Photorelease of phosphates: Mild methods for protecting phosphate derivatives.

We have developed a new photoremovable protecting group for caging phosphates in the near UV. Diethyl 2-(4-hydroxy-1-naphthyl)-2-oxoethyl phosphate (14a) quantitatively releases diethyl phosphate upon irradiation in aq MeOH or aq MeCN at 350 nm, with quantum efficiencies ranging from 0.021 to 0.067 depending on the solvent composition. The deprotection reactions originate from the triplet excited state, are robust under ambient conditions and can be carried on to 100% conversion. Similar results were found with diethyl 2-(4-methoxy-1-naphthyl)-2-oxoethyl phosphate (14b), although it was significantly less efficient compared with 14a. A key step in the deprotection reaction in aq MeOH is considered to be a Favorskii rearrangement of the naphthyl ketone motif of 14a,b to naphthylacetate esters 25 and 26. Disruption of the ketone-naphthyl ring conjugation significantly shifts the photoproduct absorption away from the effective incident wavelength for decaging of 14, driving the reaction to completion. The Favorskii rearrangement does not occur in aqueous acetonitrile although diethyl phosphate is released. Other substitution patterns on the naphthyl or quinolin-5-yl core, such as the 2,6-naphthyl 10 or 8-benzyloxyquinolin-5-yl 24 platforms, also do not rearrange by aryl migration upon photolysis and, therefore, do not proceed to completion. The 2,6-naphthyl ketone platform instead remains intact whereas the quinolin-5-yl ketone fragments to a much more complex, highly absorbing reaction mixture that competes for the incident light.

p-Hydroxyphenacyl photoremovable protecting groups - Robust photochemistry despite substituent diversity.

A broadly based investigation of the effects of a diverse array of substituents on the photochemical rearrangement of p-hydroxyphenacyl esters has demonstrated that common substituents such as F, MeO, CN, CO2R, CONH2, and CH3 have little effect on the rate and quantum efficiencies for the photo-Favorskii rearrangement and the release of the acid leaving group or on the lifetimes of the reactive triplet state. A decrease in the quantum yields across all substituents was observed for the release and rearrangement when the photolyses were carried out in buffered aqueous media at pHs that exceeded the ground-state pKa of the chromophore where the conjugate base is the predominant form. Otherwise, substituents have only a very modest effect on the photoreaction of these robust chromophores.

Collagen cross linking agents: design and development of a multifunctional cross linker.

A new cross linking reagent based on the first-generation polyamidoamine dendrimer (G.1 PAMAM) has been synthesized by reaction of the PAMAM with eight equivalents of p-nitrophenyl diazopyruvate. The resulting water-soluble octadiazopyruvoyl PAMAM (8G.1 DAP, 1.3) was shown to undergo Wolff rearrangements upon photolysis in methanol at lambda > 300 nm to yield the methyl esters of the ketenes formed from the loss of nitrogen. 8G.1 DAP also forms strong bonds with dehydrated collagen with glass as high as 36 N cm(-2). Collagen to collagen bonds with tensile strengths as high as 92 N cm(-2) were observed with fully dehydrated tissues. The bonding decreased rapidly with increasingly hydrated tissue possibly due to the increased distance between the collagen fibrils and the competition of H2O for the free ketene functions.

Bonding corneal tissue: applications of photoactivated diazopyruvoyl cross-linking agent.

Photoactivated bis-diazopyruvamide-N,N'-bis(3-diazopyruvoyl)-2,2'-(ethylenedioxy)bis-(ethylamine), (DPD)-was previously shown to bond materials containing type I collagen. However, tensile strength of bonded collagenous tissue ( approximately 78% water) was low compared with that of dehydrated collagenous gelatin ( approximately 14% water). Here we investigated the role of water in corneal tissue bond strength and in bonding corneal tissue to glass. Bonding corneal tissue to glass may be of value in surgically anchoring keratoprostheses to corneas to alleviate problems with extrusion. Bovine corneal samples were lyophilized for various times resulting in tissue hydrations of zero (no water content) to approximately 3.7 (normal water content). The lyophilized corneal tissue was bonded to solid gelatin sheets, to other corneal samples and to glass using 0.3M DPD in chloroform. Control runs used chloroform only. Samples were irradiated with 100 or 200 J of 320-500 nm light. Strong bonds formed with all three materials when corneal tissue hydration was 1. No bonding occurred with chloroform alone. Formation of strong bonds only occurs with hydration levels

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.