Photogenerated lectin sensors produced by thiol-ene/yne photo-click chemistry in aqueous solution

Abstract

The photoinitiated radical reactions between thiols and alkenes/alkynes (thiol-ene and thiol-yne chemistry) have been applied to a functionalization methodology to produce carbohydrate-presenting surfaces for analyses of biomolecular interactions. Polymer-coated quartz surfaces were functionalized with alkenes or alkynes in a straightforward photochemical procedure utilizing perfluorophenylazide (PFPA) chemistry. The alkene/alkyne surfaces were subsequently allowed to react with carbohydrate thiols in water under UV-irradiation. The reaction can be carried out in a drop of water directly on the surface without photoinitiator, and any disulfide side products were easily washed away after the functionalization process. The resulting carbohydrate-presenting surfaces were evaluated in real-time studies of protein-carbohydrate interactions using a quartz crystal microbalance (QCM) flow-through system with recurring injections of selected lectins, with intermediate regeneration steps using low pH buffer. The resulting methodology proved fast, efficient and scalable to high-throughput analysis formats, and the produced surfaces showed significant protein binding with expected selectivities of the lectins used in the study.

Figure 1

General surface functionalization procedure. The polymeric surfaces were spincoated with a solution of compound 15 and irradiated with UV-light (254 nm) for 5 min. The NHS-activated ester surfaces were linked through an amidation process with amine 14 or 16 to produce the alkene/alkyne functionalized surfaces. The alkene/alkyne surfaces were then differentiated with thiol-functionalized molecules (4, 8, 17, 18 or 19) through radical photo-click chemistry.

Figure 2

¹H-NMR-spectra of model thiol-ene and thiol-yne reactions in water; a) 2-mercaptoethanol; b) allyl alcohol; c) thiol-ene reaction mixture (1:1 ratio) after 30 min UV-irradiation at 350 nm; d) propargyl alcohol; e) thiol-yne reaction mixture (1:2.1 alkyne:thiol) after 2 h UV-irradiation at 350 nm.

Figure 3

Illustration of thiol-ene/-yne reactions performed directly on alkene/alkyne functionalized quartz crystals in a drop of pure water.

Figure 4

Corrected lectin binding to all functionalized surfaces. Negative values are due to slightly higher binding to control surfaces. Error bars represent the standard errors of the mean (SEM) of triplicate injections of lectin at the same concentration (1 µM).

Figure 5

Illustration of referenced binding curves of triplicate injections of Con A and RCA-I to mannose-/galactose-surfaces on a) alkyne-based surfaces and b) alkene-based surfaces.

Scheme 1

Synthesis of thio-functionalized carbohydrates and alkene-linker for photo-click immobilization; a) 2- bromoethanol, BF₃·Et₂O, DCM, 0 °C, 23 h; b) KSAc, DMF, 45 °C, 17 h (72 %); c) NaOMe, MeOH, rt, 3 h (quant.); d) 2-bromoethanol, BF₃·Et₂O, DCM, −40 °C, 22 h (67 %); e) KSAc, DMF, 45 °C, 7 h (56 %); f) NaOMe, MeOH, rt, 4 h (70 %); g) NaOH, DMF, 45 °C, 2 h (30 %); h) TsCl + KOH, DCM, 0 °C, 5 h (quant.); i) NaN₃ + TBAI, DMF, 45 °C, 22 h (77 %); j) PPh₃, THF + H₂O, 0 °C, 24 h (32 %).