SPECIAL FOCUS y Nitric oxide-releasing materials Special Report For reprint orders, please contact [email protected] 1 Special Report 2015/06/30 Nitric oxide: a brief overview of chemical and physical properties relevant to therapeutic applications Future Sci. OA Nitric oxide (nitrogen monoxide, •NO) has been intensively studied by chemists and Jack R Lancaster Jr physicists for over 200 years and thus there is an extensive database of information Departments of Pharmacology & that determines its biological actions. This is a very brief overview of the chemical and Chemical Biology, Medicine, & Surgery, School of Medicine, University of physical properties of •NO that are most relevant to Biology in general and to the Pittsburgh, 4200 Fifth Ave, Pittsburgh, 1(1), FSO59 development of •NO releasing materials in particular. PA 15260, USA [email protected] Keywords: diffusion • oxidation-reduction • radical • transition metals In 1987 the startling discovery was made electron. This is due to the fact that it has that the small reactive radical molecule an odd total number of electrons (15). The nitric oxide (•NO; nitrogen monoxide) is property of possessing an unpaired electron specifically produced and functions as a is called paramagnetism and it is this prop­ major messenger in the mammalian cardio­ erty that most determines its chemical reac­ vascular system, for which the 1998 Nobel tivity. However, the small uncharged nature Prize in Physiology or Medicine was awarded of •NO is especially important in determin­ to Robert Furchgott, Louis Ignarro and Ferid ing mechanisms for its selective delivery, the Murad [1] . This discovery represented a classi­ topic of this monograph. cal paradigm shift in chemical biology, reveal­ This brief overview is organized into two ing the profound concept that Nature utilizes general topics, the chemical and the physical to its advantage the properties of molecules, properties of •NO relevant to its biological even if they can also be injurious or toxic. actions. Prior to this realization, •NO was known to biologists almost exclusively as only a Chemical properties of •NO: a lone toxic pollutant and we now know it is truly electron in search of a stable home 2 a versatile player in virtually every aspect of Unpaired electrons in σ or π molecular physiology and pathophysiology. orbitals are inherently unstable. The two The proposition that the fundamental methods for the stabilization of this elec­ principles of biology must ultimately be tron are pair with another unpaired electron 2015 determined by the interactions of atoms and take up partial residence in a d orbital and molecules with each other is no better of a transition metal ion. These two mecha­ illustrated than examining the mechanis­ nisms dominate the chemistry of •NO, as tic foundations of the biological actions of described below. •NO. Because •NO has been studied since the dawn of chemistry and is one of the Pairing of the unpaired electron ten smallest stable molecules in Nature [2], One of the most important principles in understanding of its basic chemistry is rela­ chemistry is that electrons exist in atoms and tively well developed. The three properties molecules almost exclusively in pairs. This that most determine this chemistry are that means of course that the unpaired electron •NO is small, uncharged and (unlike virtu­ in •NO will require another unpaired elec­ ally all other molecules) contains an unpaired tron, which explains the rich chemistry of the part of 10.4155/FSO.15.59 © Jack R Lancaster Jr Future Sci. OA (2015) 1(1), FSO59 eISSN 2056-5623 Special Report Lancaster the rate decreases exponentially with decreasing •NO O 2 (B) concentration, and at the nanomolar concentrations of (A) NO2 ·NO2 •NO biologically (except perhaps under some inflam­ ·NO RSH matory conditions) this reaction is extremely slow and likely to be of very limited importance [4]. There how­ ONOO RSOH + NO 2 ever may be specific cellular microenvironments (spec- O •- 2 ifically the hydrophobic phases of membranes and CO 2 lipid particles) where the reaction is accelerated due to increased solubilities of both •NO and O2 in these phases [6]. A much more important route quantita­ •- - tively to biological formation of nitrite from •NO is an ·NO2 + CO3 NO3 (1/3) (2/3) ill­defined process(Figure 1B), whereby cells consume •NO and O2 [7]. This reaction may well be the sec­ Figure 1. The preponderant covalent reactions of •NO ond most important biological reaction of •NO quan­ and progeny under biological conditions. Reactions titatively (second to reaction with oxyhemo globin, not balanced chemically. (A) should be very minor vide infra). The mechanism(s) and intermediates are except at very high (•NO), but could be more important unknown. in specific microenvironments; product may not be ‘authentic’ •NO . (B) Mechanism(s) unknown; probably •NO also reacts with the one electron reduced form 2 •­ second most preponderant biological •NO reaction of oxygen, superoxide (O2 ), and unlike the O2 reac­ (behind O2Hb reaction). tion is extremely rapid and kinetically first order with •­ [O2 ]. In this case, the immediate product, peroxy­ reaction of •NO with other radical species. This also nitrite (ONOO­), is not a radical but it is a moderately explains why •NO is relatively unreactive to virtually strong two-electron oxidant. Under biological condi­ ­ all molecules in biology. These principles provide clar­ tions the major reaction of ONOO is with CO2. This ity to statements that •NO is ‘reactive’ or ‘unreactive.’ reaction produces a very transient species (nitroso­ ­ In reality, •NO does not react with almost all mol­ peroxocarbonate, ONOOCO2 ), 2/3 of which forms ­ ecules (in that sense being ‘unreactive’) but reaction the relatively unreactive nitrate anion (NO3 ) and also with other radical molecules is very rapid (‘reactive’) regenerates CO2 and 1/3 rapidly homolyzes (breaks the because it involves the very favorable pairing of two peroxo O-O bond with each electron going to separate Figure 1 • unpaired electrons. diagrams the quantita­ products) to form two radicals, NO2 and carbonate •­ tively most important biological reactions of •NO with radical anion (CO3 ). Thus, •NO essentially converts •­ nonmetals and its subsequent reactive nitrogen species 1/3 of a relatively mild oxidant (O2 ) into two highly (RNS) [3]. oxidizing radicals. Under biological conditions another Among the biological molecules with unpaired elec­ reaction of peroxynitrite that occurs, although second trons, molecular oxygen (O2) is pre-eminent. Although quantitatively to CO2, is with cellular thiols (RSH) to O2 has an even total number of electrons, in the most result in a two­electron oxidation to form the moder­ common form (the ground state) two of the electrons ately reactive sulfenic acid (RSOH). Finally, in certain are unpaired (occupy separate molecular orbitals) and microenvironments protonated peroxynitrite (peroxy­ (Figure 1A) so the reaction of •NO with O2 is with­ nitrous acid, ONOOH; pKa = 6.8) homolyzes to • out doubt the single most studied reaction of •NO. produce NO2 and the very highly oxidizing hydroxyl However, in spite of this attention (extending back to radical (•OH). John Dalton, the ‘Father of Chemistry,’ who used it The overall ‘take home lesson’ is that the initial reac­ to perform the first determination of the 2O content tions of •NO with nonmetals under biological condi­ of the atmosphere [4]) there are many aspects of this tions results in two major effects, oxidation of cellu­ complex reaction that are not understood. The imme­ lar nucleophiles and formation of the relatively stable diate product is generally (although not universally [5]) nitrite and nitrate anions. The two major nucleophiles believed to be another small radical molecule, nitro­ that are best studied are cysteine thiol (proteins and • gen dioxide ( NO2). Although •NO is not an oxidant, also the small peptide glutathione, GSH) and the • NO2 definitely is and it reacts rapidly with many phenolic ring of tyrosine. One-electron oxidation • •­ molecules ‘rich’ in electrons (nucleophiles) to extract by the radicals ( NO2, CO3 and, to a lesser extent, one electron, generating another radical and form­ •OH) generates the thiyl and tyrosyl radicals. These ­ ing nitrite anion (NO2 ) from the •NO2. This •NO/ can be considered ‘first generation’ radical products O2 reaction exhibits unusual kinetics in that the rate because they, like •NO, become stabilized primarily is second order with •NO concentration. This means by reaction with other molecules with unpaired elec­ 10.4155/FSO.15.59 Future Sci. OA (2015) 1(1), FSO59 future science group Nitric oxide: a brief overview of chemical & physical properties relevant to therapeutic applications Special Report trons. Thiyl radical reaction with •NO will result in reductions of •NO. In fact, undoubtedly the most nitrosothiol, a covalent compound containing the important reaction of •NO biologically (quan­ nitroso functional group (R-S-N = O). Tyrosyl radi­ titatively) is the extremely rapid reaction with cal reaction with •NO2 will result in nitrotyrosine, a oxyferrohemoglobin to form nitrate and ferriheme: covalent compound with the nitro functional group 2++3 : ++" - NO Fe ON23OFe Equation 1 (R-NO2). These are by far the two best studied nitro­ gen oxide adducts formed from •NO biologically. This electron transfer can also occur in reverse, with Another commonly cited mechanism for RSNO for­ electron flow to the ferriheme