Aliphatic beta-nitroalcohols for therapeutic corneoscleral cross-linking: chemical mechanisms and higher order nitroalcohols

• Published in: Investigative ophthalmology & visual science 2010-02, Vol.51 (2), p.836-843
• Main Authors: Paik, David C, Solomon, Marissa R, Wen, Quan, Turro, Nicholas J, Trokel, Stephen L
• Format: Article
• Language: English
• Subjects: Animals; Collagen - metabolism; Colorimetry; Cornea - metabolism; Cross-Linking Reagents; Ethanol - chemistry; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Nitrites - metabolism; Nitro Compounds - chemistry; Pentanols - chemistry; Propanols - chemistry; Sclera - metabolism; Swine
• ISSN: 1552-5783; 0146-0404; 1552-5783
• DOI: 10.1167/iovs.09-3937

The recent tissue cross-linking studies indicate that aliphatic beta-nitroalcohols (BNAs) may be useful as pharmacologic corneoscleral cross-linking agents. The present study was performed to identify the specific chemistry involved under physiologic conditions, with the intent of identifying more effective agents. The mechanism of chemical cross-linking at pH 7.4 and 37 degrees C was studied using three techniques. The colorimetric Griess assay was used to follow the release of nitrite from three mono-nitroalcohols (2-nitroethanol [2ne], 2-nitro-1-propanol [2nprop]), and 3-nitro-2-pentanol [3n2pent]). Second, the evolution of 2nprop in 0.2 M NaH(2)PO(4)/Na(2)HPO(4)/D(2)O was studied using (1)H-NMR. Third, thermal shrinkage temperature analysis (T(s)), a measure of tissue cross-linking, was used to support information from (1)the H-NMR studies. A time-dependent release of nitrite was observed for all three mono-nitroalcohols studied. The maximum levels were comparable using either 2ne or 2nprop (approximately 30%). However, much less (approximately 10%) was observed from 3n2pent. Using (1)H-NMR, 2nprop evolved into a unique splitting pattern. No match was observed with reference spectra from three possible products of denitration. In contrast, 2-methyl-2-nitro-1,3-propanediol (MNPD), a nitro-diol, was identified, implying the formation of formaldehyde from a retro-nitroaldol (i.e., reverse Henry) reaction. In support of this mechanism, T(s) shifts induced by the nitro-triol 2-hydroxymethyl-2-nitro-1,3-propanediol (HNPD) were superior to the nitro-diol MNPD which were superior to the mono nitroalcohol 2nprop. BNAs function as both formaldehyde and nitrite donors under physiologic conditions to cross-link collagenous tissue. Higher order BNAs are more effective than mono nitroalcohols, raising the possibility of using these agents for therapeutic corneoscleral cross-linking.