Communication pubs.acs.org/bc
Novel Conjugation of Norvancomycin−Fluorescein for Photodynamic Inactivation of Bacillus subtilis Hui-Zhou Gao,† Ke-Wu Yang,*,† Xiang-Long Wu,† Jia-Yun Liu,‡ Lei Feng,† Jian-Min Xiao,† Li-Sheng Zhou,† Chao Jia,† and Zhen Shi† †
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education; College of Chemistry and Materials Science, Northwest University, Xi’an 710069, P. R. China ‡ Clinical Laboratory, Xijing Hospital, The Fourth Military Medical University of Chinese PLA, Xi’an 710032, P. R. China S Supporting Information *
ABSTRACT: A simple and unique conjugation of norvancomycin−fluorescein (VanF) has been achieved. It was characterized by UV−vis and fluorescence spectra and confirmed by MALDI-TOF mass spectrum. The photodynamic assay indicated that VanF effectively inactivated the Gram-positive Bacillus subtilis (ATCC 6633) from clinic with inactivation rate of 30−70% within 1−7.5 μM. In vitro, VanF showed low antimicrobial activity with value of >128 μg/mL, binding affinity with value of 180 nM per 108 cells/mL against the bacteria strains. The fluorescence imaging showed that VanF could label the B. subtilis strain, but not the Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC 51299, VanD), and VRE strains from clinic.
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VanF was prepared according to Scheme 1. Norvancomycin hydrochloride (32 mg) and chlorinated fluorescein11 (18 mg) were dissolved in 2 mL 0.1 M phosphate-buffered saline (PBS) buffer (pH 8.5). The pH of the resulting mixture was adjusted to 9.5 and stirred for 20 h at 4 °C; the reaction mixture was loaded onto a silica gel column, eluted with methanol, collected, and the fractions containing the target compound (monitored by UV−vis) pooled, concentrated, and loaded onto a Sephadex G-25 column to offer the purified VanF with yield 54%. VanF was characterized by UV−vis and fluorescence spectra and confirmed by MALDI-TOF (matrix-assisted laser desorption ionization, time-of-flight) mass spectrum, the peak at m/z 2023.15 [M+Na-2H]− corresponding to VanF was clearly observed (Figure 2). The spectroscopic characterization showed that VanF had a nearly 4-fold decrease in fluorescence emission and a nearly 40% decrease in absorbance compared to chlorinated fluorescein (Figure 3). The photodynamic inactivation of VanF against the Grampositive bacteria B. subtilis was assayed as previously described.12 Briefly, the B. subtilis culture was washed with PBS buffer, incubated with the tested compounds in the dark at 37 °C for 20 min with shaking, and illuminated by light with wavelength of 400−800 nm (350 mW), which was produced by Xenon lamp and isolated by means of the optical filters, for 5 min to offer the phototreated samples. The assays showed that the phototreated VanF effectively inactivates B. subtilis; with the increase of VanF concentration, the bacterial lethality increased (Figure 4a). When the concentration of VanF was 7.5 μM, the bacterial lethality
lycopeptide antibiotics, such as vancomycin and norvancomycin, were used to combat infection of the Gram-positive bacteria by inhibiting the bacterial cell wall synthesis.1 However, the overuse of glycopeptide antibiotics in the clinical setting has resulted in the vancomycin-resistant Enterococcus (VRE), which is typically due to the mutation of peptidoglycan sequence from N-acyl-D-Ala-D-Ala to N-acyl-D-Ala-D-Lac, resulting in the substantial decrease of binding affinity to vancomycin.2,3 There are no useful antibiotics for treatment of vancomycin-resistant bacteria,4 but photodynamic therapy (PDT) may have such potential. PDT is a photochemistry-based emerging technology that relies on the wavelength-specific light activation of certain nontoxic photosensitizers (PSs) to produce active molecular species that are toxic to surrounding logical targets.5,6 PSs have been conjugated to antibodies or peptide for treatment of the area of bacteria infection, but the molecules of these targets are so big and labeling PS to antibodies or peptide is relatively complicated.7 So, developing easier and smaller targets is very necessary. Xing and co-workers reported a simple and specific conjugation of vancomycin−porphyrins to use for fluorescent imaging and antibacterial studies of VRE.2 Eosin has been employed as PS to treat basal cell carcinoma.8 The chlorinated fluoresceins, synthesized by us earlier,9−11 has the same parent structure as eosin (Figure 1). The similarity suggests that the new compound could be linked to antibiotic to construct an efficient conjugation of drug−fluorescein that may apply to a biological target. On the basis of this idea, we constructed a novel compound norvancomycin-chlorinated fluorescein (VanF), and explored photodynamic inactivation experiments, as well as antimicrobial activity, binding affinity, and fluorescence imaging against B. subtilis. © 2011 American Chemical Society
Received: July 18, 2011 Revised: October 16, 2011 Published: October 17, 2011 2217
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Figure 1. Structure of eosin, the chlorinated fluoresceins, and VanF.
Scheme 1. Conjugation of Chlorinated Fluorescein and Norvancomycin
Figure 2. MALDI-TOF mass spectrum of VanF.
reached to 70%. Also, the bacterial lethality was dependent on the irradiation time; with the increase in irradiation time, the bacterial lethality increased (Figure 4b), and it reached 68% after 5 min irradiation with concentration of 5 μM VanF. Eosin and fluoresceins could successfully produce the singlet oxygen (1O2) to inactivate the living cells;13 this suggests that the photodynamic inactivity of VanF against B. subtilis is due to the effect of the singlet oxygen species.14,15 Furthermore, we tested VanF with the Gram-negative bacteria Escherichia coli, which contained the antibiotic-resistant plasmid; it was induced with IPTG to produce the metallo-β-lactamase CcrA (B1 subgroup),16 ImiS (B2 subgroup),17 and L1 (B3 subgroup),18 respectively. The results showed that VanF had no obvious
photodynamic inactivation toward the tested bacteria. This may be ascribed to the large fluorescence group, which blocked VanF from attaching to the bacteria, or the outer membrane of Gram-negative bacteria, which blocked PS from entering the bacteria.15 In vitro antimicrobial activities of VanF were evaluated by determination of MIC according to the Clinical and Laboratory Standards Institute (CLSI) macrodilution (tube) method.19 The MIC value of VanF against B. subtilis (ATCC 6633) was larger than that of norvancomycin (Table 1), indicating that the antimicrobial efficacy of VanF decreased. The decreased activity of VanF implied that chlorinated fluorescein conjugated to norvancomycin (Figure 1), in which the large fluorescence group blocked VanF to bind to the peptidoglycan sequence 2218
dx.doi.org/10.1021/bc200382d | Bioconjugate Chem. 2011, 22, 2217−2221
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Table 1. MIC Values of VanF and Norvancomycin compounds
B. subtilis
norvancomycin VanF
4 μg/mL >128 μg/mL
N-acyl-D-Ala-D-Ala. Further, the binding affinity of VanF to B. subtilis was quantified. The results showed that both VanF (7.5 μM, saturation concentration) and chlorinated fluorescein (3.75 μM, saturation concentration) weakly bond to the bacteria (Figure 5) with values of 180 and 46 nM per 108 cells/mL,
Figure 3. UV−vis (a) and fluorescence (b) spectra of VanF and chlorinated fluorescein. The concentrations of the tested compounds were 8 μM (for UV−vis spectra) and 3.75 μM (for fluorescence spectra) in 10 mM PBS buffer at pH 7.5 (λ ex = 400 nm).
Figure 5. Binding abilities of VanF (a) and chlorinated fluorescein (b) to B. subtilis. The concentration of VanF was 7.5 μM in 10 mM PBS buffer at pH 7.5 and the concentration of chlorinated fluorescein was 3.75 μM in 10 mM PBS buffer at pH 7.5. Both the concentrations of VanF and chlorinated fluorescein are saturation concentration.
respectively, while VanF had 4-fold stronger binding ability than the chlorinated fluorescein. In this investigation, the low antimicrobial activity and low binding affinity of VanF are consistent. Imaging was conducted using VanF (Figure 6). Both VanF and chlorinated fluorescein showed obvious fluorescent imaging of B. subtilis, indicating that both of them could be used as fluorescent probes. Meanwhile, VanF was also tested to label the Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC 51299, VanD), and VRE from clinic, but the results indicated that it could label neither Gram-negative bacteria nor vancomycin-resistant bacteria. In conclusion, we successfully constructed a novel fluorescence antibiotic VanF by conjugation of the chlorinated
Figure 4. (a) Photodynamic inactivation of B. subtilis strains with different concentrations of VanF. (b) Time-dependent bacterial lethality with incubation of 5 μM VanF. 2219
dx.doi.org/10.1021/bc200382d | Bioconjugate Chem. 2011, 22, 2217−2221
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Figure 6. Fluorescent imaging of B. subtilis labeled with VanF (a) and chlorinated fluorescein (b). The concentrations of the tested compounds were 10 μM in 10 mM PBS buffer at pH 7.5.
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fluorescein and norvancomycin. VanF was characterized by UV−vis and fluorescence spectra and confirmed by MALDITOF mass spectrum. The biological assay showed that VanF had effective photodynamic inactivation against B. subtilis , low antimicrobial activity with value of >128 μg/mL and binding affinity with value of 180 nM per 108 cells/mL to the tested bacteria. The fluorescence imaging showed that VanF could label the B. subtilis strain, but not the Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC 51299, VanD), and VRE strains from clinic.
ASSOCIATED CONTENT * Supporting Information Materials and instruments, methods and experimental procedures. This material is available free of charge via the Internet at http://pubs.acs.org. S
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AUTHOR INFORMATION Corresponding Author *Tel/Fax: +8629-8830-2429; E-mail:
[email protected] (K. W. Yang). 2220
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lactamase that provides multiple antibiotic resistance. J. Biol. Chem. 273, 22402−22408. (17) Crawford, P. A., Sharma, N., Chandrasekar, S., Sigdel, T., Walsh, T. R., Spencer, J., and Crowder, M. W. (2004) Over-expression, purification, and characterization of metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria. Protein Expr. Purif. 36, 272−279. (18) Crowder, M. W., Walsh, T. R., Banovic, L., Pettit, M., and Spencer, J. (1998) Overexpression, purification, and characterization of the cloned metallo-β-lactamase L1 from Stenotrophomonas maltophilia. Antimicrob. Agents Chemother. 42, 921−926. (19) CLSI. (2009) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically: approved standard-8th ed., CLSI M07-A8, Clinical and Laboratory Standards Institute, Wayne, PA.
ACKNOWLEDGMENTS We are grateful for assistance in the photodynamic studies from Dr. Dan Sun at the Laboratory of Photonics and Photon Technique in Northwest University. We thank Dr. Michael Crowder at Miami University for the plasmids containing metallo-β-lactamase genes. This work was supported by grants (to K. W. Y.) from National Natural Science Fund of China (20972127), Doctoral Fund of China (200806970005), Natural Science Fund of Shaanxi Province (2009JM2002), and Key Fund for International Cooperation of Shaanxi Province (2010KW-16).
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