TCEP-Enabled Click Modification of Glycidyl-Bearing Polymers with Biorelevant Sulfhydryl Molecules: Toward Chemoselective Bioconjugation Strategies

Abstract

Thiol-epoxy ring opening is a highly efficient and versatile click reaction for postpolymerization modification, ideal for the conjugation of sulfhydryl-containing biomolecules. This study investigated the reactivity of thiols, disulfides, and amines toward glycidyl-bearing polymers, aiming to optimize thiol conjugation using tris(2-carboxyethyl)phosphine (TCEP) as a disulfide-reducing agent. Epoxide groups were introduced via glycidyl methacrylate (GMA) polymerized by ATRP to yield PGMA homopolymers and poly(ε-caprolactone) (PCL)-based block copolymers. ¹H NMR confirmed quantitative thiol functionalization, while amines showed poor reactivity. l-cysteine conjugation further demonstrated the reaction's chemoselectivity. Thioglycerol conjugation yielded poly(2-hydroxy-3-(thioglycerol)propyl methacrylate) (PTGMA), a highly hydroxylated PEG alternative. Functionalization was extended to PCL-b-PGMA and PEGMA-based copolymers, forming amphiphilic nanoparticles via nanoprecipitation. Sequential modification with thioglycerol and the cRGD peptide yielded bioactive, size-controlled nanocarriers. Overall, a robust strategy has emerged for synthesizing multifunctional polymeric nanomaterials. Its compatibility with equimolar reactants under ambient conditions makes it particularly suited for the efficient incorporation of sensitive, high-value biomolecules into targeted drug delivery systems.

1

Comparative analysis of reaction pathways for glycidyl modification via click ring-opening reactions with thiols, disulfides, and amines. While thiols and disulfides induce efficient functionalization, amines do not react effectively under the same mild conditions.

2

(a) Macromolecular structures of the PGMA-containing polymers tested for postpolymerization modification with thiols, disulfides, and amines. (b) List and classification of the reactive molecules and their molecular structures tested as modifying agents for the glycidyl ring-opening reaction.

3

Comparison of ¹H NMR spectra (in DMSO-d ₆) recorded: before PGMA_n modification; after the oxirane opening reaction carried out with thioacetic acid at 25 °C (epoxy ring/thioacetic acid/TEA = 1/1/3) (A); after the epoxy ring-opening reaction performed with thioglycerol at 25 °C (epoxy ring/thioglycerol/TEA = 1/1/3) (B).

4

a) ¹H NMR spectra (in DMSO-d₆) of PGMA_n treated with diphenyl disulfide in the presence of TEA at 25 °C (epoxy ring/TEA = 1/1), when reduced by TCEP. b)Kinetic profile of the functionalization reaction occurring in DMF (squares) and THF (circles), in the presence of TCEP (orange) or not (blue), highlighting the inability to functionalize PGMA with diphenyl disulfide without a reducing agent.

5

¹H NMR spectra (in DMSO-d ₆ and in D₂O) of PGMA_n reacted with l-cysteine in the presence of TEA at 25 °C (epoxy ring/l-cysteine/TEA = 1/1/1) for 18 h, formerly treating l-cysteine with TCEP to reduce possible S–S bonds present in the system. ¹H NMR spectra (in DMSO-d ₆) of cysteine-functionalized PGMA_n were acetylated with an excess of acetyl chloride to induce the formation of an amide and an ester group.

6

SEC chromatograms of native PGMA₃₀ and of the modified polymer with thioglycerol and l-cysteine.

7

¹H NMR spectra (in DMSO-d ₆) of PCL_r-b-[P­(PEGMA)_m-co-PGMA_n] reacted with (A) thiophenol in the presence of TEA at 25 °C (epoxy ring/thiophenol/TEA = 1/1/3) for 18 h (PGMA_n represents 10% of the P­(PEGMA)_m-co-PGMA_n block (n = 3, m = 29)); (B) cRGD peptide in the presence of TEA at 25 °C (epoxy ring/cRGD/TEA = 1/1/3) for 18 h (PGMA_n represents 20% of the P­(PEGMA)_m-co-PGMA_n block (n = 6, m = 29)).

8

PCL_r-b-PGMA_n modification with thioglycerol and cRGD peptide in two steps (18 h each, 25 °C) where TEA was inserted in excess at the first stage and not in the second one (epoxy ring/thioglycerol/cRGD/TEA = 1/0.9/0.1/3; p = 0.9·n and q = 0.1·n). Amphiphilic and biologically active molecules were obtained and able to produce core–shell micelles in aqueous suspension (10 mg/mL in PBS 10 mM, pH 7.4). Size distribution curve, Z-average size, and PdI were evaluated by DLS analysis (top). In the bottom, ¹H NMR spectra (in DMSO-d ₆) of the resulting macromolecule.