Functionalization and Synthesis of Difunctional Folate-targeted Polymeric Conjugates for Potential Diagnostic Applications

The aim of this research was to synthesize polymer-diagnostic agent conjugates with two folate functionalities for potential diagnostic applications. Conjugates with fluorescein (FL) as an imaging agent, poly(ethylene glycol) (PEG) as a hydrophilic linker and two folic acid (FA) as targeting agents were synthesized by chemo-enzymatic method using Candida antarctica lipase B (CALB) catalyst for the multivalent targeting of folate receptors (FRs) overexpressed on cancer cells. In this dissertation work, imaging agent FL was first acrylated using acryloyl chloride (AcrCl) in the presence of triethylamine (TEA) to precisely synthesize fluorescein o-acrylate (FL-A, yield: 52.49%) and fluorescein o,o’-diacrylate (FL-DA, yield: 63.9%). A kinetic study of FL-DA synthesis was conducted using a Nuclear Magnetic Resonance (NMR)-750 MHz spectrophotometer instrument which demonstrated the formation FL-DA in 13 seconds reaction time. Hence, our FL-DA synthesis was extremely fast and easy to purify using silica gel. Acrylate moieties of FL-DA and FL-A allow the CALB-catalyzed Michael addition of thiol and amine to develop the conjugates.

First, FL-A with single acrylate moiety was reacted with PEG-diamine (H₂N-PEG-NH₂) by the CALB-catalyzed Michael addition. However, the nucleophilic secondary amine of FL-NH-PEG-NH-FL interfered with the acrylation of 'OH' of FL-A. Hence, a new synthetic strategy was developed where H₂N-PEG-NH₂ was replaced by dithiol-functionalized PEG (HS-PEG-SH, M_n =899 g/mol, Ð=1.00, M_n =1160 g/mol, Ð=1.14 and M_n =2200 g/mol, Ð=1.09) and tetraethylene glycol (HS-TEG-SH, FW= 370.48 g/mol) which were synthesized in Dr. Puskas' lab to avoid the interference of the amine group in the acrylation reaction. Michael addition between FL-A and HS-TEG-SH, by CALB-catalysis was extremely fast and completed in 1 minute at 52°C. Reaction between FL-A and HS-PEG-SH without CALB catalysis did not go to completion even after 18 hours at 52°C but completed in 2 minutes when CALB was added. CALB catalysis was found to be extremely useful to synthesize FL-TEG-FL and FL-PEG-FL compounds. Further acrylation of the 'OH' of FL-A, followed by the attachment of FA-SH by the Michael addition resulted in successful development of the difunctional FL-PEG conjugate with different molecular weights. PEGylation of FL using brominated PEG was also attempted by lithium chemistry to develop the FL-PEG-FL conjugate which can be further acrylated for the attachment of FA-SH. However, due to the presence of impurities during the PEGylation, no further reactions were performed.

Next, tetrafunctional FL compounds were synthesized by the aza-Michael addition reaction between FL-DA and secondary amines by studying the effect of CALB-catalysis. These multifunctional FL compounds can be used as precursors to develop polymeric conjugates. The aza-Michael addition reactions of small molecules, such as diethanolamine (DEA) and diallylamine (DAA) to FL-DA were completed in 1 minute at room temperature without CALB-catalysis in dimethyl sulfoxide (DMSO), while the reactions were extremely slow in chloroform. CALB catalysis was found to be extremely useful with large sterically hindered molecules, such as diethyl iminodiacetate (DIDA) as the reaction time was three times less than the reaction without CALB catalysis. The aza-Michael addition product of DEA with FL-DA (tetrahydroxy-functionalized FL) was highly unstable, it hydrolyzed back to FL due to neighboring group (OH) participation. Hence to avoid the hydrolysis, new dihydroxy-functionalized secondary amines with longer alkyl chains were synthesized by ultraviolet (UV)-mediated thiol-ene click reaction between DAA and 4-mercapto-1-butanol and with 9-mercapto-1-nonanol. CALB-catalyzed Michael addition of these newly synthesized dihydroxy secondary amines to FL-DA resulted in successful synthesis of tetrahydroxy functionalized FL.

Finally, a scale-up of FL-DA synthesis is discussed in this dissertation, where FL-DA was successfully scaled up in a 5-liter capacity jacketed reactor with overhead stirrer and a pure product with 54.32% yield was obtained. The cost to synthesize 1 g of FL-DA in the lab was 41.29 USD.

The structures of all products were confirmed by Proton NMR (¹H-NMR), Carbon-13 NMR (¹³C-NMR) and Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-ToF MS).