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Synthesis of amphiphilic aminated inulin via ‘click chemistry’ and evaluation for its antibacterial activity
Bioorganic & Medicinal Chemistry Letters 24 (2014) 4590–4593
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis of amphiphilic aminated inulin via ‘click chemistry’ and evaluation for its antibacterial activity Fang Dong a, Jun Zhang b, Chunwei Yu b, Qing Li a, Jianming Ren a, Gang Wang a, Guodong Gu c, Zhanyong Guo a,⇑ a b c
Key Laboratory of Coastal Biology and Bioresource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China Laboratory of Environmental Monitoring, School of Tropical and Laboratory Medicine, Hainan Medical College, Haikou 571199, China Alliance Pharma, Inc. 17 Lee Boulevard Malvern, PA 19355, USA
a r t i c l e
i n f o
Article history: Received 8 April 2014 Revised 20 June 2014 Accepted 11 July 2014 Available online 17 July 2014 Keywords: Inulin Click chemistry Selective modification Reactive precursor Antibacterial activity
a b s t r a c t Inulins are a group of abundant, water-soluble, renewable polysaccharides, which exhibit attractive bioactivities and natural properties. Improvement such as chemical modification of inulin is often performed prior to further utilization. We hereby presented a method to modify inulin at its primary hydroxyls to synthesize amphiphilic aminated inulin via ‘click chemistry’ to facilitate its chemical manipulation. Additionally, its antibacterial property against Staphylococcus aureus (S. aureus) was also evaluated and the best inhibitory index against S. aureus was 58% at 1 mg/mL. As the amphiphilic aminated inulin is easy to prepare and exhibits improved bioactivity, this material may represent as an attractive new platform for chemical modifications of inulin. Ó 2014 Elsevier Ltd. All rights reserved.
Inulins are a group of polysaccharides consist of multiple bfructosyl fructose units and usually glucopyranose unit reducing ends (GFn).1 They are mainly extracted from low-requirement crops such as Jerusalem artichoke, chicory, and yacon. Several interesting properties such as beneficial attributes for human health, moderate average degree of polymerization, and readiness of being obtained have been shown by inulin.2,3 As a source of renewable, biodegradable, and environmentally benign polysaccharide, inulin is also a promising candidate for increasing demand for biodegradable and environmentally benign polymeric material.4,5 Inulin often needs improvements over its bioactivities and natural properties prior to further utilization.6–8 A powerful tool to achieve this would be chemical modification. Like many other polysaccharides, the tedious (polyol) functionality of inulin inevitably limits the chemical modifications of inulin.4 It is safe to propose that a reactive precursor of inulin would steer the chemical manipulations of this polysaccharide. Amination of inulin is an ideal solution to synthesize a reactive precursor of inulin that could be modified and adapted ‘on demand’. First, amino groups are active enough and play important parts in chemical reactions.9 For instance, Roman synthesized fluorescently labeled cellulose nanocrystals through amino groups.10 Second, ⇑ Corresponding author. Tel.: +86 535 2109171; fax: +86 535 2109000. E-mail address: [email protected] (Z. Guo). http://dx.doi.org/10.1016/j.bmcl.2014.07.029 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
aminoglycosides always exhibit excellent bioactivities. Some aminoglycosides have been reported to work as antibiotics via binding to the RNA.11 The copper(I) catalyzed azide-alkyne [3+2] cycloaddition (CuAAC) or ‘click chemistry’, developed by Sharpless, has emerged as one of the most popular methods for modifications of numerous biologically relevant molecules.12 It has been widely employed to build up polymers with complex architectures.13–15 The team of Saimoto prepared new chitosan derivatives containing triazolyl moieties at the C6 position of glucosamine units by coupling between azide and propargyl groups of chitosan via a 1,3-dipolar cycloaddition.16,17 The modification of carbohydrate polymers by ‘click chemistry’ will help to overcome their disadvantages, such as low selectivity, complicated reaction conditions, various side reactions, and low yields, and remarkably improve their substitution efficiency.18 Moreover, the reaction generated 1,4-disubstituted 1,2,3-triazolyl functional groups also have many appealing features such as antibacterial, herbicidal, fungicidal, and antiallergic.19,20 Some aminated polysaccharides can be developed as novel potential antibacterial agents as they exhibit excellent antibacterial properties.21 The property is related to interaction between positive charged aminated polysaccharides and negative charged bacterial cell membranes, which leads to the deformations of cell structure.22 To our knowledge, there are few studies focused on the highly regioselective and quantitative modifications of inulin through
4591
F. Dong et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4590–4593
HO
Br
O
OH
O O H3C C O C CH3
O
n
O
O NBS Ph3P
OH
O
n
O O OR OR
OH
OH
Br
O
n
O
NaN3 N N N
N3
O O H 2N
OR
OR OR
OR O
R= H3C
O O
NH2
n
O
n
O C
Scheme 1. Synthetic pathway of AAMINL. 23
‘click chemistry’. In this Letter we presented the preparation of amphiphilic aminated inulin via ‘click chemistry’. C-6-Br inulin derivative was synthesized firstly by reaction between the primary hydroxyls with NBS and Ph3P (Scheme 1). Then the secondary hydroxyls of the C-6-Br inulin derivative were protected with acetic anhydride. HAZDINL could be got through a nucleophilic substitution at C-6 with sodium azide. Subsequently the triazolyl groups were introduced into inulin through the ‘click reaction’. The chemical structures of the derivatives were characterized by FT-IR and 13 C NMR.24 Meanwhile, the rising incidence of drug resistant pathogens emphasizes the urgent need for new approaches to antimicrobial killing.25 One alternative to traditional antibiotics for topical microbial killing is natural polysaccharides. Herein, a common bacterium, Staphylococcus aureus (S. aureus) was selected to evaluate the antibacterial property of synthesized amphiphilic aminated inulin by hypa measurement in vitro. It was reported that NBS and Ph3P could selectively replace primary hydroxyl groups of polysaccharide with bromine.26 Therefore C-6-Br inulin derivative was selected as intermediate to activate the primary hydroxyls of inulin for azide to replace with. Then we protected the hydroxyls of the C-6-Br inulin derivative with acetic anhydride to bring hydrophobic groups into inulin backbone (hydrophobic 6-bromo-6-deoxyinulin (HBDINL)).27 The FT-IR spectrum of inulin shows peaks of saccharide at 852 cm1, 1029 cm1, and 3041 cm1. In spectrum of HBDINL, a new strong peak at 1730 cm1 appeared comparing with spectrum of inulin, which was assigned to the vibration of the C@O bonds of the acetyl ester (Fig. 1). In the 13C NMR spectrum of HBDINL (Fig. 2), methyl carbon of CH3AC@O was observed at 21 ppm, and carbonyl carbon of CH3AC@O was observed as two peaks at 162 and 170 ppm. Moreover, carbon of C-6-Br was clearly observed at 33 ppm. Azidation of HBDINL could be conveniently achieved through a nucleophilic substitution at C-6 with sodium azide to get hydrophobic 6-azido-6-deoxyinulin (HAZDINL).28 Characteristic peak of C-6-azido was observed at 2105 cm1 in HAZDINL spectrum (Fig. 1).29 As shown in Figure 2, the peak of C-6 carbon was completely shifted downfield to 54 ppm as a single signal, supporting regioselective and complete C-6 substitution of the azide moiety.30
Figure 1. FTIR spectra of inulin and inulin derivatives.
4592
F. Dong et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4590–4593 0.1mg/mL 0.5mg/mL
inhibition index (%)
1mg/mL
time (h) Figure 3. The antibacterial activities of AAMINL against S. aureus.
Figure 2.
13
C NMR spectra of inulin and inulin derivatives.
As long as we got HAZDINL, the ‘click chemistry’ could be performed in an elegant way with propynyl amine to synthesize the aimed amphiphilic aminated inulin.31 The peak at 2105 cm1 in HAZDINL spectrum disappeared when the C-6-azido in HAZDINL was transformed to 1,2,3-triazoles and a new sorption band at about 1540 cm1 appeared (Fig. 1). The 1,4-triazole linker is clearly observed at 123 and 144 ppm as two new peaks in the 13C NMR spectrum of AAMINL.30 As seen in Figure 2, the signals of carbons in 13C NMR spectra were well attributed to the structure of the inulin derivatives. The 13C NMR spectra of HBDINL, HAZDINL, and AAMINL further confirmed the success of the preparation. The test of antibacterial activity was carried out according to method described by Liu.32,33 Data were analyzed by an analysis of variance (P <0.05) and the mean values were separated by
Duncan’s multiple range test. The results were processed by computer programs (Excel and SPSS) and reported as mean ± SD. The antibacterial activity of the synthesized AAMINL and inulin against S. aureus were determined by optical density method. The results turned out that inulin did not inhibit the growth of S. aureus at the tested concentrations. When inulin was transformed to AAMINL, it could inhibit the growth of the tested bacterial at 0.5 mg/mL and 1.0 mg/mL (Fig. 3). Based on Figure 3, we could conclude that AAMINL exhibited concentration-dependent inhibitory index on the growth of S. aureus. AAMINL could inhibit the growth S. aureus at 0.5 mg/mL and 1.0 mg/mL evidently and the best inhibitory index against S. aureus could reach 58% at 1.0 mg/mL when the culture time was 16 hours. It is reasonable to propose that the obtained antibacterial activity of AAMINL may mainly benefit from amino groups and amphiphiles. It was reported that cationic amino-containing amphiphiles showed good antimicrobial activity as they could disrupt the bacterial membrane by a hydrophobic and electrostatic adsorption at membrane and water interface. Moreover, the 1,2,3-triazolyl functional groups in AAMINL could also have a synergies effect as they exhibited a variety of biological activities including antibacterial activity. In this Letter, the preparation of AAMINL, a precursor for facile chemical modification of inulin, has been established and the investigation of its potential antibacterial activity against S. aureus at series concentrations has also been reported. With active amino groups, the AAMINL is a suitable precursor for facile chemical manipulation of inulin. For the investigation of antibacterial activity against S. aureus, the data obtained in in vitro models clearly suggested the antibacterial potency of the substance. The mechanism of the obtained antibacterial activity was also discussed. It may be the result of hydrophilic/hydrophobic balance of the aminated inulin derivative, which led to the interaction with bacterial cell surface. The 1,2,3-triazolyl functional groups in AAMINL may also contribute to the antibacterial activity against the tested bacterial since triazoles are effective antibacterial functional groups. Acknowledgments We thank the Project of the 12th Five-Year Science and Technology Plan for Agriculture (2012BAD32B09), the Science and Technology Project of Yantai (2012131), and the Taishan Scholar Program of Shandong Province for financial support of this work. References and notes 1. Rogge, T. M.; Stevens, C. V. Biomacromolecules 2004, 5(5), 1799. 2. Beylot, M. Br. J. Nutr. 2006, 93, S163.
4593
F. Dong et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4590–4593 3. Causey, J. L.; Feirtag, J. M.; Gallaher, D. D.; Tungland, B. C.; Slavin, J. L. Nutr. Res. 2000, 20(2), 191. 4. Stevens, C. V.; Meriggi, A.; Booten, K. Biomacromolecules 2001, 2(1), 1. 5. Stevens, C. V.; Meriggi, A.; Peristeropoulou, M.; Christov, P. P.; Booten, K.; Levecke, B.; Vandamme, A.; Pittevils, N.; Tadros, T. F. Biomacromolecules 2001, 2(4), 1256. 6. Kim, J.-B.; Carpita, N. C. Plant Physiol. 1992, 98(2), 646. 7. Morros, J.; Levecke, B.; Infante, M. R. Carbohydr. Polym. 2011, 84(3), 1110. 8. Verraest, D. L.; Peters, J. A.; Kuzee, H. C.; Raaijmakers, H. W. C.; van Bekkum, H. Carbohydr. Polym. 1998, 37(3), 209. 9. Guo, Z.; Chen, R.; Xing, R.; Liu, S.; Yu, H.; Wang, P.; Li, C.; Li, P. Carbohydr. Res. 2006, 341(3), 351. 10. Dong, S.; Roman, M. J. Am. Chem. Soc. 2007, 129(45), 13810. 11. Ma, C.; Baker, N. A.; Joseph, S.; McCammon, J. A. J. Am. Chem. Soc. 2002, 124(7), 1438. 12. Kolb, H. C.; Finn, M.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40(11), 2004. 13. Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radi, Z.; Carlier, P. R.; Taylor, P.; Finn, M.; Sharpless, K. B. Angew. Chem. 2002, 114(6), 1095. 14. Zampano, G.; Bertoldo, M.; Ciardelli, F. React. Funct. Polym. 2010, 70(5), 272. 15. Uttamapinant, C.; Tangpeerachaikul, A.; Grecian, S.; Clarke, S.; Singh, U.; Slade, P.; Gee, K. R.; Ting, A. Y. Angew. Chem. 2012, 124(24), 5954. 16. Ifuku, S.; Wada, M.; Morimoto, M.; Saimoto, H. Carbohydr. Polym. 2012, 90(2), 1182. 17. Ifuku, S.; Matsumoto, C.; Wada, M.; Morimoto, M.; Saimoto, H. Int. J. Biol. Macromol. 2013, 52, 72. 18. Chen, Y.; Wang, F.; Yun, D.; Guo, Y.; Ye, Y.; Wang, Y.; Tan, H. J. Appl. Polym. Sci. 2013, 129(6), 3185. 19. Aizpurua, J. M.; Azcune, I.; Fratila, R. M.; Balentova, E.; Sagartzazu-Aizpurua, M.; Miranda, J. I. Org. Lett. 2010, 12(7), 1584. 20. Chen, Y.; Wang, F.; Yun, D.; Guo, Y.; Ye, Y.; Wang, Y.; Tan, H. Fiber Polym. 2013, 14(7), 1058. 21. Muhizi, T.; Grelier, S.; Coma, V. J. Agric. Food Chem. 2009, 57(19), 8770. 22. Hoque, J.; Akkapeddi, P.; Yarlagadda, V.; Uppu, D. S. S. M.; Kumar, P.; Haldar, J. Langmuir 2012, 28(33), 12225. 23. Izawa, K.; Hasegawa, T. Bioorg. Med. Chem. Lett. 2012, 22(2), 1189. 24. Inulin was purchased from Wede biological Corp. (Beijing. China) and was employed without further purification. Its average degree of polymerization was around 20 fructosyl fructose units. Triethylamine (Et3N), Nbromosuccinimide (NBS), triphenylphosphine (Ph3P), acetic anhydride, sodium azide, and propargyl amine were purchased from the Sigma–Aldrich Chemical Co. Other reagents were analytical grade and were purified and dried by standard procedures. FT-IR spectra were recorded on a Jasco-4100 (Tokyo, Japan, provided by JASCO China (Shanghai), Co., Ltd Shanghai, China.) and Solid-state 13C CP/MAS NMR spectra were obtained from a Bruker AC-300 spectrometer. (Fällanden, Switzerland, provided by Bruker BioSpin CN / Bruker (Beijing) Tech. and Serv. Co., Ltd Beijing, China.). ICP-MS spectra were recorded on a Elan DRC II (America, provided by PerkinElmer). 25. Kempf, M.; Rolain, J.-M. Int. J. Antimicrob. Agents 2012, 39, 105. 26. Matsui, Y.; Ishikawa, J.; Kamitakahara, H.; Takano, T.; Nakatsubo, F. Carbohydr. Res. 2005, 340(7), 1403. 27. Hydrophobic 6-bromo-6-deoxyinulin (HBDINL). Inulin (10 mmol) was dissolved in 150 mL anhydrous N,N-dimethylformamide (DMF) and stirred at 25 °C. When the solution was cooled to room temperature, Nbromosuccinimide (NBS) (50 mmol) and triphenylphosphine (Ph3P) (50 mmol) were added. The mixture was stirred at 80 °C for 3 h under Ar. The mixture was precipitated in 600 mL acetone and filtered by suction. The unreacted NBS, Ph3P, and other outgrowth were extracted in a Soxhlet
28.
29. 30. 31.
32. 33.
apparatus with ethanol and acetone for 48 h, respectively. The product was dried at 60 °C for 24 h, yield: 63.2%. The product obtained was dissolved in 150 mL anhydrous pyridine, and 15 mL acetic anhydride was added dropwise over 15 min. After being stirred at 25 °C for 8 h under Ar, the mixture was poured to ice water, filtered and the product was washed with water and acetone, respectively. After Soxhlet extraction with acetone for 48 h, the obtained product was freeze dried in vaccum, yield: 65.1%; 13C NMR (solidNMR): d 21 ppm (methyl carbon of CH3AC@O). d 33 ppm (carbon of C-6-Br). d 60–103 ppm (carbon of C-1-2-3-4 and -5). d 162 and 170 ppm (carbonyl carbon of CH3AC@O); FT-IR (thin film): v 1730 (C@O). v 3041, 1029, 852 (saccharide ring). Hydrophobic 6-azido-6-deoxyinulin (HAZDINL). HBDINL (10 mmol) was dissolved in 200 mL DMF at room temperature, and sodium azide (15 mmol) was added. After being stirred at 80 °C for 4 h under Ar, the mixture was cooled to room temperature and slowly poured to 400 mL ice water. The precipitate was collected, washed by water and ethanol. After Soxhlet extraction with water for 48 h to remove the probable remained sodium azide, the obtained product was freeze dried in vaccum, yields: 57.3%; 13C NMR (solid-NMR): d 21 ppm (methyl carbon of CH3AC@O). d 54 ppm (carbon of C-6-N3). d 60104 ppm (carbon of C-1-2-3-4 and -5). d 162 and 170 ppm (carbonyl carbon of CH3AC@O); FT-IR (thin film): v 2105 (C-6-azido). Cimecioglu, A. L.; Ball, D. H.; Kaplan, D. L.; Huang, S. H. Macromolecules 1994, 27(11), 2917. Ifuku, S.; Wada, M.; Morimoto, M.; Saimoto, H. Carbohydr. Polym. 2011, 85(3), 653. Amphiphilic aminated inulin (AAMINL). HAZDINL (10 mmol), CuI (1 mmol), and Et3N (10 mmol) were dissolved in 200 mL DMF. Propynyl amine (15 mmol) was added to the mixture and the solution was stirred at 70 °C for 48 h under Ar. The mixture was filtered and the filtrate was collected and precipitated into diethyl ether. The precipitate was collected by filtration and the probable remained reagents was extracted in a Soxhlet apparatus with acetone for two days. The obtained product was freeze dried in vaccum, yields: 53.4%; 13C NMR (solid-NMR): d 21 ppm (methyl carbon of CH3AC@O). d 57–104 ppm (carbon of C-1-2-3-4-5 and NH2–CH2–). d 123-144 ppm (carbon of triazoles). d 162 and 170 ppm (carbonyl carbon of CH3AC@O); FT-IR (thin film): v 1730 (C@O). v 1540 (N–H). An ICP-MS of AAMINL was also performed and the concentration of Cu in the sample is 0.8 ppm. Liu, H.; Bao, J.; Du, Y.; Zhou, X.; Kennedy, J. F. Carbohydr. Polym. 2006, 64(4), 553. S. aureus was incubated in nutrient broth (peptone 10 g, beef extracts 5 g, and NaCl 10 g in deionized water) at 37 °C for 24 h. To prepare bacterial suspensions, the culture was diluted to obtain a bacterial suspension containing 105–106cells/mL. Samples of inulin and AAMINL were added to the mixture of bacterial suspensions (20 lL) and nutrient broth (19.8 mL) to give a final concentrations at 0.1 mg/mL, 0.5 mg/mL, and 1.0 mg/mL. The obtained mixture was incubated in a shaking bed (150 rpm) at 37 °C. During the incubation the turbidity of the medium was measured at 600 nm using a 721-E visible Spectrophotometer. The inhibition index was calculated as follows:
Inhibition index ð%Þ ¼ 1
Asample Asample0 100% Amedium Amedium0
Where Asample is the absorbance of bacterial medium with sample after incubation, Asample0 is the absorbance of bacterial medium with sample before incubation, Amedium is the absorbance of bacterial medium after incubation and Amedium0 is the absorbance of bacterial medium before incubation.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis of amphiphilic aminated inulin via ‘click chemistry’ and evaluation for its antibacterial activity Fang Dong a, Jun Zhang b, Chunwei Yu b, Qing Li a, Jianming Ren a, Gang Wang a, Guodong Gu c, Zhanyong Guo a,⇑ a b c
Key Laboratory of Coastal Biology and Bioresource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China Laboratory of Environmental Monitoring, School of Tropical and Laboratory Medicine, Hainan Medical College, Haikou 571199, China Alliance Pharma, Inc. 17 Lee Boulevard Malvern, PA 19355, USA
a r t i c l e
i n f o
Article history: Received 8 April 2014 Revised 20 June 2014 Accepted 11 July 2014 Available online 17 July 2014 Keywords: Inulin Click chemistry Selective modification Reactive precursor Antibacterial activity
a b s t r a c t Inulins are a group of abundant, water-soluble, renewable polysaccharides, which exhibit attractive bioactivities and natural properties. Improvement such as chemical modification of inulin is often performed prior to further utilization. We hereby presented a method to modify inulin at its primary hydroxyls to synthesize amphiphilic aminated inulin via ‘click chemistry’ to facilitate its chemical manipulation. Additionally, its antibacterial property against Staphylococcus aureus (S. aureus) was also evaluated and the best inhibitory index against S. aureus was 58% at 1 mg/mL. As the amphiphilic aminated inulin is easy to prepare and exhibits improved bioactivity, this material may represent as an attractive new platform for chemical modifications of inulin. Ó 2014 Elsevier Ltd. All rights reserved.
Inulins are a group of polysaccharides consist of multiple bfructosyl fructose units and usually glucopyranose unit reducing ends (GFn).1 They are mainly extracted from low-requirement crops such as Jerusalem artichoke, chicory, and yacon. Several interesting properties such as beneficial attributes for human health, moderate average degree of polymerization, and readiness of being obtained have been shown by inulin.2,3 As a source of renewable, biodegradable, and environmentally benign polysaccharide, inulin is also a promising candidate for increasing demand for biodegradable and environmentally benign polymeric material.4,5 Inulin often needs improvements over its bioactivities and natural properties prior to further utilization.6–8 A powerful tool to achieve this would be chemical modification. Like many other polysaccharides, the tedious (polyol) functionality of inulin inevitably limits the chemical modifications of inulin.4 It is safe to propose that a reactive precursor of inulin would steer the chemical manipulations of this polysaccharide. Amination of inulin is an ideal solution to synthesize a reactive precursor of inulin that could be modified and adapted ‘on demand’. First, amino groups are active enough and play important parts in chemical reactions.9 For instance, Roman synthesized fluorescently labeled cellulose nanocrystals through amino groups.10 Second, ⇑ Corresponding author. Tel.: +86 535 2109171; fax: +86 535 2109000. E-mail address: [email protected] (Z. Guo). http://dx.doi.org/10.1016/j.bmcl.2014.07.029 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
aminoglycosides always exhibit excellent bioactivities. Some aminoglycosides have been reported to work as antibiotics via binding to the RNA.11 The copper(I) catalyzed azide-alkyne [3+2] cycloaddition (CuAAC) or ‘click chemistry’, developed by Sharpless, has emerged as one of the most popular methods for modifications of numerous biologically relevant molecules.12 It has been widely employed to build up polymers with complex architectures.13–15 The team of Saimoto prepared new chitosan derivatives containing triazolyl moieties at the C6 position of glucosamine units by coupling between azide and propargyl groups of chitosan via a 1,3-dipolar cycloaddition.16,17 The modification of carbohydrate polymers by ‘click chemistry’ will help to overcome their disadvantages, such as low selectivity, complicated reaction conditions, various side reactions, and low yields, and remarkably improve their substitution efficiency.18 Moreover, the reaction generated 1,4-disubstituted 1,2,3-triazolyl functional groups also have many appealing features such as antibacterial, herbicidal, fungicidal, and antiallergic.19,20 Some aminated polysaccharides can be developed as novel potential antibacterial agents as they exhibit excellent antibacterial properties.21 The property is related to interaction between positive charged aminated polysaccharides and negative charged bacterial cell membranes, which leads to the deformations of cell structure.22 To our knowledge, there are few studies focused on the highly regioselective and quantitative modifications of inulin through
4591
F. Dong et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4590–4593
HO
Br
O
OH
O O H3C C O C CH3
O
n
O
O NBS Ph3P
OH
O
n
O O OR OR
OH
OH
Br
O
n
O
NaN3 N N N
N3
O O H 2N
OR
OR OR
OR O
R= H3C
O O
NH2
n
O
n
O C
Scheme 1. Synthetic pathway of AAMINL. 23
‘click chemistry’. In this Letter we presented the preparation of amphiphilic aminated inulin via ‘click chemistry’. C-6-Br inulin derivative was synthesized firstly by reaction between the primary hydroxyls with NBS and Ph3P (Scheme 1). Then the secondary hydroxyls of the C-6-Br inulin derivative were protected with acetic anhydride. HAZDINL could be got through a nucleophilic substitution at C-6 with sodium azide. Subsequently the triazolyl groups were introduced into inulin through the ‘click reaction’. The chemical structures of the derivatives were characterized by FT-IR and 13 C NMR.24 Meanwhile, the rising incidence of drug resistant pathogens emphasizes the urgent need for new approaches to antimicrobial killing.25 One alternative to traditional antibiotics for topical microbial killing is natural polysaccharides. Herein, a common bacterium, Staphylococcus aureus (S. aureus) was selected to evaluate the antibacterial property of synthesized amphiphilic aminated inulin by hypa measurement in vitro. It was reported that NBS and Ph3P could selectively replace primary hydroxyl groups of polysaccharide with bromine.26 Therefore C-6-Br inulin derivative was selected as intermediate to activate the primary hydroxyls of inulin for azide to replace with. Then we protected the hydroxyls of the C-6-Br inulin derivative with acetic anhydride to bring hydrophobic groups into inulin backbone (hydrophobic 6-bromo-6-deoxyinulin (HBDINL)).27 The FT-IR spectrum of inulin shows peaks of saccharide at 852 cm1, 1029 cm1, and 3041 cm1. In spectrum of HBDINL, a new strong peak at 1730 cm1 appeared comparing with spectrum of inulin, which was assigned to the vibration of the C@O bonds of the acetyl ester (Fig. 1). In the 13C NMR spectrum of HBDINL (Fig. 2), methyl carbon of CH3AC@O was observed at 21 ppm, and carbonyl carbon of CH3AC@O was observed as two peaks at 162 and 170 ppm. Moreover, carbon of C-6-Br was clearly observed at 33 ppm. Azidation of HBDINL could be conveniently achieved through a nucleophilic substitution at C-6 with sodium azide to get hydrophobic 6-azido-6-deoxyinulin (HAZDINL).28 Characteristic peak of C-6-azido was observed at 2105 cm1 in HAZDINL spectrum (Fig. 1).29 As shown in Figure 2, the peak of C-6 carbon was completely shifted downfield to 54 ppm as a single signal, supporting regioselective and complete C-6 substitution of the azide moiety.30
Figure 1. FTIR spectra of inulin and inulin derivatives.
4592
F. Dong et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4590–4593 0.1mg/mL 0.5mg/mL
inhibition index (%)
1mg/mL
time (h) Figure 3. The antibacterial activities of AAMINL against S. aureus.
Figure 2.
13
C NMR spectra of inulin and inulin derivatives.
As long as we got HAZDINL, the ‘click chemistry’ could be performed in an elegant way with propynyl amine to synthesize the aimed amphiphilic aminated inulin.31 The peak at 2105 cm1 in HAZDINL spectrum disappeared when the C-6-azido in HAZDINL was transformed to 1,2,3-triazoles and a new sorption band at about 1540 cm1 appeared (Fig. 1). The 1,4-triazole linker is clearly observed at 123 and 144 ppm as two new peaks in the 13C NMR spectrum of AAMINL.30 As seen in Figure 2, the signals of carbons in 13C NMR spectra were well attributed to the structure of the inulin derivatives. The 13C NMR spectra of HBDINL, HAZDINL, and AAMINL further confirmed the success of the preparation. The test of antibacterial activity was carried out according to method described by Liu.32,33 Data were analyzed by an analysis of variance (P <0.05) and the mean values were separated by
Duncan’s multiple range test. The results were processed by computer programs (Excel and SPSS) and reported as mean ± SD. The antibacterial activity of the synthesized AAMINL and inulin against S. aureus were determined by optical density method. The results turned out that inulin did not inhibit the growth of S. aureus at the tested concentrations. When inulin was transformed to AAMINL, it could inhibit the growth of the tested bacterial at 0.5 mg/mL and 1.0 mg/mL (Fig. 3). Based on Figure 3, we could conclude that AAMINL exhibited concentration-dependent inhibitory index on the growth of S. aureus. AAMINL could inhibit the growth S. aureus at 0.5 mg/mL and 1.0 mg/mL evidently and the best inhibitory index against S. aureus could reach 58% at 1.0 mg/mL when the culture time was 16 hours. It is reasonable to propose that the obtained antibacterial activity of AAMINL may mainly benefit from amino groups and amphiphiles. It was reported that cationic amino-containing amphiphiles showed good antimicrobial activity as they could disrupt the bacterial membrane by a hydrophobic and electrostatic adsorption at membrane and water interface. Moreover, the 1,2,3-triazolyl functional groups in AAMINL could also have a synergies effect as they exhibited a variety of biological activities including antibacterial activity. In this Letter, the preparation of AAMINL, a precursor for facile chemical modification of inulin, has been established and the investigation of its potential antibacterial activity against S. aureus at series concentrations has also been reported. With active amino groups, the AAMINL is a suitable precursor for facile chemical manipulation of inulin. For the investigation of antibacterial activity against S. aureus, the data obtained in in vitro models clearly suggested the antibacterial potency of the substance. The mechanism of the obtained antibacterial activity was also discussed. It may be the result of hydrophilic/hydrophobic balance of the aminated inulin derivative, which led to the interaction with bacterial cell surface. The 1,2,3-triazolyl functional groups in AAMINL may also contribute to the antibacterial activity against the tested bacterial since triazoles are effective antibacterial functional groups. Acknowledgments We thank the Project of the 12th Five-Year Science and Technology Plan for Agriculture (2012BAD32B09), the Science and Technology Project of Yantai (2012131), and the Taishan Scholar Program of Shandong Province for financial support of this work. References and notes 1. Rogge, T. M.; Stevens, C. V. Biomacromolecules 2004, 5(5), 1799. 2. Beylot, M. Br. J. Nutr. 2006, 93, S163.
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F. Dong et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4590–4593 3. Causey, J. L.; Feirtag, J. M.; Gallaher, D. D.; Tungland, B. C.; Slavin, J. L. Nutr. Res. 2000, 20(2), 191. 4. Stevens, C. V.; Meriggi, A.; Booten, K. Biomacromolecules 2001, 2(1), 1. 5. Stevens, C. V.; Meriggi, A.; Peristeropoulou, M.; Christov, P. P.; Booten, K.; Levecke, B.; Vandamme, A.; Pittevils, N.; Tadros, T. F. Biomacromolecules 2001, 2(4), 1256. 6. Kim, J.-B.; Carpita, N. C. Plant Physiol. 1992, 98(2), 646. 7. Morros, J.; Levecke, B.; Infante, M. R. Carbohydr. Polym. 2011, 84(3), 1110. 8. Verraest, D. L.; Peters, J. A.; Kuzee, H. C.; Raaijmakers, H. W. C.; van Bekkum, H. Carbohydr. Polym. 1998, 37(3), 209. 9. Guo, Z.; Chen, R.; Xing, R.; Liu, S.; Yu, H.; Wang, P.; Li, C.; Li, P. Carbohydr. Res. 2006, 341(3), 351. 10. Dong, S.; Roman, M. J. Am. Chem. Soc. 2007, 129(45), 13810. 11. Ma, C.; Baker, N. A.; Joseph, S.; McCammon, J. A. J. Am. Chem. Soc. 2002, 124(7), 1438. 12. Kolb, H. C.; Finn, M.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40(11), 2004. 13. Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radi, Z.; Carlier, P. R.; Taylor, P.; Finn, M.; Sharpless, K. B. Angew. Chem. 2002, 114(6), 1095. 14. Zampano, G.; Bertoldo, M.; Ciardelli, F. React. Funct. Polym. 2010, 70(5), 272. 15. Uttamapinant, C.; Tangpeerachaikul, A.; Grecian, S.; Clarke, S.; Singh, U.; Slade, P.; Gee, K. R.; Ting, A. Y. Angew. Chem. 2012, 124(24), 5954. 16. Ifuku, S.; Wada, M.; Morimoto, M.; Saimoto, H. Carbohydr. Polym. 2012, 90(2), 1182. 17. Ifuku, S.; Matsumoto, C.; Wada, M.; Morimoto, M.; Saimoto, H. Int. J. Biol. Macromol. 2013, 52, 72. 18. Chen, Y.; Wang, F.; Yun, D.; Guo, Y.; Ye, Y.; Wang, Y.; Tan, H. J. Appl. Polym. Sci. 2013, 129(6), 3185. 19. Aizpurua, J. M.; Azcune, I.; Fratila, R. M.; Balentova, E.; Sagartzazu-Aizpurua, M.; Miranda, J. I. Org. Lett. 2010, 12(7), 1584. 20. Chen, Y.; Wang, F.; Yun, D.; Guo, Y.; Ye, Y.; Wang, Y.; Tan, H. Fiber Polym. 2013, 14(7), 1058. 21. Muhizi, T.; Grelier, S.; Coma, V. J. Agric. Food Chem. 2009, 57(19), 8770. 22. Hoque, J.; Akkapeddi, P.; Yarlagadda, V.; Uppu, D. S. S. M.; Kumar, P.; Haldar, J. Langmuir 2012, 28(33), 12225. 23. Izawa, K.; Hasegawa, T. Bioorg. Med. Chem. Lett. 2012, 22(2), 1189. 24. Inulin was purchased from Wede biological Corp. (Beijing. China) and was employed without further purification. Its average degree of polymerization was around 20 fructosyl fructose units. Triethylamine (Et3N), Nbromosuccinimide (NBS), triphenylphosphine (Ph3P), acetic anhydride, sodium azide, and propargyl amine were purchased from the Sigma–Aldrich Chemical Co. Other reagents were analytical grade and were purified and dried by standard procedures. FT-IR spectra were recorded on a Jasco-4100 (Tokyo, Japan, provided by JASCO China (Shanghai), Co., Ltd Shanghai, China.) and Solid-state 13C CP/MAS NMR spectra were obtained from a Bruker AC-300 spectrometer. (Fällanden, Switzerland, provided by Bruker BioSpin CN / Bruker (Beijing) Tech. and Serv. Co., Ltd Beijing, China.). ICP-MS spectra were recorded on a Elan DRC II (America, provided by PerkinElmer). 25. Kempf, M.; Rolain, J.-M. Int. J. Antimicrob. Agents 2012, 39, 105. 26. Matsui, Y.; Ishikawa, J.; Kamitakahara, H.; Takano, T.; Nakatsubo, F. Carbohydr. Res. 2005, 340(7), 1403. 27. Hydrophobic 6-bromo-6-deoxyinulin (HBDINL). Inulin (10 mmol) was dissolved in 150 mL anhydrous N,N-dimethylformamide (DMF) and stirred at 25 °C. When the solution was cooled to room temperature, Nbromosuccinimide (NBS) (50 mmol) and triphenylphosphine (Ph3P) (50 mmol) were added. The mixture was stirred at 80 °C for 3 h under Ar. The mixture was precipitated in 600 mL acetone and filtered by suction. The unreacted NBS, Ph3P, and other outgrowth were extracted in a Soxhlet
28.
29. 30. 31.
32. 33.
apparatus with ethanol and acetone for 48 h, respectively. The product was dried at 60 °C for 24 h, yield: 63.2%. The product obtained was dissolved in 150 mL anhydrous pyridine, and 15 mL acetic anhydride was added dropwise over 15 min. After being stirred at 25 °C for 8 h under Ar, the mixture was poured to ice water, filtered and the product was washed with water and acetone, respectively. After Soxhlet extraction with acetone for 48 h, the obtained product was freeze dried in vaccum, yield: 65.1%; 13C NMR (solidNMR): d 21 ppm (methyl carbon of CH3AC@O). d 33 ppm (carbon of C-6-Br). d 60–103 ppm (carbon of C-1-2-3-4 and -5). d 162 and 170 ppm (carbonyl carbon of CH3AC@O); FT-IR (thin film): v 1730 (C@O). v 3041, 1029, 852 (saccharide ring). Hydrophobic 6-azido-6-deoxyinulin (HAZDINL). HBDINL (10 mmol) was dissolved in 200 mL DMF at room temperature, and sodium azide (15 mmol) was added. After being stirred at 80 °C for 4 h under Ar, the mixture was cooled to room temperature and slowly poured to 400 mL ice water. The precipitate was collected, washed by water and ethanol. After Soxhlet extraction with water for 48 h to remove the probable remained sodium azide, the obtained product was freeze dried in vaccum, yields: 57.3%; 13C NMR (solid-NMR): d 21 ppm (methyl carbon of CH3AC@O). d 54 ppm (carbon of C-6-N3). d 60104 ppm (carbon of C-1-2-3-4 and -5). d 162 and 170 ppm (carbonyl carbon of CH3AC@O); FT-IR (thin film): v 2105 (C-6-azido). Cimecioglu, A. L.; Ball, D. H.; Kaplan, D. L.; Huang, S. H. Macromolecules 1994, 27(11), 2917. Ifuku, S.; Wada, M.; Morimoto, M.; Saimoto, H. Carbohydr. Polym. 2011, 85(3), 653. Amphiphilic aminated inulin (AAMINL). HAZDINL (10 mmol), CuI (1 mmol), and Et3N (10 mmol) were dissolved in 200 mL DMF. Propynyl amine (15 mmol) was added to the mixture and the solution was stirred at 70 °C for 48 h under Ar. The mixture was filtered and the filtrate was collected and precipitated into diethyl ether. The precipitate was collected by filtration and the probable remained reagents was extracted in a Soxhlet apparatus with acetone for two days. The obtained product was freeze dried in vaccum, yields: 53.4%; 13C NMR (solid-NMR): d 21 ppm (methyl carbon of CH3AC@O). d 57–104 ppm (carbon of C-1-2-3-4-5 and NH2–CH2–). d 123-144 ppm (carbon of triazoles). d 162 and 170 ppm (carbonyl carbon of CH3AC@O); FT-IR (thin film): v 1730 (C@O). v 1540 (N–H). An ICP-MS of AAMINL was also performed and the concentration of Cu in the sample is 0.8 ppm. Liu, H.; Bao, J.; Du, Y.; Zhou, X.; Kennedy, J. F. Carbohydr. Polym. 2006, 64(4), 553. S. aureus was incubated in nutrient broth (peptone 10 g, beef extracts 5 g, and NaCl 10 g in deionized water) at 37 °C for 24 h. To prepare bacterial suspensions, the culture was diluted to obtain a bacterial suspension containing 105–106cells/mL. Samples of inulin and AAMINL were added to the mixture of bacterial suspensions (20 lL) and nutrient broth (19.8 mL) to give a final concentrations at 0.1 mg/mL, 0.5 mg/mL, and 1.0 mg/mL. The obtained mixture was incubated in a shaking bed (150 rpm) at 37 °C. During the incubation the turbidity of the medium was measured at 600 nm using a 721-E visible Spectrophotometer. The inhibition index was calculated as follows:
Inhibition index ð%Þ ¼ 1
Asample Asample0 100% Amedium Amedium0
Where Asample is the absorbance of bacterial medium with sample after incubation, Asample0 is the absorbance of bacterial medium with sample before incubation, Amedium is the absorbance of bacterial medium after incubation and Amedium0 is the absorbance of bacterial medium before incubation.