A photoactivated diazopyruvoyl cross-linking agent for bonding tissue containing type-I collagen.
On the basis of the earlier examples of diazopyruvoyl (DAP) groups reported by Lawton for covalent binding and cross-linking of proteins and oligopeptides and our recent demonstration that a coumaryl diazopyruvamide was used to label Type-I collagen, we have extended our investigations to the synthesis and cross-linking capabilities of a bis-DAP polyethylene glycol to cross-link Type-I collagen. The new photoactivated cross-linking agent, N,N'-bis(3-diazopyruvoyl)-2,2'-(ethylenedioxy)bis(ethylamine) (DPD, 2), has been designed and synthesized specifically to "weld" collagenous tissues by cross-linking Type-I collagen. A working model for the photochemical welding studies of collagenous tissues was developed using gelatin strips (gel strips) composed of denatured Type-I collagen. Gel strips are transparent to near-UV and visible light, uniform in thickness, and have reproducible composition. Furthermore, the availability of nucleophilic amine sites in gel strips was demonstrated by reaction with o-phthalaldehyde, producing a fluorescent derivative of the protein. Gel strips were coated with a solution of DPD in chloroform 7 irradiated at 320-390 nm, and the resulting bonded gel strips were tested for the strength of the weld. The welds were generally brittle and had average tensile strengths that exceeded 100 N/cm2. Welds were not formed in the absence of light or DPD. Scanning electron microscopy studies revealed a pockmarked surface from severed welds. Welds of rabbit Achilles tendon were also obtained using the tethered diazopyruvamide. These welds were much weaker, having an average tensile strength of 11.95 N/cm2 for DPD-2,2'-ethylenedioxy(bis)ethylamine comonomers in the cross-linking reaction. In both studies the welds obtained by this method were significantly stronger than the controls.
Givens
Richard S
RS
Department of Chemistry, University of Kansas, Lawrence, KS 66045-7582, USA. givensr@ku.edu
Timberlake
George T
GT
Conrad
Peter G
PG
Yousef
Abraham L
AL
Weber
Jörg F
JF
Amslinger
Sabine
S
D000125:Achilles Tendon / Q000378:metabolism; D000588:Amines / Q000737:chemistry; D000818:Animals; D024042:Collagen Type I / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D005021:Ethylamines / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D005780:Gelatin / Q000737:chemistry; D008027:Light; D009596:Nitrophenols / Q000737:chemistry; D010455:Peptides / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011773:Pyruvates / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D011817:Rabbits; D013718:Tensile Strength
D000588:Amines; D024042:Collagen Type I; D003432:Cross-Linking Reagents; D005021:Ethylamines; C483534:N,N'-bis(3-diazopyruvoyl)-2,2'-(ethylenedioxy)bis(ethylamine); D009596:Nitrophenols; D010455:Peptides; D011506:Proteins; D011773:Pyruvates; C061076:4-nitrophenyl 3-diazopyruvate; D005780:Gelatin