Journal of Modern Chemical Sciences Category: Chemistry Type: Research Article
Recovery of the Antibiotic Activity against Resistant Bacterial Strains by Selective Guanidinylation of the 3-Methylamino Group of Gentamicin
- Julia Revuelta1*, Andrés G Santana1*, Sandra G Zárate2, Agatha Bastida2
- 1 Departmento De Quimica Bioorganica, Instituto De Química Orgánica General (CSIC) Juan De La Cierva, 3. 28006-Madrid, Spain
- 2 Departmento De Quimica Bioorganica, Instituto De Química Orgánica General (CSIC), Juan De La Cierva, Spain
*Corresponding Author:Julia Revuelta
Departmento De Quimica Bioorganica, Instituto De Química Orgánica General (CSIC) Juan De La Cierva, 3. 28006-Madrid, Spain
Andrés G Santana
Departmento De Quimica Bioorganica, Instituto De Química Orgánica General (CSIC) Juan De La Cierva, 3. 28006-Madrid, Spain
Tel:+34 91 2587455,
Received Date: Jul 11, 2017 Accepted Date: Sep 12, 2017 Published Date: Sep 26, 2017
Acquired resistance to aminoglycoside antibiotics can occur via three different mechanisms: Mutation of the ribosomal target, reduced permeability for the antibiotics, and enzymatic modification of the drugs, thus leading to inactivation. From a clinical point of view, the most relevant source of resistance is the enzymatic inactivation of the drugs by modification of its amine or hydroxyl groups. Antibiotic Modifying Enzymes (AMEs) can be broadly classified as N-Acetyltransferases (AACs), O-Adenyl Transferases (ANTs) and O-Phosphotransferases (APHs). Each family involves enzymes that catalyse the same reactions but with different regioselectivity and substrate specificity (Figure 1).
With this in mind, and given the urgent demand for the discovery of new antibiotics to overcome the resistance to these compounds, considerable effort has been devoted to designing new semisynthetic aminoglycoside antibiotics that are immune to inactivation . The intrinsic potency of aminoglycosides makes them excellent candidates to explore new ways to avoid bacterial resistance and diminish toxicity. Our group has been actively involved in the design of new antibiotics that are not susceptible to inactivation by AMEs. With this purpose, different design strategies have been employed (Figure 1) .
These derivatives included changes in the distribution of the amino groups (Figure 1a-c) , conformational restriction of the drug (Figure 1d) , the simultaneous incorporation of kanamycin and ribostamycin fragments within the same antibiotic scaffold (Figure 1e) , aminoglycoside dimerisation (Figure 1f)  and the introduction of bulky substituent’s properly positioned to interfere with drug recognition (Figure 1g) . Unfortunately, despite their structural diversity, these compounds are substrates of the AMEs to some extent and some of them even lost their antibiotic activity. More recently, we have demonstrated that the A site shows a clear tolerance for modification at the N-3″ position of the aminoglycosides [14,15] and that the guanidinylation of this position maintains the antibiotic activity against aminoglycoside 6′-acetyl-transferase and 4′-nucleotidyl-transfersase-expressing strains (Figure 1h) [16, 17].
Figure 1: Representation of the different aminoglycoside systems (a-g) designed and synthesized by our group to prevent AME inactivation. Addition of a substituent at the N-3″ position of kanamycin A (strategy highlighted with a red square) is well tolerated by the rRNA and, particularly, guanidinylation of this position prevents the inactivation of the antibiotic by AMEs.
Herein, we propose the synthesis and evaluation of the new gentamicin derivatives 1a and 1b with the aim of expanding the usefulness of our strategy, demonstrating its broad applicability against other resistance enzymes. Gentamicin is a mixture of various congeners: C1, C1a and C2 (Figure 2). Structural differences between them are minor, differing only by a methyl or a hydrogen substitution in two R groups (R1 or R2) on the purpurosamine residue. This mixture has been the only aminoglycoside antibiotic used to date in clinic and recently it has demonstrated utility for the treatment of sepsis caused by diverse strains of MRSA bacteria . The MIC of gentamicin (2a-c) typically ranges from 6 to 48 ?g/mL. However, many strains have acquired aminoglycoside resistance genes that encode various AMEs, which eventually result in very high resistance to gentamicin (MICs usually higher than 200 ?g/mL). The most clinically relevant of these genes are aac (6′), aac(2′), aph(2″) and ant(2″).
Figure 2: (Left) the structures of gentamicin C1, C1a and C2 components (2a-c) are shown, indicating the positions susceptible to enzymatic modification.
MATERIALS AND METHODS
SYNTHESIS OF COMPOUNDS 3A-3B
To a well stirred solution of gentamicin C1 (2a), C1a (2b) and C2 (2c) (ratio = 25:35:40) free base (0.3 g, 0.64 mmol) in a saturated aqueous solution of Na2CO3 (4 ml) at 0°C was added a solution of CbzCl (benzyl chloroformate) (0.65 g, 3.84 mmol) in acetone (1.5 ml) drop by drop. The mixture was vigorously stirred for 2 hours at this temperature and then 8 hours at room temperature. Subsequently, the solvent was removed under reduced pressure and the residue was pulverized in a mortar. This solid was added to an aqueous solution of HCl (1M) until neutralization and the formed solid product was filtered and dried under vacuum, obtaining the mixture of compounds 3a-c (0.36 g, 50%). MS-API-ES: 1148 (M+H)+ (3a), 1134 (M+H)+ (3b), 1120 (M+H)+ (3c).
1,3, 2′,6′,-(Cbz)4-2″,3″-carbamate-gentamicin C1 (4a), 1,3,2′,6′,-(Cbz)4-2″,3″-carbamate-gentamicin C1a (4b) and 1,3, 2′,6′,-(Cbz)4-2″,3″-carbamate-gentamicin C2 (4c).
To a stirred solution of the mixture of compounds 3a-c (0.3g, 0.32 mmol) in a mixture of 1,4-dioxane/H2O (30 ml/10 ml) was added an aqueous solution of NaOH (2.5M). The mixture was stirred at 50°C for 24 hours, after which was added an aqueous solution of HCl (1M) until pH 10. The mixture was concentrated under reduced pressure and subsequently water was added (25 ml). The obtained suspension was filtered and the solid was dried under vacuum. Finally, the residue was purified through a column chromatography (CHCl3/MeOH/NH4OH, 8:2:0.2 → 6:3:1), yielding the mixture of products 4a, 4b and 4c as a white solid (60%). MS-API-ES: 1040 (M+H)+ (4a), 1026 (M+H)+ (4b), 1012 (M+H)+ (4c).
1,3,2′,6′-(Cbz)4-gentamicin C1a (5a), 1,3,2′,6′-(Cbz)4-gentamicin C2 (5b) and 1,3,2′-(Cbz)3-gentamicin C1 (6).
To a solution of free base gentamicin C1, C1a and C2 (2a-c) (ratio = 25:35:40) (0.3 g, 0.64 mmol) in DMSO (4 ml) at 0°C was added N-benzyloxycarbonyl succinimide (0.52 g, 2.11 mmol). The mixture was stirred vigorously for 2 hours at room temperature. Subsequently, Et2O was added (100 ml), observing the formation of a colorless oil. After solvent decantation the residue was purified by resin chromatography column Amberlite™ IRA-120-H+ (5 g), yielding a mixture of compounds 5a and 5b (0.17 g, 37%) when the resin was eluted with a 0.5M solution of NH4OH in 1,4-dioxane/H2O (1:1) and compound 6 (73 mg, 13%) when the concentration of NH4OH was increased to 1M. MS-API-ES: 985 (M+H)+ (5a), 1000 (M+H)+ (5b) and MS-API-ES: 880 (M+H)+ (6).
1,3,2′,6′-(Cbz)4-3″-(Boc)2-guanidine-gentamicin C1a (7a) and 1,3,2′,6′-(Cbz)4-3″-(Boc)2-guanidinio-gentamicina C2 (7b).
To a stirred solution of compounds 5a and 5b (0.17 g, 0.23 mmol) in 1,4-dioxane (11.7 ml) was added 1,3-(Boc)2-2-(trifluoromethylsulfonyl)guanidine (0.134 g, 0.34 mmol) and Et3N (95 ? L, 0.69 mmol). The mixture was stirred at room temperature for 5 days. Then, the solvent was removed under reduced pressure and finally the residue was purified in silica gel column chromatography (AcOEt →AcOEt/MeOH 9:1) to yield a mixture of 7a and 7b (0.20 g, 72%) as a white foam. 1H NMR (DMSO-d6, 400 MHz) (selected signals): 7.60-7.20 (m, 40H), 5.35-5.27 (m, 4H), 5.25-4.95 (m, 10H), 2.10-1.70 (m, 4H), 1.50-1.40 (s, 18H), 1.20 (s, 6H). MS-API-ES: 1228 (M+H)+ (7a), 1244 (M+H)+ (7b).
3″-guanidine-gentamicin C1a (1a) and 3″-guanidine-gentamicin C2 (1b).
To a solution of compounds 7a and 7b (0.20 g, 0.165 mmol) in MeOH (3.2 ml) was added palladium on carbon (37 mg, 20% w/w) and acetic acid (1.0 ml). The reaction flask was purged three times and the mixture was stirred under a H2 atmosphere overnight, then filtered over a Celite® pad, washed with methanol and concentrated to dryness. The crude residue was used in the following reaction without further purification. Finally, this material was subjected to deprotection under acidic conditions by dissolving it in DCM/TFA (2.5 ml, 4:1 v/v). The reaction mixture was stirred at r.t. for 5 h, then evaporated to dryness and co-evaporated twice with toluene. The residue thus obtained was dissolved in distilled water, and the clear supernatant was taken and freeze-dried to yield a white fluffy powder 1a and 1b (67 mg, 82% two steps) as their corresponding TFA salts. 1H-NMR (D2O, 400 MHz): ? 5.19 (d, J = 3.6, 2H), 5.05 (d, J = 3.9 Hz, 2H), 3.83 – 3.79 (m, 2H), 3.79 – 3.61 (m, 6H), 3.47-3.24 (m, 6H), 3.16 (t, J = 5.0 Hz, 2H), 3.02 (t, J = 5.8 Hz, 2H), 2.82 – 2.72 (m, 5H), 2.78 (s, 3H), 2.68 (s, 3H), 2.52 – 2.21 (m, 2H), 1.68-1.48 (m, 4H), 1.48 - 1.39 (m, 2H), 1.19 (d, J = 6.8 Hz, 3H), 1.16 (d, J = 6.9 Hz). 13C-NMR (D2O, 100 MHz): ? 157.5, 103.1, 90.1, 89.5, 89.3, 77.1, 76.0, 75.1, 72.9, 72.0, 70.4, 67.0, 53.4, 52.5, 52.3, 52.2, 52.1, 51.9, 51.5, 47.5, 38.3, 39.5, 30.1, 29.2, 28.5, 27.7, 25.2, 24.2, 20.3. MS-API-ES (m/z): 492 (M+H)+ (1a), 506 (M+H)+ (1b). HRMS (m/z) 461.5631 (C19H39N7O6, 461.5640) (1a), 475.3115 (C20H41N7O6, 475.3118) (1b).
In the case of the resistance strain expressing AAC (6′)-Ib, E. coli BL21 (DE3) containing the pET-AAC(6′)-Ib plasmid, enzyme production was induced with IPTG prior to addition of the antibiotics.
RESULTS AND DISCUSSION
Figure 3: Reagents and conditions: (a) CbzCl, Acetone/Na2CO3 aq. sat., 0°C, 10 h, 50%; (b) NaOH (2.5M), 1,4-dioxane/H20, 50°C, 24 h, 60%.
In view of these results a slightly different approach was employed (Figure 4). Treatment of gentamicin 2a-c with N-(benzyloxycarbonyloxy)succinimide in DMSO afforded a mixture of 6′,2′,1,3-(Cbz)4-gentamicin C1a (5a) and 6′,2′,1,3-(Cbz)4-gentamicin C2 (5b) in 37% (45:55) which was employed in the next reaction and 2′,1,3-(Cbz)3-gentamicin C1 (6) in 13% yield that was discarded . Conversion of 3″-methyl amino groups to the corresponding guanidine was achieved by treatment of the mixture (5a-b) with 1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine and Et3N , obtaining 6′,2′,1,3-(Cbz)4-3″-guanidino(Boc)2 gentamicins C1 and C2 (7a-b) in 72% yield. Catalytic hydrogenolysis of the Cbz groups and finally, acidic deprotection of the guanidine Boc groups, afforded a mixture of 3″-guanidino-gentamicins C1 and C2 (1a-b) in 82% yield as their TFA salt (Figure 4).
Figure 4: Reagents and conditions: (a) N-(benzyloxycarbonyloxy)succinimide, DMSO, 0°C, 2h, 37% (5a and 5b) and 13% (6); (b) 1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine, Et3N, 1,4-dioxane, 5 d, 72%; (c) i. H2, Pd•C, AcOH, MeOH, 12 h. ii. TFA/DCM (1:4), r.t., 5 h, 82% (two steps).
Figure 5: MIC values of commercial gentamicin (2a-c) (red) and novel 3″-guanidine gentamicin (1a-b) (blue). The antibiotic threshold (50??g/ml) is shown with a dashed line.
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Citation:Zárate SG, Bastida A, Santana AG, Revuelta J (2017) Recovery of the Antibiotic Activity against Resistant Bacterial Strains by Selective Guanidinylation of the 3″-Methylamino Group of Gentamicin. J Mod Chem Sci 1: 002.
Copyright: © 2017 Julia Revuelta, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.