Method for the Synthesis of γ-PEGylated Folic Acid and Its Fluorescein-Labeled Derivative

Abstract

This paper reports exclusive γ-conjugation of poly­(ethylene glycol) (PEG) to folic acid (FA) and the synthesis of its fluorescein (Fl)-labeled derivative. FA is first reacted with 1 equiv of n-BuLi that forms the metal salt exclusively at the γ-position. Model reactions between γ-lithiated FA and bromo-alcohols were used to demonstrate exclusive γ-conjugation. γ-PEGylated FA was then synthesized by reacting γ-lithiated FA with a bromine-functionalized methoxy-PEG (Br-PEGM) that is prepared by Candida antarctica Lipase B (CALB)-catalyzed transesterification of ethyl 5-bromovalerate (EBV) with methoxy-PEG (HO-PEGM). Fl-labeled γ-PEGylated FA was also synthesized by CALB-catalyzed Michael addition of FA-γ-SH to acrylate-PEG-Fl that was made by a series of CALB-catalyzed transformations. NMR spectroscopy and MALDI-ToF spectrometry verified the structure and purity of the compounds. These lithiation-based methods are superior compared to both the currently used activated ester method that gives a mixture of γ, α, and double-substituted FA and the multistep retrosynthesis methods.

Synthesis of Mono- and Dithiols of Tetraethylene Glycol and Poly(ethylene glycol)s via Enzyme Catalysis

“…PEG 1000 -dithiol was obtained by reacting PEG 1000 -monothiol with fresh CALB and MP-SH for 24 h. PEG 2050 -monothiol was formed in 16 h, and dithiol formation required additional CALB and MP-SH. Based on our data, it can be concluded that enzyme catalyzed transesterification is a convenient and green method to effectively synthesize PEG mono-and di-thiols that are suitable candidates for thiol-ene click reactions and Michael addition type of reactions [4].…”

“…Poly(ethylene glycol) (PEG) is the most frequently used polymer for biomedical research and applications because it is soluble in organic as well as aqueous media [1], is not cytotoxic and immunogenic [2], and is easily excreted from living organisms [3]. Click chemistries and Michael addition reactions are often used for PEGylation of drugs to make them more water soluble [4]. Thiol-functionalized PEGs have an important role in these reactions [5][6][7][8][9][10] and can be used as a 'Michael donor' or in thiol-ene click reactions to synthesize conjugates for targeted drug delivery [7].…”

Folic acid conjugated polymeric drug delivery vehicle for targeted cancer detection in hepatocellular carcinoma

“…A polymeric DDV composed of fluorescein (FL) and exactly two FA attached to a central PEG molecule was synthesized to target overexpressed FRs in neoplastic cells (Puskas et al, ). FA served as a ligand for the overexpressed cell surface FRs, which is internalized through receptor‐mediated endocytosis.…”

“…First, diamine‐terminated poly(ethylene glycol) (H 2 N‐PEG‐NH 2 ) ( M n = 2,000 g/mol) underwent a Michael addition with two moles of fluorescein diacrylate (FLDA) in the presence of Candida antarctica Lipase B (CALB) yielding Acrylate‐FL‐PEG‐FL‐Acrylate ( M n = 2,880 g/mol). Then a second Michael addition was carried out with two moles of thiol‐functionalized folic acid (FA‐γ‐SH) to get the folic acid end groups, as reported (Puskas et al, ). This synthetic methodology allows the replacement of fluorescein with a functional group to which doxorubicin (DOX) can be conjugated: FA‐(DOX)‐PEG‐(DOX)‐FA.…”

“…In this study, we explored the feasibility of using a precision‐synthesized FA‐containing PEG‐based drug delivery vehicle (DDV) using new chemistry for targeted cancer detection with two FAs (Puskas, Castano, Mulay, Dudipala, & Wesdemiotis, ). The increase in FAs in the nanocarrier increases the avidity towards FRs in the cancer cells when compared to molecules containing single FA.…”