Photoactivated Coumaryl-diazopyruvate Fluorescent Label for Amine Functional Groups of Tissues Containing Type-I Collagen¶

Abstract

The design, synthesis and application of a new fluorescent-labeling reagent for collagen has been developed as a prerequisite for the design of a photoactivated collagen-crosslinking compound for surgical wound closure. The amine groups in collagen are the targets of a rational design for a new fluorophore because natural collagen crosslinks are formed between primary (1°) amine groups of lysine and hydroxylysine. The availability of 1° amines for crosslinking in native collagenous tissues was evaluated by reacting tendon and corneal samples with o-phthalaldehyde and dansyl chloride, fluorophores commonly used for the detection of 1° and 2° amines. The resulting fluorescent collagen fibrils indicated the presence of amines in native tissue. Subsequently, a photoactivated fluorescent label for 1° and 2° amines, coumaryl gamma-amino-butyric acid diazopyruvate (CGDP), was designed and synthesized. CGDP was first used to photolabel poly-l-lysine, forming a fluorescent, covalent bond to the 1° amine. CGDP was then photoreacted with corneal and tendon tissue samples to produce CGDP fluorescent–labeled samples that were statistically significantly more fluorescent than were the controls. These experiments support the postulate that 1° or 2° (or both) amines in native collagenous tissues are available to serve as targets for photoactivated collagen crosslinkers for wound closure.