candida and three strains of Cryptococcus neoformans.
Table 2. The MIC values a (in μg/mL) determined for TOB; its derivatives 3, 4, 7a, 7b, 9, and 16; as well as CAS, against a panel of nine non-albicans Candida and three strains of Cryptococcus neoformans.
Yeast Strains
Non-albicans CandidaCryptococcus neoformans
Cpd #CG1CG2CG3HIJCP1CP2CP3CN1CN2CN3
TOB>62.5>62.5>62.5>62.5>62.5>62.5>62.5>62.5>62.5>62.5>62.5>62.5
315.67.815.67.83.97.815.67.815.60.9750.480.48
47.83.93.915.67.87.83.93.97.81.950.480.975
7a31.315.615.662.515.615.615.615.631.30.9750.9750.48
7b3.93.93.97.83.91.953.91.957.80.480.480.975
915.615.615.631.315.67.831.315.631.30.9750.9750.975
16>62.562.562.562.531.331.362.562.562.51.950.9753.9
CAS1.950.240.9750.060.481.950.480.480.4815.631.315.6
a MIC-0 values are reported for all compounds tested. Yeast strains: CG13 = Candida glabrata clinical isolates, CP13 = Candida parapsilosis clinical isolates, CN13 = Cryptococcus neoformans clinical isolates, H = Candida glabrata ATCC 2001, I = Candida krusei ATCC 6258, J = Candida parapsilosis ATCC 22019.
Table 3. The MIC values a (in μg/mL) determined for TOB; its derivatives 3, 4, 7a, 7b, 9, and 16; as well as CAS, against a panel of three Aspergillus strains.
Table 3. The MIC values a (in μg/mL) determined for TOB; its derivatives 3, 4, 7a, 7b, 9, and 16; as well as CAS, against a panel of three Aspergillus strains.
Filamentous Strains
Cpd #KLM
TOB>62.5>62.5>62.5
315.63.9>62.5
431.33.962.5
7a31.33.9>62.5
7b7.81.9562.5
931.33.9>62.5
16>62.53.9>62.5
CAS>31.3>31.3>31.3
a MIC-0 values are reported for all compounds tested. Filamentous strains: K = Aspergillus flavus ATCC MYA-3631, L = Aspergillus nidulans ATCC 38163, M = Aspergillus terreus ATCC MYA-3633.

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