Effect of Solvent Type Approaches on Ester Formation in the Synthesis of Folic Acid N-Hydroxy Succinimide Ester

Yıl 2025, Cilt: 15 Sayı: 2, 93 - 111, 31.12.2025

Sümeyye İdil Çelikkaya , Şahin Yıldırım , Ahmet Altun

https://doi.org/10.37094/adyujsci.1661151

https://izlik.org/JA93DB79PG

Öz

Folic acid is a crucial substituent compound for vital functions of cell known as vitamin B9. Folic acid, which is taken into the cell by folic acid receptors, is used for the pH-dependent Michaelis-Menten kinetics of the drug into the cell in diseases where these receptors are overexpressed. Using the ester form of folic acid in targeted treatments is important in terms of folic acid binding to the carrier drug. N-hydroxy succinimide is an agent used to form esters and contributes to the formation of esters from folic acid with Steglich esterification. In many syntheses, dimethyl sulfoxide (DMSO) is used as a solvent and N, N'-dicyclohexylcarbodiimide (DCC) represents a reagent. Although an effective synthesis is carried out with these substances, other analyses should be performed in subsequent studies due to possible impurities. Solvents such as dichloromethane or dimethyl formamide can be more suitable for this reaction as they produce fewer impurities and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) represents a reagent and forms a more simply removable intermediate product. As a result of our research, the presence of excessive amounts of folic acid in the synthesis carried out with dimethyl formamide indicates that the molar ratios of N-hydroxy succinimide and EDC should be increased in subsequent studies. In the synthesis carried out with dichloromethane, ester formation was detected, but the result could not be matched with UV-Vis experiment. In addition, it was observed that the use of EDC did not form intermediate products, and this contributed to the impurity of the reaction. As a result, EDC has played an effective role to get rid of the impurities as intermediate product instead of DCC. Thus, the combination of DCC/EDC in reaction discussed as more efficient reaction purity and yield profile.

Anahtar Kelimeler

Esterification , N-hydroxy succinimide , Folic Acid , 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide. , 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide.

Folik Asit N-Hidroksisüksinimid Ester Sentezinde Farklı Çözücü Tiplerinin Ester Oluşumuna Etkisi

https://izlik.org/JA93DB79PG

Öz

Folik asit, hücrelerin hayati fonksiyonları için önemli bir B9 vitaminidir. Folik asit reseptörleri tarafından hücre içine pH'a bağlı Michaelis-Menten kinetiği ile alınan folik asit, bu reseptörlerin aşırı ifade edildiği hastalıklarda ilacın hücre içine kolay alınabilmesine imkan tanır. Folik asidin ester formunun hedefli tedavilerde kullanılması, folik asidin taşıyıcı ilaca bağlanması açısından önemlidir. N-hidroksi süksinimid, ester oluşturmak için kullanılan bir ajandır ve Steglich esterifikasyonu ile folik asitten ester oluşumuna katkıda bulunur. Birçok sentezde, çözücü olarak dimetil sülfoksit (DMSO) ve reaktif olarak ise N, N'-disiklohekzilkarbodiimid (DCC) kullanılır. Bu maddelerle etkili bir sentez gerçekleştirilmesine rağmen, olası safsızlıklar nedeniyle sonraki çalışmalarda başka analizler yapılmalıdır. Diklorometan veya dimetil formamid gibi çözücüler daha az safsızlık üretmeleri ve 1-Etil-3-(3-dimetilaminopropil) karbodiimid (EDC)’in reaktif olarak daha kolay bir şekilde uzaklaştırılabilen ara ürün oluşturması sebebiyle bu reaksiyon için daha uygun olabilir. Araştırmamız sonucunda dimetil formamid ile yapılan sentezde aşırı miktarda folik asit bulunması, sonraki çalışmalarda N-hidroksi süksinimid ve EDC'nin molar oranlarının artırılması gerektiğini göstermektedir. Diklorometan ile yapılan sentezde ester oluşumu tespit edilmiş ancak sonuç UV-Vis deneyi ile doğrulanamamıştır. Ayrıca EDC kullanımının ara ürün oluşturmadığı ve bunun reaksiyonun safsızlığına katkıda bulunduğu gözlenmiştir. Sonuç olarak EDC, DCC yerine ara ürün olarak safsızlıklardan kurtulmada etkili bir rol oynamıştır. Bu nedenle reaksiyonda DCC/EDC kombinasyonunun daha verimli reaksiyon saflığı oluşturduğu tartışılmıştır.

Anahtar Kelimeler

Esterifikasyon , N-hidroksisüksinimid , Folik Asit , 1-Etil-3-(3-dimetilaminopropil)karbodiimid.

Kaynakça

• [1] Angelopoulou, A., Kolokithas-Ntoukas, A., Fytas, C., Avgoustakis, K., Folic Acid-Functionalized, Condensed Magnetic Nanoparticles for Targeted Delivery of Doxorubicin to Tumor Cancer Cells Overexpressing the Folate Receptor, ACS Omega, 4(26), 22214–27, 2019.
• [2] Bhushan, B., Gopinath, P., Tumor-targeted folate-decorated albumin-stabilised silver nanoparticles induce apoptosis at low concentration in human breast cancer cells, RSC Advances, 5(105), 86242–53, 2015.
• [3] Fernández, M., Javaid, F., Chudasama, V., Advances in targeting the folate receptor in the treatment/imaging of cancers, Chemical Science, 9(4), 790–810, 2018.
• [4] Chen, C., Ke, J., Zhou, X.E., Yi, W., Brunzelle, J.S., Li, J., Yong, E.-L., Xu, H.E., Melcher, K., Structural basis for molecular recognition of folic acid by folate receptors, Nature, 500(7463), 486–9, 2013.
• [5] Siengalewicz, P., Mulzer, J., Rinner, U., 6.09 Synthesis of Esters and Lactones. In: Knochel, P., (Ed.), Comprehensive Organic Synthesis Second Edition, Elsevier, Amsterdam, 355p., 2014.
• [6] Pérez-Carreón, K., Martínez, L.M., Videa, M., Cruz-Angeles, J., Gómez, J., Ramírez, E., Effect of Basic Amino Acids on Folic Acid Solubility, Pharmaceutics, 2544, 15(11), 2023.
• [7] Shulpekova, Y., Nechaev, V., Kardasheva, S., Sedova, A., Kurbatova, A., Bueverova, E., Kopylov, A., Malsagova, K., Dlamini, J.C., Ivashkin, V., The Concept of Folic Acid in Health and Disease, Molecules, 3731, 26(12), 2021.
• [8] Singh, S.K., Singh, R., Surface Plasmon Resonance, a Novel Technique for Sensing Cancer Biomarker: Folate Receptor and Nanoparticles Interface., Cancer Biomarkers: Methods and Protocols, Springer US, 2413, 211–28, 2022.
• [9] Alexander, C.M., Hamner, K.L., Maye, M.M., Dabrowiak, J.C., Multifunctional DNA-gold nanoparticles for targeted doxorubicin delivery, Bioconjugate Chemistry, 25(7), 1261–71, 2014.
• [10] Figliola, C., Marchal, E., Groves, B.R., Thompson, A., A step-wise synthetic approach is necessary to access γ-conjugates of folate: folate-conjugated prodigiosenes, RSC Advances, 9(25), 14078–92, 2019.
• [11] Atkinson, S.F., Bettinger, T., Seymour, L.W., Behr, J.-P., Ward, C.M., Conjugation of Folate via Gelonin Carbohydrate Residues Retains Ribosomal-inactivating Properties of the Toxin and Permits Targeting to Folate Receptor Positive Cells, Journal of Biological Chemistry, 276(30), 27930–5, 2001.
• [12] Hermanson, Greg T. Bioconjugate techniques. Academic press, 3 ed., 1146p. 2013.
• [13] Jordan, A., Whymark, K.D., Sydenham, J., Sneddon, H.F., A solvent-reagent selection guide for Steglich-type esterification of carboxylic acids, Green Chemistry, 23(17), 6405–13, 2021.
• [14] Grumezescu, Alexandru Mihai, ed. Organic materials as smart nanocarriers for drug delivery. William Andrew, 738p. 2018.
• [15] Clark, T., Murray, J.S., Lane, P., Politzer, P., Why are dimethyl sulfoxide and dimethyl sulfone such good solvents?, Journal of Molecular Modeling, 14(8), 689–97, 2008.
• [16] Ha, N.T.N., Gopakumar, T.G., Yen, N.D.C., Mende, C., Smykalla, L., Schlesinger, M., Buschbeck, R., Rüffer, T., Lang, H., Mehring, M., Hietschold, M., Ester formation at the liquid–solid interface, Beilstein Journal of Nanotechnology, 8, 2139–50, 2017.
• [17] Parekh, V. J., V. K. Rathod, and A. B. Pandit. Substrate Hydrolysis: Methods, Mechanism, and Industrial Applications. Academic Press, 2 ed., 655p. 2011.
• [18] Wang, R., Chen, C., Yang, W., Zhou, P., Zhu, F., Xu, H., Hu, G., Sun, W., Shen, W., Hu, Y., Solubility determination and thermodynamic characterization of orotic acid in twelve pure solvents and four binary mixed solvents, Journal of Molecular Liquids, 341, 117335, 2021.
• [19] Yang, N., Dichloromethane. In: Wexler, P., (Ed.), Encyclopedia of Toxicology Third Edition, Academic Press, 3 ed., 5220p. 2005.
• [20] Klykov, O., Weller, M.G., Quantification of N-hydroxysuccinimide and N-hydroxysulfosuccinimide by hydrophilic interaction chromatography (HILIC), Anal. Methods, 7(15), 6443–8, 2015.
• [21] Pottorf, R.S., Szeto, P., Srinivasarao, M., 1-Ethyl-3-(3′-dimethylaminopropyl)carbodiimide Hydrochloride, Encyclopedia of Reagents for Organic Synthesis, John Wiley & Sons, Ltd, 1–5, 2017.
• [22] Wang, R., Chen, C., Yang, W., Zhou, P., Zhu, F., Xu, H., Hu, G., Sun, W., Shen, W., Hu, Y., Solubility determination and thermodynamic characterization of orotic acid in twelve pure solvents and four binary mixed solvents, Journal of Molecular Liquids, 341, 117335, 2021.
• [23] Nam, C.T., Yang, W.-D., Duc, L.M., Solvothermal Synthesis of TiO2 Photocatalysts in Ketone Solvents with Low Boiling Points, Journal of Nanomaterials, 2013(1), 627385, 2013.
• [24] Wilkinson, T.J., Recycling Solvent Mixtures of Ethyl Acetate and Hexanes, Journal of Chemical Education, 75(12), 1640, 1998.
• [25] Oluwasanmi, A., Lindsay, S., Curtis, A., Perrie, Y., Hoskins, C., Chain length impact on the retro Diels-Alder mediated release of gemcitabine from hybrid nanoparticles towards pancreatic cancer therapy, International Journal of Pharmaceutics, 644, 123304, 2023.
• [26] Becker, E.D., High Resolution NMR Third Edition, Chapter 4-Chemical Shifts, Academic Press, 3 ed., 441p. 2000.
• [27] Lee, Y., Matviychuk, Y., Holland, D.J., Quantitative analysis using external standards with a benchtop NMR spectrometer, Journal of Magnetic Resonance, 320, 106826, 2020.
• [28] Pauli, G.F., Jaki, B.U., Lankin, D.C., A routine experimental protocol for qHNMR illustrated with taxol, Journal of Natural Products, 70(4), 589–95, 2007.
• [29] Pinciroli, V., Biancardi, R., Colombo, N., Colombo, M., Rizzo, V., Characterization of small combinatorial chemistry libraries by 1H NMR. Quantitation with a convenient and novel internal standard, Journal of Combinatorial Chemistry, 3(5), 434–40, 2001.
• [30] Bai, M.-Y., Tang, S.-L., Chuang, M.-H., Wang, T.-Y., Hong, P., Evaluation of chitosan derivative microparticles encapsulating superparamagnetic iron oxide and doxorubicin as a pH-sensitive delivery carrier in hepatic carcinoma treatment: an in vitro comparison study, Frontiers in Pharmacology, 9, 1025, 2018.
• [31] Castillo, J.J., Torres, M.H., Molina, D.R., Castillo-León, J., Svendsen, W.E., Escobar, P., Martínez, F., Monitoring the functionalization of single-walled carbon nanotubes with chitosan and folic acid by two-dimensional diffusion-ordered NMR spectroscopy, Carbon, 50(8), 2691–7, 2012.
• [32] Motsch, S., Tremmel, P., Richert, C., Regioselective formation of RNA strands in the absence of magnesium ions, Nucleic Acids Research, 48(3), 1097–107, 2020.
• [33] Budavari, S., O’Neil, M.J., Smith, A., Heckelman, P.E., The merck index. vol. 11, Merck & Company Incorporated, 938p. 1989.
• [34] Bai, M.-Y., Tang, S.-L., Chuang, M.-H., Wang, T.-Y., Hong, P., Evaluation of chitosan derivative microparticles encapsulating superparamagnetic iron oxide and doxorubicin as a pH-sensitive delivery carrier in hepatic carcinoma treatment: an in vitro comparison study, Frontiers in Pharmacology, 9, 1025, 2018.
• [35] Zu, Y., Zhao, Q., Zhao, X., Zu, S., Meng, L., Process optimization for the preparation of oligomycin-loaded folate-conjugated chitosan nanoparticles as a tumor-targeted drug delivery system using a two-level factorial design method, International Journal of Nanomedicine, 3429–41, 2011.
• [36] Jain, S., Rathi, V. V., Jain, A.K., Das, M., Godugu, C., Folate-decorated PLGA nanoparticles as a rationally designed vehicle for the oral delivery of insulin, Nanomedicine, 7(9), 1311–37, 2012.