Applications of functional dyes in biomedicine and life sciences
Huang, Jingyi
DOI:
10.33612/diss.123828369
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Publication date: 2020
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Huang, J. (2020). Applications of functional dyes in biomedicine and life sciences. University of Groningen. https://doi.org/10.33612/diss.123828369
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101
Chapter 4
Overcoming Bacterial Resistance by
Photo-Switchable Antibiotic
102
4.1 Introduction
The recent studies on antimicrobials made by the World Health Organization (WHO)1,2 and the European Union3 stress the importance of delivering novel
antimicrobial compounds, and strategies to avoid the further spread of antibiotic resistance. During the last 60 years, a vast amount of research has been directed towards unveiling different ways to eradicate antibacterial drug resistance, leading to the development of new classes of antibiotics and numerous analogues of the existing classes4. Aminoglycoside antibiotics (AG), which are known as broad
spectrum antibiotics, exert antibacterial activity by targeting the ribosome and hindering the bacterial translation process5,6.
The different mechanisms adopted by bacteria to withstand aminoglycosides activity have been addressed in several reviews5,7. Their protection mechanisms
include: mutations of the ribosome target, enhancing cell wall impermeability and active efflux pumps. Alongside these mechanisms, aminoglycoside modifying enzymes (AME) are the major tools evolved by microorganisms to prevent aminoglycosides’ toxicity8,9. AMEs modify the amino sugar scaffolds at specific
positions and thus decrease their affinity to the ribosome. The genes of AMEs are very mobile as they can be transmitted through plasmids, transposons and other transposable genetic elements resulting in genotypes carrying multiple resistance genes. In the past decades, several chemical and semi-enzymatic methodologies were applied to develop new AG derivatives to evade structural modifications by AMEs10,11. Unfortunately, once these active drugs are placed in contact with their
bacterial targets, the evolution of new drug-resistant bacteria starts12.
A recently investigated approach to tackle the above mentioned problem, has been the use of light as external trigger in order to reversibly control drug activity13–15.
This novel approach not only enables the activation of drugs with high spatial and temporal precision but also allows auto-inactivation of drugs in a reasonable time scale. Based on the assumption that the inactive form of the drug does not harm bacteria, the use of such ‘smart’ drugs could prevent the accumulation of active antimicrobial materials in the environment16 and hence slow down the emergence
of resistance. In spite of these promising prospects, the interaction of such drugs with resistance enzymes or resistant bacteria has never been investigated.
In this chapter, we present a novel approach in the context of photo-pharmacology with the introduction of a photo-responsive AG whose conformation can be modulated using light as an external trigger. In contrast to previous photoswitchable antibiotics where the antimicrobial activity was switched between
103 “on” and “off” state13, we present here a novel opto-drug that maintains continuous
antibiotic activity towards its target, however, its susceptibility towards resistance enzymes can be modulated by light.
4.2 Results and discussion
4.2.1 Design of photoswitchable aminoglycoside antibiotics
Paromomycin was chosen as the parent drug to introduce this concept because it contains a unique aminomethyl group in position 6’’’ (Figure 4.1a), which can be regioselectively modified and, at the same time, can accommodate bulky substituents due to its minor role in the binding to the ribosome17. Moreover, the
ring bearing this group is less important for the binding to the active pocket of AMEs that actively modify paromomycin, which is supported by crystallographic data as evidenced by a binary complex of O-phosphotransferase APH(3’)Ia and paromomycin (see supporting information, figure S1) and the previously known structure of N-acetyltransferase AAC(3)-IIIb in complex with the same antibiotic18.
Two types of photo-switchable dimeric paromomycin (H-dimer and F-dimer, see Figure 4.1) containing azobenzene or o-fluoroazobenzene moieties as the photo-responsive linker were synthesized. Fluorine substituents in the ortho-positions, as σ-electron withdrawing groups, can lower the n-orbital energy of the Z-isomer, resulting in a separation of the n→π* absorption bands for the E and Z isomers. This effect allows for selective excitation of the two isomers in the visible range of the spectrum19. Therefore, the introduction of fluorine atoms at the ortho-positions not
only allows the use of light in the visible range to trigger the E→Z isomerization, but also dramatically enhances the thermal stability of the Z-isomers19.
We hypothesized that the antibacterial properties of the dimers might be retained in spite of the introduction of the azobenzene units into the AG (Figure 4.1). Moreover, we envisioned that the Z-isomers would be able to overcome bacterial resistance caused by AME since the amino sugar rings in the Z isomers are arranged in a more crowded configuration than in the E isomer, thus introducing steric hindrance and eventually blocking the binding to the active sites of the enzymes.
104
Figure 4.1 Overview of photoswitchable paromomycin antibacterial agents. a) Structure of
paromomycin (1) and molecular structures of azobenzene-containing paromomycins, with two paromomycin substituents (2), (3). (b) Schematic of azobenzene E-Z isomerization. The photoisomerization of azobenzene (R=H) is traditionally triggered by UV and visible light irradiation. Irradiation at 365 nm leads to conversion of the E isomer to the Z isomer and visible-light switches the Z isomer back to the E form. The Z to E isomerization occurs also thermally at ambient temperature, albeit at a slower rate than the photoisomerization. In ortho-fluoroazobenzenes (R=F), visible light is used for both E to Z (λ> 500 nm) and Z back to E (λ =450 nm) isomerizations.
4.2.2 Syntheses and characterization of photoswitchable aminoglycoside antibiotics
The photo-switchable antibiotic dimers were synthesized from azobenzenes containing two carboxylic acids by well-established amide bond formation procedures20,21. Following this procedure, azobenzene-4,4’-dicarboxylic acid as well
as symmetrical fluoro-azobenzene-dicarboxylic acid were synthesized with moderate yields (typically 20–35%). These two compounds were further activated to form the corresponding N-hydroxysuccinimide (NHS) esters, which were readily modified with amine bearing functionalities of the AG. The aminomethyl group in position 6’’’ of paromomycin has been functionalized by mildly activated acylating reagents with high regioselectivities. Similarly, the activated azobenzene NHS esters were selectively reacted with the amino group in position 6’’’ in the presence of
a)
105 excess of paromomycin to form the photoswitchable antibiotic dimers. The obtained crude products, i.e. H-dimer and F-dimer, were purified by high performance liquid chromatography (HPLC) and further characterized by 1D- and 2D-NMR (heteronuclear single quantum coherence (HSQC)) spectroscopy (Figure 4.2b and S4.7). Due to the fast Z to E isomerization, only the E isomer of H-dimer’s NMR was showed (Figure S4.9).
Scheme 4.1: Fabrication of antibiotic dimers by functionalization of paromomycin with
azobenzene as a linker at ring IV.
As shown in Figure 4.2 b and Figure 4.2 c, HSQC spectrum of H-dimer (2) and F-dimer (3) show a remarkable chemical shift of the J(C6´´´-H) coupling of ring IV in comparison to the 2D-spectrum of paromomycin (Figure 4.2a) proving the regioselective acylation of amino group in C6 position of ring IV. Moreover, fluorine substituents in the ortho-positions, as σ-electron withdrawing groups, renders the ortho-tetrafluorinated azobenzenes with extraordinarily slow thermal cis-to-trans relaxation rate (see below), so that they are truly bistable on biological time scales. Therefore, the E and Z isomers of the resulting F-dimer were isolated by HPLC with >95% purity, as determined by NMR and UPLC. (Figure 4.5 and Figure S 4.2)
106
Figure 4.2 Sections of HSQC-spectrum of a) paromomycin antibiotic; b) H-dimer and c) F-dimer.
Arrows indicate chemical shift of J(C6´´´-H) (red) coupling comparing to the unmodified paromomycin.
4.2.3 Photochemical behavior of H-dimer and F-dimer
The photo-physical properties of H-dimer and F-dimer were studied using UV-vis spectroscopy and ultra-performance liquid chromatography (UPLC). The UV−vis spectrum of E isomer of H-dimer (Figure 4.3) has a characteristic strong π→π* absorption band at short wavelength (≈ 329 nm) in water. Irradiation with UV light at 365 nm causes E→Z isomerization resulting in decrease of the π→π* band producing a photo-stationary state (PSS) containing 30% of the corresponding Z isomer calculated according to the UV-vis spectroscopy. The absorption peaks at 329 nm from the E isomer decreased over a period of up to 120 min until the photo-stationary state was reached. Importantly, throughout the isomerization process, the H-dimer maintained two isosbestic points at 286 and 371 nm, respectively. This means that the E and Z isomers can be photo-switched without any side reaction under this irradiation conditions.
107
Figure 4.3. Changes in the absorption spectra of a solution of H-dimer in MQ water (10 µM), a)
upon irradiation at 365nm for the given time periods; b) upon irradiation at 450nm for the given time periods. Arrows indicate the nature of changes in the observed absorption region of π-π* transitions of the azobenzene units.
The UV-vis absorption spectra of the F-dimer were measured in water (Figure 4.4). The E isomer of the F-dimer exhibits a characteristic strong π→π* absorption band at short wavelength (λ ≈ 316 nm) and a weaker n→ π* band centered around 440 nm. The solution was then irradiated with a 500-Watt xenon lamp equipped with a thermal cut-off filter to exclude wavelengths less than 500 nm. During the E→Z photo-isomerization, hypochromism is observed in the spectra of the E isomer at 316 nm. The absorption peaks at 316 nm from the E isomer decreased over a period of 240 min until the photo-stationary state was reached. Under this illumination, two isosbestic points were found at 276 and 378 nm. While during the Z→E photo-isomerization upon irradiation at 450 nm, the absorption peaks at 316 nm from the E isomer increased over a period of 60 min until the photo-stationary state was reached. Using UPLC the ratio of the two photo-isomers at the photo-stationary state were determined from the elution profile. Irradiation with visible light was used to isomerize F-dimer in both directions, producing PSSs containing 78% of Z isomer with green light (λ > 500 nm) and 73% of E isomer with blue light (450 nm) (Figure
4.5 and Figure S 4.2). Multiple E/Z isomerization cycles did not result in any
noticeable degradation, highlighting the robustness of the F-dimer switch (Figure S
4.4). As a consequence of lowering the energy of the n-orbital through the
introduction of ortho-fluoro substituents, thermal cis-trans isomerization of F-dimer shows a half-life of ca. 8 days at 37 °C in the dark. That is remarkably longer than the half-life of the H-dimer (t ½ = 1h) at the same conditions (Figure S 4.5 and Figure S 4.6).
108
Figure 4.4. Changes in the absorption spectra of a solution of F-dimer in MQ water (10 µM), a)
upon irradiation at >500 nm for the given time periods (indicative for formation of Z isomer); b) upon irradiation at 450nm for the given time periods (formation of E isomer). Arrows indicate the absorbance increase or decrease over time.
Figure 4.5 1H-NMR (500MHz, D2O) spectrum of F-dimer. a) PSS after green light (>500 nm)
irradiation; b) PSS after blue light ( 450 nm) irradiation; c) pure cis-F-dimer after HPLC purification; d) pure trans-F-dimer after HPLC purification.
109 4.2.4 Evaluation of antibacterial activity
To investigate the antibacterial activity of the H- and F-dimers we determined the minimal inhibitory concentration (MIC) prior to and after irradiation with UV (365nm) and green light (>500 nm) against Escherichia coli (E.coli) strain ATCC 25922 and the same strain carrying different AMEs (see table 4.1). For this purpose
E.coli (ATCC) was transformed with the plasmids pET9b(+) and pAT21-1 carrying
the genes encoding for the two O-phosphotransferases APH(3’)Ia and APH(3’)IIIa, respectively. The two different enzymes both catalyze the transfer of a phosphate group to the hydroxyl group in position 3’ of paromomycin (Figure 4.1,a). Furthermore, the E.coli (ATCC) strain was transformed with the plasmid pBluescript KS(+) encoding for the N-acetyltransferase AAC(3)IV. The enzyme is responsible for the transfer of an acetyl group from its cofactor, acetyl coenzyme A, to the amino group in position 3 of paromomycin (Figure 4.1,a).
Figure 4.6. Growth curves of E.coli carrying the gene encoding for (a) APH(3’)Ia, (b) APH(3’)IIIa
and (c) AAC(3)IV in presence of different concentration of E (cis) and Z (trans) isomers of the F-dimer. The OD was measured after 16 h of growth in LB media at 37°C.
110
Upon inspection of the growth curve of E.coli carrying APH(3’)IIIa (Figure 4.6,b), it becomes obvious that the E isomer of the F-dimer significantly inhibited the bacterial growth at a concentration of 256 μM. In stark contrast the same growth inhibition in case of the Z isomer was achieved only at a concentration of 1024 μM. Similar results were obtained for the other strain harbouring the APH(3’)Ia gene (Figure 4.6,a), although with a less pronounced difference given the MIC of 512 μM and 1024 μM for the E and Z isomers, respectively. Besides phosphorylation, acetylation catalyzed by the AAC(3)IV was inhibited by the E isomer at a concentration of 512 μM while the strain was still able to slowly grow at a concentration of 1024 μM (Figure 4.6,c). The difference in the activity of the two isomers is probably due to the high steric hindrance of the Z conformation, which may decrease the recognition by AMEs, therefore retaining most of its antibiotic activity toward the ribosome. Compared to pristine paromomycin, both H-dimer and F-dimer show an 8-fold lower antibacterial performance against wild type E.coli, resulting in a MIC of 32 µM (Table 4.1). We assume that this decrease in activity is due to a decreased affinity of modified paromomycin derivatives for the bacterial ribosome. The Z isomer of H-dimer could not be tested due to its limited half-life as already stated.
Table 4.1 MIC values determined against Escherichia coli ATCC 25922 wild-type and the same
strain carrying different aminoglycoside modifying enzymes. MIC value for the E isomer of the H-dimer was evaluated only against wt E.coli to compare it with F-dimer.
Resistance
enzyme Wild Type APH(3’)Ia APH(3’)IIIa AAC(3)IV
Paromomycin 4 >512 >512 >512
H-dimer (trans) 32 - - -
F-dimer (trans) 32 512 512 512
F-dimer (cis) 32 256 128 256
4.3 Conlusion
In this chapter, we synthesized two compounds, H-dimer and F-dimer, which consist of two paromomycin molecules that are connected via an azbenzene and an o-fluoroazobenzene linker, respectively. In this regard, we obtained for the first time two photoswitchable aminoglycosides. With the help of these molecules a new
111 concept in the context of photopharmacology was realized. For the F-dimer, it was proven that in both states the E and the Z conformation the activity towards the target remains similar. In contrast, towards resistance enzymes the two conformations exhibit different activity.
The two compounds were characterized by means of NMR and UV-vis spectroscopy and their antimicrobial activity was evaluated against three strains of E.coli carrying genes encoding for three different aminoglycoside modifying enzymes.
We proposed an explanation for the different antibiotic activity of the two conformational states, which can be further proved by x-ray crystallography and ITC measurements. In particular, the F-dimer showed promising results with the Z isomer exhibiting two-fourfold lower bactericidal activity compared to the E isomer, thus overcoming, at least partly, the resistances induced by AMEs. The fluorinated azobenzene functionalized selectively with two paromomycin molecules has the potential to be optimized further and, eventually, result in antibiotic candidates able to overcome aminoglycoside resistance. At the same time, the conformer of the antibiotic that is able to overcome AMEs relaxes over a reasonable period of time into a state where it is susceptible to AMEs. Hence, this molecule might pose minimal evolutionary pressure towards the evolution of new resistance mechanisms.
4.4 Experimental General
1H-NMR and heteronuclear single-quantum correlation (HSQC) spectra were
recorded on a Varian Unity Inova (500 MHz for 1H-NMR, 13C-NMR and HSQC) NMR
spectrometer at 25 °C. High resolution mass spectrometry (HRMS) was carried out on a LTQ ORBITRAP XL instrument (Thermo Scientific) employing electron impact ionization in positive ion mode (EI+). Chromatographic separations were carried out
on a Shimadzu VP series HPLC modular system (DGU-14A3 Online Vacuum-Degasser, two LC-20 AT pumps, SIL-20A auto sampler, CTP-20 A column oven, RID-10 refractive detector, FRC-RID-10 A fraction collector and Shimadzu LCsolution software). HPLC purification was performed with a Waters Spherisorb ODS-2 C18 analytical (250 x 4.6 mm) and semi-preparative column (250 x 10mm) (spherical particles of 5 µm and 80 Å pore size) using isocratic elution at 30 °C. A pH-meter (Hanna Instruments pH 209) equipped with a glass combination electrode was used for pH adjustments of the reaction buffers.
Materials
112
without further purification, unless otherwise noted. Paromomycin sulfate salt (98 %) was purchased from Sigma Aldrich and used as received. For reversed phase HPLC (rp-HPLC) purification, trifluoroacetic acid (TFA) (Sigma-Aldrich, HPLC grade) and acetonitrile (Sigma-Aldrich, HPLC grade) were used. Ultrapure water (specific resistance > 18.4 MΩ cm) was obtained by Milli-Q water purification system (Sartorius®). Chromatographic separations were performed using a Shimadzu VP series high performance liquid chromatography (HPLC) modular system (DGU- 14A3 Online Vacuum-Degasser, two LC-20 AT pumps, SIL-20A auto sampler, CTP-20 A column oven, RID-10 refractive detector, FRC-10 A fraction collector and Shimadzu LCsolution software). HPLC purifications were performed with a Gemini NX-C18- semi-preparative column (250 x 10 mm, spherical particles of 5 µm and 110 Å pore size) using isocratic elution at 30 °C. Solvent A: H2O (HPLC grade) + 0.028% v/v trifluoroacetic acid (TFA); solvent B: H2O/MeCN 2:1+ 0.028% v/v TFA; detection at 314 nm and 275nm (if not stated otherwise). All compounds (including intermediates) were stored in a refrigerator at about +5 °C, unless otherwise stated. The reactions were monitored by TLC on MERCK ready-to-use plates with silica gel 60 (F254). Column chromatography: MERCK silica gel, grade 60, 0.04–0.063 mm. Synthesis
Scheme 4.2. Synthetic routes to compounds (6).
Azobenzene-4, 4'-dicarboxylic acid (5).
Product 5 was synthesized following previous work.22 4-Nitrobenzoic acid (4)
(15.00 g, 89.8 mmol) was added to a solution of sodium hydroxide (50 g in 225 mL water), and the solution was heated until the solid dissolved. A hot aqueous glucose
113 solution (100 g in 150 mL water) was added into the above solution dropwise at 50 ℃, whereupon a yellow precipitate initially formed, which immediately turned into a brown solution upon further addition of glucose. The solution was allowed to react at room temperature overnight to result in a dark solution. After aging for several hours, a bright brown precipitate formed. It was filtered, dissolved in water and acidified with acetic acid (20 mL), whereupon a light pink precipitate was obtained. The final product was filtered, washed with plenty of water (300 mL) and dried to yield 5 (6.18 g, 22.9 mmol, 51 %) 1 H NMR (DMSO-d6, 500 MHz): δ 8.01-8.03
(d, 4H), δ 8.16-8.18 (d, 4H), δ 13.25 (brs, 1H). 13C NMR (DMSO-d6, 100 MHz):122.84,
130.70, 132.87, 154.18, 167.75. HRMS‐ESI: m/z = 269.2054 (calcd for [M-H]-,
269.2440).
4,4'-Bis( 2,5-dioxo-pyrrolidin-1-yl ester) azobenzene (6).
Azobenzene-4,4'-dicarboxylic acid (5) (3.00 g, 11.1 mmol) was dissolved in dimethylformamide (DMF) at 60 ℃. NHS (3.07 g, 26.7 mmol), EDC-HCl (5.12 g, 26.7 mmol) and a catalytic amount of DMAP were added sequentially to the solution. The reaction was allowed to react at room temperature for 12 hours. Pure water (200 mL) was added to the solution until red precipitate was obtained and filtered. The solid was washed with pure water (100 mL) for many times, dried to yield a light red product 7 (4.85 g, 10.4 mmol, 94 %). 1H NMR (DMSO-d6, 500 MHz): δ 2.93 (s,
8H), δ 8.17-8.19 (d, 4H), δ 8.35-8.37(d, 4H). 13C NMR (DMSO-d6, 100 MHz): 25.59,
124.57, 126.47, 129.23, 152.70, 161.23, 168.00. Paromomycin-azobenzene conjugate synthesis
The designed structures 2 and 3 were synthesized according to the general strategy (Scheme 4.1). Paramomycin sulfate salt was first treated with Ion-exchange Resin to obtain paromomycin free base. In order to synthesize the photoswichable paromomycin, azobenzenes were introduced via their carboxylic acid function. They were dissolved in dry DMF and activated using N‐hydroxysuccinimide (NHS) in the presence of N,N’‐dicyclohexyl carbodiimide (DCC). These activated azobenzene ester (1 equiv) reacted with an excess of paromomycin (10 equiv) in a water/DMF mixture. As shown in Scheme 4.1, the transformation of paromomycin with azo-NHS resulted in the formation of dimeric photoswitchable 6’’’N-acetyl paromomycin. Finally, HPLC was used to purify these photoswitchable antibiotics.
114
H-dimer (2). In a 5 ml round bottom flask,
azobenzene-4,4'-dicarboxylic acid (16.2 mg, 0.06 mmol) and N‐ hydroxysuccinimide (13.8 mg, 0.12 mmol) were dissolved in 20 mL dry DMF. After the addition of N,N' ‐ dicyclohexylcarbodiimide (24.8 mg, 0.12 mmol), the reaction was carried out for 12 h under inert atmosphere at room temperature. Precipitated dicyclohexylurea (DCU) was removed by filtration. 4,4'-Bis( 2,5-dioxo-pyrrolidin-1-yl ester) azobenzene was used for coupling without further purification.
Paromomycin (184 mg, 0.3 mmol) was dissolved in 20 mM sodium phosphate buffer (20 mL, pH 7.0), a solution of compound 6 (14 mg, 0.03mmol) in DMF (2.5 mL) was added drop by drop in 3h. The reaction was continued overnight under inert atmosphere at room temperature. The solution was evaporated. The obtained crude mixture was purified by column chromatography using a homogeneous dichloromethane/methanol/aq. 25% ammonia (from 2:2:1 to 2:3:2 v/v/v) mixture. After evaporation of the solvent the residue was resolved in water (3 mL) and traces of silica were removed by filtration through 0.45 mm syringe filters. Lyophilization yielded 2 as orange solid. Yield: 53.6 mg (0.03 mmol, 61%). 1H NMR (500 MHz, D2O)
(E‐isomer) δ ppm 8.07-8.09 (d, 4H, azo), 8.00-8.01 (d, 4H,azo), 5.71 (d, J = 3.5 Hz, 2H, 1-H´), 5.40 (d, J = 1.5 Hz, 2H, 1-H´´), 5.27 (s, 2H, 1-H´´´), 4.43 (t, J = 5.5 Hz, 2H, 3-H´´), 4.33 (m, 2H, 2-3-H´´), 4.32 (m, 2H, 3-H´´´), 4.31 (t, J = 5.5 Hz 2H, 5-H´´´), 4.24 (m, 2H, 4-H´´), 4.04 (T, J= 7.8 Hz, 2H, 4-H), 3.95-3.81 (m, 10H, 6a-H´´´, 5a-H´´, 6a-H´, 3-H´, 5-H), 3.83-3.71 (m, 8H, 5-H´, 5b-H´´, 6b-H´, 4-H´´´), 3.75 (t, J = 9.5 Hz, 2H, 6-H), 3.64 (m, 2H, 6b-H´´´), 3.61(m, 2H, 2-H´´´), 3.62 (t, J = 9.75 Hz, 2H, 3-H), 3.46 (t, J = 7.75 Hz, 2H, 4-H´), 3.38 (dd, J = 11.0 Hz, J = 4.0 Hz, m, 2H, 2-H´), 3.41(m, 2H , 1-H), 2.54(dt, J = 12.5 Hz; J = 4.0 Hz, 2H, 2-He), 1.90 (dd, J = 12.0 Hz, 2H, 2-Ha). 13C-signals based on
HSQC (D2O, 500 MHz) δ (ppm.) 127.48 (azo), 122.49 (azo), 109.67 (C-1´´),
96.23(C-1´), 93.25 (C-1´´´), 84.19 (C-5), 82.05 (C- 4´´), 77.67 (C-4), 76.49 (C-3´´), 73.98 (C-5´), 73.90 (C-2´´), 72.56 (C-5´´´), 72.30 (C-6), 69.43 (C-4´), 69.03 (C-3´), 67.74(C-3´´´), 66.46 (C-4´´´), 60.50 (2C, C-5´´, C-6´), 53.82 (C-2´), 51.17 (C-2´´´), 49.78 (C-1), 49.02 (C-3), 40.35 (C-6´´´), 28.15 (C-2). HRMS‐ESI: m/z = 1466.4542 (calcd for [M+H]+,
115
Scheme 4.3. Synthetic routes to F4-diacid (13).
Product 13 was synthesized following previous work.20
2,6‐difluoro‐4‐bromoaniline (8). NBS (35.6 g, 200 mmol) was added to a
solution of 2,6‐difluoroaniline (7) (25.8 g, 200 mmol) in acetonitrile (300 mL) and stirred overnight at room temperature. The mixture was diluted with water and hexane. After separation of the layers, the organic phase was dried over MgSO4,
filtrated and the solvent was evaporated in vacuo. The resulting mixture was purified by column chromatography (DCM/hexanes: 1/1) to give 8 as a purple solid (33.5 g, 80%). 1H NMR (500 MHz, CDCl3) δ ppm 7.00 (dd, J = 6.1, 1.3 Hz, 2 H), 3. 75
(br s, 2 H). 13C NMR (100 MHz, CDCl3) δ ppm 153.36, 150.12, 123.50, 114.72, 107.09. 4‐amino‐3,5‐difluorobenzonitrile (9). Compound 8 (20 g, 96 mmol) and
CuCN (25.6 g, 288 mmol) were dissolved in DMF (100 mL). The mixture was refluxed overnight. After the solution was cooled down, the mixture was poured into a NH3 12% aqueous solution and extracted with ethyl acetate. The organic phase
was dried over MgSO4, filtered, and the solvent was evaporated in vacuo. The
product was purified by column chromatography (DCM/hexanes: 2/1) to give 9 as white solid (9.2 g, 62%). 1H NMR (500 MHz, CDCl3) δ ppm 7.16 (dd, J = 6.1, 2.2 Hz, 2
116
4‐amino‐3,5‐difluorobenzoic acid (10). Compound 9 (6.9 g, 45 mmol) was
suspended in NaOH 1M (150 mL) and refluxed overnight. After the solution was cooled down, 1M HCl was added until hydrochloric salt 10 precipitates. The precipitate was then dissolved in EtOAc, dried over MgSO4, filtered, and the solvent
was evaporated in vacuo. The product was obtained as white solid and used without purification in the next step. 1H NMR (500 MHz, DMSO‐d6) δ ppm 7.40 (dd, J = 4.2,
1.4 Hz, 2 H), 3.81 (br s). 13C NMR (100 MHz, DMSO‐d6) δ ppm 165.96, 150.65,
148.74, 130.65, 115.59, 112.22. HRMS‐ESI: m/z = 172.0278 (calcd for [M‐H]‐
, 172.0204).
Ethyl 4‐amino‐3,5‐difluorobenzoate (11). To a solution of acid 10 (5.92 g, 28
mmol) in EtOH (100 mL) was added H2SO4 (2 mL) and refluxed for 5 h. Saturated
NaHCO3 was added until the solution was neutralized (pH 7). The mixture was
extracted with DCM and the organic phase was dried over MgSO4 and filtered. The
solvent was evaporated under reduced pressure to give 11 as a pale brown solid (4.2 g, 69% over two steps). 1H NMR (500 MHz, CDCl3) δ ppm 7.50 (dd, J = 7.2, 2.2
Hz, 2 H), 4.30 (q, J = 7.1 Hz, 2 H), 4.07 (br s, 2 H), 1.34 (t, J = 7.1 Hz, 3H). 13C NMR
(100 MHz, CDCl3) δ ppm 165.19, 152.21, 149.03, 128.78, 118.38, 112.55, 61.04,
14.25. HRMS‐ESI: m/z = 202.0535 (calcd for [M+H]+, 202.0680).
Diethyl ‐ 4,4’ ‐ (2,2’,6,6’ ‐ tetrafluoro)azobenzene dicarboxylate (12).
Compound 11 (600 mg, 3 mmol) was dissolved in DCM (100 mL) and a freshly grinded mixture of KMnO4 (5.1 g) and FeSO4∙7H2O (5.1 g) was suspended in this
solution. The solution was refluxed overnight, filtered through celite, dried over MgSO4, filtered, and the solvent was evaporated in vacuo. The product was purified
by column chromatography (DCM/hexanes : 1/1) to give 12 as an orange/red solid (136 mg, 23%). 1H NMR (500 MHz, CDCl3) (E‐isomer) δ ppm 7.76 (m, 4 H), 4.45 (q,
J = 7.0 Hz, 4 H), 1.45 (t, J = 7.0 Hz, 6 H). 13C NMR (100 MHz, CDCl3) (E‐isomer) δ
ppm 163.63, 156.75, 153.24, 133.80, 113.90, 62.18, 14.20. HRMS ‐ ESI: m/z = 399.0999(calcd for [M+H]+, 399.0968).
117
F4-diacid (13). Compound F4-diester (12)
(136 mg, 0.34 mmol) and KOH (67 mg, 1.19 mmol) were dissolved in H2O/THF 2:1 (4 mL)
and the solution was heated at 70 °C for 2 h. The solution was diluted with water and ethyl acetate, the two layers were separated, and the aqueous layer was washed twice with ethyl acetate. The aqueous layer was acidified with HCl (1M) until the orange precipitate fell out of solution. The orange precipitate was filtered to give 13 as an orange solid (113 mg, 97%). HRMS‐ESI: m/z = 342.0264. (calcd for [M‐H]-, 342.2056).
Into a solution of paramomycin sulfate salt (0.3 mmol) in water/methanol (1:1 10 mL), Ion-exchange Resin was added and the solution was stirred for 2 h at room temperature. Ion-exchange Resin was removed by filtration and the aqueous solution was concentrated under reduced pressure to remove methanol and freeze dried.
F-dimer (3). In a 5 ml round bottom flask, F4-diacid (13) (20.4 mg, 0.06 mmol) and
N‐hydroxysuccinimide (13.8 mg, 0.12 mmol) were dissolved in 20 mL dry DMF. After the addition of N,N'‐dicyclohexylcarbodiimide (24.8 mg, 0.12 mmol), the reaction was continued for 12 h under inert atmosphere at room temperature. Precipitated dicyclohexylurea (DCU) was removed by filtration. F4-diacid‐NHS was used for coupling without further purification.
Paromomycin (0.3 mmol) was dissolved in 20 mM sodium phosphate buffer (20 mL, pH 7.0), a solution of F4-diacid‐NHS in DMF (2.5 mL) was added drop by drop in 3 h. The reaction was carried out overnight under inert atmosphere at room temperature. The solution was evaporated. The obtained crude mixture was purified by column chromatography by using homogeneous mobile phase consisting of dichloromethane/methanol/aq. 25% ammonia (from 2:2:1 to 2:3:2 v/v/v) mixture. After evaporation of the solvent, the residue was re-dissolved in water (3 mL) and traces of silica were removed by filtration through 0.45 mm syringe filters. Lyophilization yielded the F-dimer (3) as orange solid. Yield: 51.2 mg (0.03 mmol, 56%). 1H NMR (500 MHz, D2O) (E‐isomer) δ ppm 7.69 (m, 4 H,azo), 5.73 (d, J = 3.5
Hz, 2H, 1-H´), 5.40 (d, J = 1.5 Hz, 2H, 1-H´´), 5.27 (s, 2H, 1-H´´´), 4.44 (t, J = 5.5 Hz, 2H, 3-H´´), 4.34 (m, 2H, 2-H´´), 4.31 (m, 2H, 3-H´´´), 4.26 (t, J = 5.5 Hz 2H, 5-H´´´), 4.22 (m,
118
2H, 4-H´´), 4.01 (t, J= 7.8 Hz, 2H, 4-H), 3.92-3.82 (m, 10H, 6a-H´´´, 5a-H´´, 6a-H´, 3-H´, 5-H), 3.81-3.68 (m, 8H, 5b-H´´, 5-H´, 6b-H´, 4-H´´´), 3.72 (t, J = 9.5 Hz, 2H, 6-H), 3.64 (m, 2H, 6b-H´´´), 3.60(m, 2H, 2-H´´´), 3.58 (t, J = 9.75 Hz, 2H, 3-H), 3.44 (t, J = 7.75 Hz, 2H, 4-H´), 3.40 (dd, J = 11.0 Hz, J = 4.0 Hz, m, 2H, 2- H´), 3.37(m, 2H, 1-H), 2.50 (dt, J = 13.0 Hz; J = 4.0 Hz, 2H, 2-He), 1.86 (dd, J = 12.0 Hz, 2H, 2-Ha). 13C-signals based on
HSQC (D2O, 500 MHz) δ ppm 111.76 (azo), 109.64 (C-1´´), 92.12 (C-1´), 96.02 (C-1´´´),
84.12 (C-5), 81.97 (C- 4´´), 77.59 (C-4), 76.31 (C-3´´), 73.90 (C-5´), 73.87 (C-2´´), 72.48 (C-5´´´), 72.22 (C-6), 69.25 (C-4´), 68.82 (C-3´), 67.67(C-3´´´), 66.33 (C-4´´´), 60.36 (2C, C-5´´, C-6´), 53.75 2´), 51.09 2´´´), 49.61 1), 48.81 3), 40.31 (C-6´´´), 28.07 (C-2). HRMS‐ESI: m/z = 1539.242 (calcd for [M+H]+, 1538.443).
Antimicrobial activity studies
Bacterial Strains, Plasmids, and Growth Conditions.
Escherichia coli strain ATCC 25922 was transformed with the plasmids pET9b(+)
(Novagen), pAT21.1 and pBluescript KS(+), harboring the genes encoding for APH(3')Ia, APH(3')IIIa and Aac(3)III, respectively. E.Coli were grown in Luria Broth (LB) medium (5g/l yeast extract, 10g/l tryptone, 0.5 g/l NaCl) and supplemented with the required antibiotic, 50 ug/ml Neomycin at 37°C.
Minimal Inhibitory Concentration Tests.
The H-dimer was first irradiated at 365 nm overnight to get their PSS mixture. The E and Z isomers of F-dimer were isolated by HPLC after irradiated under green light (>500 nm) and blue light (450 nm) for overnight. The antibiotics were then serial diluted from 1024 μm to 16 μm in 15 ml tubes. Overnight cultures of E. coli ATCC 25922, harboring a plasmid encoding the resistance gene, were diluted to an optical density OD600 = 0.1 at 600 nm and 100 ul of this cell suspension was added to total
volume of 500 ul LB medium containing the antibiotic at the given concentration. After overnight growth at 37 °C and shaking at 220 rpm, the optical density at 600 nm was measured. Graphs were background-corrected by subtracting the OD of the LB medium without any bacteria.
119 Author Contribution
In this chapter, Jingyi Huang and Lifei Zheng performed the synthesis of the photoswitchable aminoglycosides. Jingyi Huang characterized photochemical behavior of the resulting photoswitchable aminoglycosides by nuclear magnetic resonance and UV-vis spectroscopy. Shuo Yang quantified the ratio of photoisomers at photostationary-state by UPLC. Avishek Paul carried out the antibacterial evaluation.
120 Reference
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121
Appendix
Figure S 4.1: HPLC-Chromatogram of E/Z mixture of F-dimer (recorded at the corresponding
isosbestic point 390 nm).
Figure S 4.2: UPLC traces (recorded at the corresponding isosbestic points) of F-dimer in MQ water.
a) Pure trans isomer after HPLC purification (upper panel) and pure cis isomer after HPLC purification (lower panel); b) PSS mixtures at different wavelengths.
AU 0.000 0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 Minutes 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20 4.30 AU 0.00 0.50 1.00 1.50 2.00 2.50 Minutes 3.00 3.05 3.10 3.15 3.20 3.25 3.30 3.35 3.40 3.45 3.50 3.55 3.60 3.65 3.70 3.75 3.80 3.85 AU 0.00 0.02 0.04 0.06 0.08 Minutes 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10 AU 0.00 0.10 0.20 0.30 0.40 0.50 Minutes 2.65 2.70 2.75 2.80 2.85 2.90 2.95 3.00 3.05 3.10 3.15 3.20 3.25 3.30 3.35 3.40 3.45 3.50 3.55 3.60 3.65 3.70 3.75 3.80 PSS @ 450 nm PSS @ > 500 nm nm Z E Z isomer E isomer 94.01% 97.62% 78.48% 21.52% 72.38% 27.62%
a)
b)
E isomer Z isomer Z isomer E isomer122
Figure S 4.3: Reversible photochromism of H-dimer (20 μM) in miliQ water at 25 °C, upon
alternating irradiation with UV light (λ = 365 nm) and visible light (λ = 450 nm). Six cycles could be
performed without the observation of significant fatigue or reduction of absorbance. The
absorbance was measured at the maximum of the π-π* transition (329 nm) of the trans-isomer .
Figure S 4.4: Reversible photochromism of F-dimer (20 μM) in miliQ water at 25 °C, upon
alternating irradiation with green light (λ > 500 nm) and blue light (λ = 450 nm). Six cycles could
be performed without the observation of significant fatigue or reduction of absorbance. The
absorbance was measured at the maximum of the π-π* transition (328 nm) of the trans-isomers .
0 1 2 3 4 5 6 0.9 1.0 1.1 1.2 1.3 1.4 1.5 Absor ba nce at 32 9 n m ( AU) Switching cycles 0 1 2 3 4 5 6 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Absor ba nce at 32 8 n m ( AU) Switching cycles
123
Figure S 4.5: Determination of half-life for H-dimer at 37 °C in water in the dark. First, PSS was
reached upon 365 nm irradiation, after which the absorption was measured at λmax = 329 nm.
Line presents the fitting with single exponential.
Figure S 4.6: Determination of half-life for F-dimer at 37 °C in water in the dark. First, PSS was
reached upon >500 nm irradiation, after which the absorption was measured at λmax = 324 nm.
Line presents linear fit.
200 400 600 800 1000 1200 0.80 0.85 time (min) Absor ba nce at 32 9 n m ( AU) 500 1000 1500 2000 2500 3000 0.28 0.30 0.32 0.34 0.36 0.38 0.40 Absor ba nce at 32 4 n m ( AU) Times (min) t1/2 > 8 days t1/2 > 1 h
124
Figure S 4.7: HSQC (500 MHz, D2O) spectrum of H-dimer (2).
125
