Facile synthesis of chlorin bioconjugates by a series of click reactions

Abstract

A multifunctional chlorin platform appended with four short polyethylene glycols and a carboxylate-linker allows rapid conjugation to biotargeting motifs such as proteins and oligonucleotides. The stability and photophysical properties of the chlorin enable development of diagnostics, imaging, molecular tracking, and theranostics.

Figure 1

20% denaturing polyacrylamide gel for A) the O-PEG-chlorin-DNA conjugate, B) the S-PEG-chlorin-DNA conjugate. Gels are stained with Nuclistain to visualize DNA and conjugates: the conjugation reaction mixture and the free 14 nt DNA.

Figure 2

A) SDS-PAGE for conjugation of O-PEG-chlorin to lysozyme in DMSO. Lane 1: molecular weight marker. 2: Free lysozyme. 3: Crude product reaction (1:1) 4: Free SPEG-chlorin. 5: Crude product reaction (1:1). B) SDS-PAGE for conjugation of S-PEG-chlorin to lysozyme in DMSO. Lane 1: molecular weight marker. 2: Free lysozyme. 3: Crude product reaction (1:1) 4: Free SPEG-chlorin. 5: Crude product reaction (1:1). Lanes 1- 3 Visualization of protein after staining with Coomassie blue. Lanes 4-5 Visualization of chlorin fluorescence after excitation at 365 nm.

Figure 3

Enzymatic activity assay of A) lysozyme in DMSO; B) O-PEG-chlorin- lysozyme conjugate in DMSO using Micrococcus lysodeikticus in 66 mM potassium phosphate pH 6.2 as substrate (see ESI).

Scheme 1

Core chlorin platform in green is modified by successive click chemistries to append PEG groups that modulate solubility, and conjugate biomolecules for targeting and tracking. The chelated metal ions modulate the photophysics.

Scheme 2

(i) glycine/paraformaldehyde (1:1), added in four aliquots in chlorobenzene, 145 °C, 8 h, 36% yield. (ii) 5 equiv. succinic anhydride, CH₂Cl₂, NEt₃, 25 °C, overnight, 95% yield. (iii) for HS-PEG, 4.5 equiv. 2-(2-methoxyethoxy)ethane thiol and stoichiometric K₂CO₃ in acetone, 25 °C, 6 h, 85%; for HO-PEG 10 equiv. 2-(2-methoxyethoxy)ethanol and stoichiometric K₂CO₃ in dry acetone, reflux, overnight, 85%. (iv) 1.2 equiv. N-hydroxysuccinimide in dioxane or tetrahydrofuran with 1.2 eq. dicyclohexylcarbodiimide at 25 °C, overnight, quantitative yield. (v) bio-conjugation depends on the specific targeting molecule but is generally accomplished in buffer or DMSO at 25 °C for 2-10 hr. Chlorins were reacted in a 5 μM solution of a 14 nt DNA with an amino terminated tether (10:1 ratio; 5′-NH₂-(CH₂)₆-NH-TTCTTCTCCTTTCT-3′), pH 7.4 phosphate buffer overnight, 37 °C. For the lysozyme (hydrolases) conjugate, a 100 μM solution of 8 was allowed to react with lysozyme in DSMO over night at room temperature and purified by gel filtration on a 5 mL column of Sephadex G-25 with water as eluent.