15 1.3 Experimental
Materials and characterization.
Chemicals used in the synthesis were purchased from Aldrich, Acros, Alfa Aesar, TCI America without further purification unless otherwise specified. NMR spectra were taken in CDCl3, MeOD or DMSO-d6 in a Varian Mercury/VX 300 spectrometer and the δ values are in ppm. All chemical shifts were reported as parts per million (ppm) w.r.t. TMS and referenced by residual solvent resonances. IR spectra were studied with a Shimadzu FTIR Spectrophotometer. Mass spectra were recorded by Micromass Q-TOF I mass spectrometer. Fluorescence emission and excitation spectra were recorded using a JASCO FP-6500 Spectrofluorometer. Absorption was recorded utilizing a NANODROP 2000C Spectrophotometer. Fluorescence quantum yields were determined in ethanol using rhodamine B in ethanol (F = 0.49) as the standard.31 The path length was 1 cm with a cell volume of 3.0 mL.
Synthesis of ANQCC.
Cyanoacetic acid (2.5 g, 29.75 mmol) in round bottom flask was heated at 80℃
to dissolve. Then acenaphthoquinone (0.9 g, 4.94 mmol) was added under the protection of N2. After stirring at 110 ℃ for 3 h, TLC showed the reaction was completed. After cooling down to room temperature, the crude reaction mixture was diluted with 30 mL acetonitrile, filtered and washed twice with acetonitrile three times to afford an orange solid ANQCC (0.62 g, yield: 50%).
16 Synthesis of CCP.
To a solution of ANQCC (400 mg, 1.6 mmol) in acetonitrile (25 mL) was added K2CO3 (28.3 mg, 0.4 mmol) under the protection of N2. After 1 h of reflux, the color of the reaction mixture changed from orange to yellow and TLC showed the complete consumption of the starting material. The solvent was then removed to afford the crude product, which was purified over column chromatography on silica gel (DCM: MeOH = 7:1) to give CCP as a yellow solid (376 mg, yield 94%).
Synthesis of s2 and CCPAN.
Scheme 1.7. Synthesis for the fluorescent probe CCPAN.
To a suspended solution of 3-bromopropylamine hydrobromide (8.7 g, 40 mmol) in toluene (100 mL) was added pyridine (4.74 mL, 60 mmol). The reaction mixture was then heated at reflux for 24 h under N2. After cooling down, the crude reaction mixture was filtered and washed with ether three times to afford the white solid (10.87 g, 91.7%). 1H NMR (300 MHz, DMSO-d6) δ: 9.24 (d, 2 H, J = 5.8 Hz), 8.66 (t, 1 H, J = 7.8 Hz), 8.22 (t, 2 H, J = 7.0 Hz), 8.08 (s, 3 H), 4.82 (t, 2 H, J = 7.0 Hz), 2.89 (sext, 2 H, J = 6.3 Hz), 2.28 (quint, 2 H, J = 7.2 Hz)
17
To a solution of CCP (120 mg, 0.48 mmol) in mixture of CH3CN : H2O 5 mL (1:1) at 0 ℃ in ice bath was added a solution of 2 (120 mg, 0.4 mmol) and K2CO3 (132.5 mg, 0.96 mmol) in mixture of CH3CN : H2O 1 mL (1:1). HOBt (74 mg, 0.48 mmol) and EDCI (92 mg, 0.48 mmol) were then added to the solution at r. t. and stirred overnight. The reaction mixture was concentrated under vacuum at low temperature (35 ℃) and purified by the preparative TLC (silica gel; DCM : MeOH = 4:1 ) to afford the yellow solid (21 mg, 10%). 1H NMR (300 MHz, MeOD) δ: 9.07 (d, 2 H, J = 5.7 Hz), 8.48 (t, 1 H, J = 7.8 Hz),
To a suspended solution of 3-bromopropylamine hydrobromide (25.1 g, 115 mmol) in 1-butanol (150 mL) was added triphenylphosphine (42.1 g, 160 mmol). After heated at reflux for 24 h under N2, the transparent reaction mixture was cooled down and poured into a stirring solution of ethyl ether (450 mL) and toluene (250 mL). After stirring at r. t. for 0.5 h, the solid was collected and washed with ethyl ether three times to afford s3 as a
18
EDCI (138 mg, 0.72 mmol) were then added to the solution at r. t. and stirred overnight.
The reaction mixture was concentrated under vacuum at low temperature (35 ℃) and purified by the preparative TLC (silica gel; DCM : MeOH = 10:1 ) to afford the CCPAP as a yellow solid (36.4 mg, 10%).1H NMR (300 MHz, MeOD) δ: 8.01 (d, 1 H, J = 8.0 Hz), 7.70-7.92 (m, 16 H), 7.66 (t, 1 H, J = 7.5 Hz), 7.59 (d, 1 H, J = 8.2 Hz), 7.57 (d, 1 H, J = 6.6 Hz), 7.46 (t, 1 H, J = 7.7 Hz), 3.67 (t, 2 H, J = 6.3 Hz), 3.51-3.61 (m, 2 H), 2.10 (sext, 2 H, J = 7.4 Hz). ESI-MS: [M]+calcd for (M + H+): 542. Found: 542; calcd for (M + Na+):
564. Found: 564.13C NMR (100 MHz, MeOD) δ: 195.22, 168.97, 161.24, 136.79, 136.42, 135.79, 134.99, 133.75, 132.14, 131.75, 131.38, 129.83, 128.37, 122.72, 121.90, 119.99, 116.99, 101.67, 46.35, 25.36, 20.50.
1.4 Conclusions
Two types of positive charged moieties, i.e. triphenylphosphonium and pyridinium, were introduced to the frame structure CCP respectively by coupling carboxylic acid on the 3-position with amine to improve the cell permeability and cysteine selectivity. ESI-MS, NMR, IR were employed for the successful characterization of the novel potential cysteine sensor. The further cellular cysteine response of the probes is still under investigation. The cationic moieties triphenylphosphonium and pyridinium would enable the probes CCPAP and CCPAN to have potential improved cell permeability, which could increase the sensitivity towards cellular cysteine for a good cell imaging.
Besides, triphenylphosphonium in CCPAP is a mitochondria targeting moiety, which could increase the selectivity and be a potential mitochondria targeting sensor.
19 1.5 References
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Stromal control of cystine metabolism promotes cancer cell survival in chronic lymphocytic leukaemia. Nat. Cell Biol. 2012, 14, 276-286.
(2) Jiang, X. D.; Zhang, J.; Shao, X. M.; Zhao, W. L. A selective fluorescent turn-on NIR probe for cysteine. Org. Biomol. Chem. 2012, 10, 1966-1968.
(3) Park, S.; Imlay, J. A. High levels of intracellular cysteine promote oxidative DNA damage by driving the Fenton reaction. J. Bacteriol. 2003, 185, 1942-1950.
(4) Stipanuk, M. H.; Dominy, J. E.; Lee, J. I.; Coloso, R. M. Mammalian cysteine metabolism: New insights into regulation of cysteine metabolism. J. Nutr. 2006, 136, 1652S-1659S.
(5) Heafield, M. T.; Fearn, S.; Steventon, G. B.; Waring, R. H.; Williams, A.
C.; Sturman, S. G. Plasma cysteine and sulphate levels in patients with motor neurone, Parkinson's and Alzheimer's disease. Neurosci. Lett. 1990, 110, 216-220.
(6) El-Khairy, L.; Vollset, S. E.; Refsum, H.; Ueland, P. M. Plasma total cysteine, pregnancy complications, and adverse pregnancy outcomes: the Hordaland Homocysteine Study. Am. J. Clin. Nutr. 2003, 77, 467-472.
(7) Ozkan, Y.; Ozkan, E.; Simsek, B. Plasma total homocysteine and cysteine levels as cardiovascular risk factors in coronary heart disease. Int. J. Cardiol. 2002, 82, 269-277.
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(8) Niu, L. Y.; Guan, Y. S.; Chen, Y. Z.; Wu, L. Z.; Tung, C. H.; Yang, Q. Z.
BODIPY-Based Ratiometric Fluorescent Sensor for Highly Selective Detection of Glutathione over Cysteine and Homocysteine. J. Am. Chem. Soc. 2012, 134, 18928-18931.
(9) Shao, N.; Jin, J. Y.; Cheung, S. M.; Yang, R. H.; Chan, W. H.; Mo, T. A spiropyran-based ensemble for visual recognition and quantification of cysteine and homocysteine at physiological levels. Angew. Chem. Int. Ed. 2006, 45, 4944-4948.
(10) Lee, K. S.; Kim, T. K.; Lee, J. H.; Kim, H. J.; Hong, J. I. Fluorescence turn-on probe for homocysteine and cysteine in water. Chem. Commun. 2008, 6173-6175.
(11) Zeng, Y.; Zhang, G. X.; Zhang, D. Q. A selective colorimetric chemosensor for thiols based on intramolecular charge transfer mechanism. Anal. Chim. Acta 2008, 627, 254-257.
(12) Zeng, Y.; Zhang, G. X.; Zhang, D. Q.; Zhu, D. B. A dual-function colorimetric chemosensor for thiols and transition metal ions based on ICT mechanism.
Tetrahedron Lett. 2008, 49, 7391-7394.
(13) Huang, S. T.; Ting, K. N.; Wang, K. L. Development of a long-wavelength fluorescent probe based on quinone-methide-type reaction to detect physiologically significant thiols. Anal. Chim. Acta 2008, 620, 120-126.
(14) Chen, H. L.; Zhao, Q.; Wu, Y. B.; Li, F. Y.; Yang, H.; Yi, T.; Huang, C. H.
Selective phosphorescence chemosensor for homocysteine based on an iridium(III) complex. Inorg. Chem. 2007, 46, 11075-11081.
(15) Tang, B.; Xing, Y. L.; Li, P.; Zhang, N.; Yu, F. B.; Yang, G. W. A rhodamine-based fluorescent probe containing a Se-N bond for detecting thiols and its application in living cells. J. Am. Chem. Soc. 2007, 129, 11666-11667.
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(16) Tanaka, F.; Mase, N.; Barbas, C. F. Determination of cysteine concentration by fluorescence increase: reaction of cysteine with a fluorogenic aldehyde. Chem. Commun.
2004, 1762-1763.
(17) Li, T.; Ye, B.; Niu, Z. W.; Thompson, P.; Seifert, S.; Lee, B.; Wang, Q.
Closed-Packed Colloidal Assemblies from Icosahedral Plant Virus and Polymer. Chem.
Mater. 2009, 21, 1046-1050.
(18) Zhang, M.; Yu, M. X.; Li, F. Y.; Zhu, M. W.; Li, M. Y.; Gao, Y. H.; Li, L.;
Liu, Z. Q.; Zhang, J. P.; Zhang, D. Q.; Yi, T.; Huang, C. H. A highly selective fluorescence turn-on sensor for Cysteine/Homocysteine and its application in bioimaging. J. Am. Chem.
Soc. 2007, 129, 10322-10323.
(19) Xiao, Y.; Liu, F. Y.; Qian, X. H.; Cui, J. N. A new class of long-wavelength fluorophores: strong red fluorescence, convenient synthesis and easy derivation. Chem.
Commun. 2005, 239-241.
(20) Liu, F. Y.; Qian, X. H.; Cui, J. N.; Xiao, Y.; Zhang, R.; Li, G. Y. Design, synthesis, and antitumor evaluation of novel acenaphtho 1,2-b pyrrole-carboxylic acid esters with amino chain substitution. Bioorg. Med. Chem. 2006, 14, 4639-4644.
(21) Li, H. L.; Liu, F. Y.; Xiao, Y.; Pellechia, P. J.; Smith, M. D.; Qian, X. H.;
Wang, G. R.; Wang, Q. Revisit of a series of ICT fluorophores: skeletal characterization, structural modification, and spectroscopic behavior. Tetrahedron 2014, 70, 5872-5877.
(22) Liu, F. Y.; Xiao, Y.; Qian, X. H.; Zhang, Z. C.; Cui, J. N.; Zhang, R.
Versatile acenaphtho 1,2-b pyrrol-carbonitriles as a new family of heterocycles: diverse Snar(H)H reactions, cytotoxicity and spectral behavior. Tetrahedron 2005, 61, 11264-11269.
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(23) Guan, H.; Wang, Q. Applications of Organic Probes for Imaging and Analysis of Different Cancer Cell Models. Ph.D. thesis 2015.
(24) Romanelli, E.; Sorbara, C. D.; Nikic, I.; Dagkalis, A.; Misgeld, T.;
Kerschensteiner, M. Cellular, subcellular and functional in vivo labeling of the spinal cord using vital dyes. Nat. Protoc. 2013, 8, 481-490.
(25) Putey, A.; Joucla, L.; Picot, L.; Besson, T.; Joseph, B. Synthesis of latonduine derivatives via intramolecular Heck reaction. Tetrahedron 2007, 63, 867-879.
(26) Gaspari, P.; Banerjee, T.; Malachowski, W. P.; Muller, A. J.; Prendergast, G. C.; DuHadaway, J.; Bennett, S.; Donovan, A. M. Structure-activity study of Brassinin derivatives as indoleamine 2,3-dioxygenase inhibitors. J. Med. Chem. 2006, 49, 684-692.
(27) Prosser, A. R.; Banning, J. E.; Rubina, M.; Rubin, M. Formal Nucleophilic Substitution of Bromocyclopropanes with Amides en route to Conformationally Constrained beta-Amino Acid Derivatives. Org. Lett. 2010, 12, 3968-3971.
(28) Lee, M. H.; Han, J. H.; Lee, J. H.; Choi, H. G.; Kang, C.; Kim, J. S.
Mitochondrial Thioredoxin-Responding Off-On Fluorescent Probe. J. Am. Chem. Soc.
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24 CHAPTER 2
SYNTHESIS AND CHARACTERIZATION OF POLYCAPROLACTONE BASED
POLYMER FOR PROTEIN STABILITY AND ACTIVITY STUDY
2.1 Introduction
Enzyme has been widely used in food, pharmaceutical and biotechnology industry due to its mild reaction conditions, specificity, and efficiency.1 However, some enzymes have the disadvantages with inherent instability after repeated use, easy aggregation, high cost, and difficulty in biocatalyst-product separation, which limit their wide applications.2,3 Recent developments in enzyme engineering by conjugation or coassembly with polymers provided a strategy to improve the enzyme applications with extended use, recyclability, low cost, and easy separation of substrate from enzyme.4-6 This kind of enzyme-polymer nanocomposites has become a viable approach to improve the functionality of enzyme,7 optimize the procedure of separation, and minimize the cost.5 It has been reported that coassembly of protein with nanoparticles can prevent the non-optimal geometry of the enzyme conformational structure.8 This enhances the chances of the access of the substrate to the active site of the immobilized enzyme, which resulted in an enhanced catalytic activity.9 Recently, a number of enzyme/nanoparticle nanocomposites have been studied in terms of their catalytic properties, such as β-galactosidase,10 alkaline phosphatase,11 and glucose oxidase.12
25
Our group has developed stable core-shell protein-polymer nanohybrids with various bio-applications in drug delivery, antigen immunogenicity, and enzyme activity study.4,7,13 We have investigated the nanohydrids for different proteins (virus particle TMV,14 CPMV,6 TYMV,14 ferritin,6 antigen protein,7 and M13 Bacteriophage13). The polymers used in the nanohybrids included poly (4-vinylpyridine) (P4VP), poly (ε-caprolactone)-block-poly(2-vinylpyridine) (PCL-b-P2VP), and pyridine-grafted-poly(ε-caprolactone) (PCL-Py), which contained the pyridine functional group. The interactions between proteins and polymers in the nanocomposites could be van der Waals forces, hydrogen bonding, hydrophobic-hydrophilic interactions, and electrostatic interactions.6 This kind of nanocomposites with a stable core-shell structure could maintain the native structure and functionality of the protein due to the existence of the pyridine groups linked to the polymer chain.
In this work, we systematically investigated the activities of β-glucuronidase (BGus) coassembled with P4VP, PCL-Py homopolymer, and P(CL-g-Py)-ran-PCL random polymer. BGus expressed by Escherichia coli is a member of the glycosidase family 2 of enzymes. It was utilized as a model enzyme, which could hydrolyze β-glucuronides to produce a great variety of valuable chemicals. Amin et al. reported to employ BGus-Ca-alginate beads to convert glycyrrhizin (GL) into glycyrrhetinic acid monoglucuronide (GAMG), which was a superior sweetener and flavoring reagent.15 Jiang et al. reported to immobilize BGus in biomimetic alginate/protamine/silica composites to convert Baicalin into a more active anticancer and antioxidant drug Baicalein, which was expensive due to its lower availability in nature.16 Moschel et al. reported a potent DNA repair inactivator O6-benzylguanine was released from the β-glucuronidase-cleavable
26
prodrugs, which may be applied for a prodrug monotherapy.17 Herein we utilized 4-nitrophenyl β-D-glucuronide (pNPG) as the substrate of BGus to study the activity of free BGus and BGus-polymer nanocomposites. This substrate is commercially-available and its hydrolyzed product p-nitrophenol can be easily monitored by UV-Vis spectrum (405 nm) with color change from colorless to yellow.
In this study, we designed and synthesized PCL based homopolymers PCL-Py and random polymer P(CL-g-Py)-ran-PCL. These two synthetic pyridine containing polymers and a commercially available P4VP co-assembled with BGus to form nanocomposites for the activity and stability study. These polymers formed stable core-shell nanocomposites with BGus via non-covalent interactions. The activity of BGus-polymer nanocomposite was affected by the quality of freshly prepared BGus. Here we used the number of ticks to stand for the relative activity with 3 ticks representing highest activity and 1 tick representing the lowest activity. The nanocomposite formed by freshly prepared BGus with high initial activity showed almost the same activity as free BGus (Scheme 2.1a, b). However, some batches of freshly prepared BGus exhibited low activity, which might result from the expression and purification process orbacteria effect.18 The nanocomposites formed by less active BGus with polymers showed a higher activity than free BGus, which implied the polymer platform might refold the structure of the enzyme (Scheme 2.1c, d).
27
Scheme 2.1. Schematic representation of the assembled process for BGus with P4VP or