|Nurul Aina Jamaludin||Universiti Kuala Lumpur- Branch Campus Malaysian Institute of Chemical and Bioengineering Technology, Lot1988, Kawasan Perindustrian Bandar Vendor, Taboh Naning, 78000, Alor Gajah, Melaka, Malaysia|
|Noor Faizah Che Harun||Universiti Kuala Lumpur- Branch Campus Malaysian Institute of Chemical and Bioengineering Technology, Lot1988, Kawasan Perindustrian Bandar Vendor, Taboh Naning, 78000, Alor Gajah, Melaka, Malaysia|
6-Hydroxyflavone (6HF) contains attractive biological properties of significance pharmacologically and has been discovered as an effective diabetic medication. However, due to its high degradation in aqueous solution, its efficacy in biological treatment remains a considerable obstacle. Thus, conjugating a polymer, polyethylene-glycol, to 6HF by direct esterification between the carboxyl group of PEG and the hydroxyl group at the sixth carbon of the 6HF biomolecule is one of the approaches applied in this research to increase its stability while maintaining the inherent biological characteristics. This study examined the optimum esterification reaction conditions for conjugate PEG-6HF utilizing EDC and DMAP as conjugation reagents in various solvents, such as DMSO, PBS, and PBS 10 mM HEPES pH7.4 with the assistance of HOBt, including its stability in the biomimicking environment. For this purpose, PEG-6HF connected through the ester bond was validated using various analytical techniques such as FTIR, UV-Vis spectroscopy, and HPLC. , Notably, esterification at 25 °C for 24 hours in a 10 mM HEPES pH 7.4 buffer solution using EDC with HOBt resulted in the most significant conjugation percentage, 42 percent. Furthermore, PEG-6HF revealed 1.3 times lower degradation percentages of 6HF biomolecules than unconjugated-6HF following 6 hours of incubation in 10 mM HEPES pH 7.4 at 37 °C. Hence, the optimal conditions and the resulting conjugation percentage with high stability are expected to be a fundamental approach to conjugated polymer with a biomolecule.
Conjugation; Esterification; 6-Hydroxyflavone; PEG-6HF; Stability
Recently, a breakthrough has emerged in polymer
chemistry development by applying a fundamental theory of polymer phase
behavior at site-specific modification of peptides, synthetic biology, and
single-chain polymer behavior (Shu et al., 2013; Dey et al., 2015; Krisanti et al., 2020).
However, there have been slight changes in obtaining successful selective
molecular transport, hierarchical structure control, modulated responsiveness
to small perturbation, and long-term enzymatic activity (Shu et al., 2013).
Therefore, researchers have agreed that polymer-biomolecule conjugate would
improve this limitation. Currently, few conjugation methods have been discovered,
for example, click chemistry, amidation, thiol-maleimide, and esterification (Shimokawa et al., 2009; Liechty et al., 2010; Shu et al., 2013; Che-Harun et al., 2016).
Nevertheless, esterification is seen as a better alternative due to its simplicity,
eliminating the need for further biomolecule modification. Therefore, polymer-biomolecule conjugates
formed by direct covalent conjugation through the esterification process are a
new class of soft materials since each component is complementary (Shu
et al., 2013; Dey et al., 2015).
6-Hydroxyflavone (6HF) is a group of naturally derived bioactive polyphenolic compounds that possess tremendous medicinal assets that have potential roles in preventing chronic diseases, including effectiveness against some neurological disorders paraplegia or sciatica (Wang et al., 2021). 6HF has pharmacologically significant biological characteristics such as neuroprotection, antimicrobial, anti-inflammatory, anticancer, and antioxidants and has been discovered to be an effective treatment for diabetic patients against glomerulonephritis and glomerulosclerosis (Iwakiri et al., 2013; Wang et al., 2015; Das et al., 2018; Das et al., 2019; Stompor et al., 2019; Wang et al., 2021). Stompor et al. (2019) and Mikell et al. (2015) revealed that the hydroxyl group or the propionyl group located in the A ring of the flavones at the C-6 positions has an inhibitory effect on hormone production in the process of steroidogenesis and has cytotoxic solid and apoptotic activities against cancer cell (Iwakiri et al., 2013; Mikell et al., 2015; Wang et al., 2015; Stompor et al., 2019). A hydroxyl group at the sixth carbon in the 6HF compound chemical structure makes 6HF easily esterifiable. Even though much research focuses on intramolecular chemical changes of 6HF for certain specific applications, the stability of 6HF and its derivatives in an aqueous solution remains a big challenge.
Previously, Bayard et al. (2013) reported the successful conjugation between PEG with low molecular weight hydrophobic biomolecules, including hormones and antioxidants, through esterification leads to excellent pharmacokinetic properties of the drug. The objective of developing PEG conjugates was to enhance water solubility and stability while also lessening clearance through the kidney, which prolonged circulation in the bloodstream and increased the drug molecule's biocompatibility (Bayard et al., 2013; Hamley, 2014; Cui et al., 2021). Additionally, PEG has good solubility and stability, increasing membrane permeability and enhancing oil recovery (Febriasari et al., 2021; Irawan et al., 2017). Furthermore, PEG is appropriate for biological applications due to its biological inertness and low toxicity (Rashmi et al., 2020; Turecek & Siekmann, 2019). Moreover, there has been no fact-finding discussion on the conjugation of a 6HF biomolecule with a polymer unit to the authors' knowledge. Thus, researchers were inspired to direct conjugate a molecule of 6HF with a hydrophilic biodegradable polymer, polyethylene glycol (PEG), to improve 6HF stability. Researchers investigated the optimum esterification conditions of a PEG having a carboxyl functional end-group with a 6HF molecule with an active hydroxyl functional group using various solvents such as 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), dimethyl sulfoxide (DMSO), and phosphate buffer saline (PBS) to expand the compatibility of the conjugation process and evaluated PEG—6HF conjugates stability in the biomimicking environment. Figure 1 depicts the general route for the conjugation of PEG-6HF. The produced PEG-6HF conjugates were characterized by their physicochemical properties by RP-HPLC analysis, FTIR, and UV-Vis analysis. The results revealed that the conjugation of PEG-6HF in 10 mM HEPES with pH 7.4 buffer solvent with the assistance of additive peptide coupling, HoBt, demonstrated the highest percentage of conjugation. This is the first research to develop a conjugation between a 6HF biomolecule with a unit of polymer and the conjugate enhanced 6HF stability in a biological environment. Moreover, this research analyzes the optimum conjugation conditions of the PEG and 6HF through esterification, which may aid in understanding the new molecular construction, which is expected to be a new methodology for material chemistry research to provide crucial information for future bioorganic and medicinal chemistry studies.
Figure 1 Synthetic scheme of PEG-6HF
In conclusion, a successful conjugation with a high percentage yield (42 percent) between polyethylene glycol (PEG) and 6-Hydroxyflavone (6HF) synthesized by direct esterification in 10 mM HEPES with pH 7.4 at 25°C was observed in comparison to the PEG-6HF conjugate that prepared in DMSO (11 percent). Moreover, the PEG-6HF conjugate significantly improved its stability, which was 20 percent more stable than unconjugated-6HF due to 1.3 times lower degradation in physiological conditions for 6 hours. The antimicrobial activity of PEG-6HF conjugate is currently being studied. Hence, this new conjugation can be a fundamental methodology for material chemistry field study and provide critical information for future bioorganic and medicinal chemistry studies.
The authors thank the Ministry of Higher Education (MOHE) Malaysia for the financial support provided via the Fundamental Research Grant Scheme [ref no. FRGS/1/2018/STG05/UNIKL/02/5]. The authors also thank Universiti Kuala Lumpur Malaysian Institute of Chemical and Bioengineering Technology, Melaka, Malaysia, for providing resources and necessary facilities for FTIR, UV-Vis Spectrophotometer, and HPLC analyses.
Bayard, F.J.C., Thielemans, W., Pritchard, D.I., Paine, S.W., Young, S.S., Bäckman, P., Ewing, P., Bosquillon, C., 2013. Polyethylene Glycol-Drug Ester Conjugates for Prolonged Retention of Small Inhaled Drugs in the Lung. Journal of Controlled Release, Volume 171(2), pp. 234–240
Charoensit, P., Pompimon, W., Khorana, N., Sungthongjeen, S., 2019. Effect of Amide Linkage of PEG-Lipid Conjugates on the Stability and Cytotoxic Activity of Goniodiol Loaded in PEGylated Liposomes. Journal of Drug Delivery Science and Technology, Volume 50, pp. 1–8
Che-Harun, N.F., Takemoto, H., Nomoto, T., Tomoda, K., Matsui, M., Nishiyama, N., 2016. Artificial Control of Gene Silencing Activity Based on siRNA Conjugation with Polymeric Molecule Having Coil-Globule Transition Behavior. Bioconjugate Chemistry, Volume 27(9), pp. 1961–1964
Cui, Z., Luo, Q., Bannon, M.S., Gray, V.P., Bloom, T.G., Clore, M.F., Hughes, M.A., Crawford, M.A., Letteri, R.A., 2021. Molecular Engineering of Antimicrobial Peptide (AMP)-Polymer Conjugates. Biomaterials Science, Volume 9(15), pp. 5069–5091
Das, S., Karn, A., Sarmah, R., Rohman, M.A., Koley, S., Ghosh, P., Roy, A.S., 2018. Characterization of Non-Covalent Binding of 6-Hydroxyflavone and 5,7-Hydroxyflavone with Bovine Hemoglobin: Multi-Spectroscopic and Molecular Docking Analyses. Journal of Photochemistry and Photobiology B: Biology, Volume 178, pp. 40–52
Das, S., Santra, S., Rohman, M.A., Ray, M., Jana, M., Roy, A. S., 2019. An Insight into the Binding of 6-Hydroxyflavone with Hen Egg-White Lysozyme: A Combined Approach of Multi-Spectroscopic and Computational Studies. International Journal of Biomolecular Structure and Dynamics, Volume 37 (15), pp. 4019–4034
Dey, S., Ambattu, L.A., Hari, P.R., Rekha, M.R., Sreenivasan, K., 2015. Glutathione-Bearing Fluorescent Polymer-Curcumin Conjugate Enables Simultaneous Drug Delivery and Label-Free Cellular Imaging. Polymer, Volume 75, pp. 25–33
Esmaeili, Y., Bidram, E., Zarrabi, A., Amini, A., Cheng, C., 2020. Graphene Oxide and its Derivatives as Promising In-Vitro Bio-Imaging Platforms. Scientific Reports, Volume 10(1), pp. 1–13
Febriasari, A., Suhartini, M., Yunus, A.L., Rahmawati, R., Sudirman, S., Hotimah, B., Hermana, R.F., Kartohardjono, S., Fahira, A., Permatasari, I.P., 2021. Gamma Irradiation of Cellulose Acetate-Polyethylene Glycol 400 Composite Membrane and Its Performance Test for Gas Separation. International Journal of Technology, Volume 12(6), pp. 1198–1206
Hamley, I.W., 2014. PEG-Peptide Conjugates. Biomacromolecules, Volume 15(5), pp. 1543–1559
Irawan, Y., Juliana, I., Adilina, I.B., Alli, Y. F., 2017. Aqueous Stability Studies of Polyethylene Glycol and Oleic Acid-Based Anionic Surfactants for Application in Enhanced oil Recovery through Dynamic Light Scattering. International Journal of Technology, Volume 8(8), pp. 1414–1421
Iwakiri, T., Mase, S., Murakami, T., Matsumoto, M., Hamada, H., Nakayama, T., Ozaki, S.I., 2013. Glucosylation of Hydroxyflavones by Glucosyltransferases from Phytolacca Americana. Journal of Molecular Catalysis B: Enzymatic, Volume 90, pp. 61–65
Krisanti, E.A., Lazuardi, D., Kiresya, K.K., Mulia, K.,2020. Tablet Formulation Containing Chitosan-Alginate Microparticles: Characterization and Release Profile of Xanthones. International Journal of Technology, Volume 11(5), pp. 900–909
Liechty, W.B., Kryscio, D.R., Slaughter, B.V., Peppas, N.A., 2010. Polymers for Drug Delivery Systems. Annual Review of Chemical and Biomolecular Engineering, Volume 1(1), pp. 149–173
Liu, Y., Yang, L., Guo, Y., Zhang, T., Qiao, X., Wang, J., Xu, J., Xue, C., 2020. Hydrophilic Astaxanthin: PEGylated Astaxanthin Fights Diabetes by Enhancing the Solubility and Oral Absorbability. Journal of Agricultural and Food Chemistry, Volume 68(11), pp. 3649–3655
Mikell, J.R., Herath, W., Khan, I.A., 2015. Eleven Microbial Metabolites of 6-Hydroxyflavanone. Chemical and Pharmaceutical Bulletin, Volume 63(8), pp. 579–583
Rashmi, Zabihi, F., Singh, A.K., Achazi, K., Schade, B., Hedtrich, S., Haag, R., Sharma, S.K. , 2020. Non-Ionic PEG-Oligoglycerol Dendron Conjugated Nano-Carriers for Dermal Drug Delivery. International Journal of Pharmaceutics, Volume 580, p. 119212
Shimokawa, K., Yamada, K., Ohno, O., Oba, Y., Uemura, D., 2009. Design, Synthesis, and Biological Evaluation of Biotin-Labeled (-)-Ternatin, a Potent Fat-Accumulation Inhibitor Against 3T3-L1 Adipocytes. Bioorganic and Medicinal Chemistry Letters, Volume 19(1), pp. 92–95
Shu, J.Y., Panganiban, B., Xu, T., 2013. Peptide-Polymer Conjugates: From Fundamental Science to Application. Annual Review of Physical Chemistry, Volume 64(1), pp. 631–657
Stompor, M., Switalska, M., Bajek, A., Wietrzyk, J., 2019. Influence of Amide Versus Ester Linkages on the Anticancer Properties of the New Flavone-Biotin Conjugates. Zeitschrift Fur Naturforschung - Section C Journal of Biosciences, Volume 74(7–8), pp. 193–200
Ta-Aithuak, S., Loedsapchinda, N., Houngkamhang, N., 2020. Conjugation of Antibody on Gold Nanoparticles for Biosensors Application. Key Engineering Materials, Volume 853 KEM, pp. 92–96.
Turecek, P.L., Siekmann, J., 2019. PEG-Protein Conjugates: Nonclinical and Clinical Toxicity Considerations. In: Polymer-Protein Conjugates, Pegylation and Beyond, Elsevier B.V., pp. 61–101
Wang, X., Cao, Y., Chen, S., Lin, J., Bian, J., Huang, D., 2021. Anti-Inflammation Activity of Flavones and Their Structure-Activity Relationship. Journal of Agricultural and Food Chemistry, Volume 69(26), pp. 7285–7302
Wang, X., Wang, Z., Sidhu, P.S., Desai, U.R., Zhou, Q., 2015. 6-Hydroxyflavone and Derivatives Exhibit Potent Anti-Inflammatory Activity Among Mono-, Di- and Polyhydroxylated Flavones in Kidney Mesangial Cells. PLoS ONE, Volume 10(3), pp. 1–11