**5.1 Untangling oxidation behaviors arising when Fe+2 ions, H2O2, and H2O are present**

The Fenton reaction (Fe+2 + H2O2 + H2O) has been shown to generate three powerful oxidants: 1) (HO•) radical [16, 17]; 2) (Fe = O)+2 ion [18]; and 3) [Fe = O]+3 ion [86, 88].

Sugimoto & Sawyer (1985a & 1985b) proposed that both ferrous and ferric ions can abstract an •O• atom from H2O2, thus explaining how ferrous and ferric spontaneously reorganize to form the secondary oxidants ferryl (Fe = O)+2 and perferryl (Fe = O)+3 ions, respectively.

Sugimoto and Sawyer (1984) and (1985b) compared (Fe = O)+2 and (Fe = O)+3 oxidations, respectively, in anhydrous CH3CN or 90% CH3CN/10% H2O with several organic and inorganic molecules. In anhydrous CH3CN, ferryl-oxo ions oxidation produced only 2-electron oxidations, primarily dehydrations or hydroxyl additions, while perferryl-oxo ions produced both 2-, and 4- electron oxidations. Neither oxidant produced 1- electron oxidations.

In aqueous acetonitrile (CH3CN/H2O), single electron oxidations, characteristic of HO• were observed including: 1) carbon–carbon fusions; 2) oxidation of Fe+2 ions; and 3) reduction of Fe+3 ions to Fe+2 ions. The authors proposed that HO• radicals are created by ferryl-oxo and perferryl-oxo ions only when water is present, implying that H• abstraction from water produces HO• radicals via the formula: (Fe = O)+2,+3 + H2O• HO• + Fe(+3,+4)OH [59, 61].

Sawyer et al. (1993) tested the oxidizing capability of Fe+2 ions and organic peroxides (R-O-O-H) 1) under anhydrous conditions in the presence vs. absence of O2, and 2) under anoxic conditions with anhydrous (Fe+2) or partially hydrated (Fe+2(H2O)2) conditions. The authors found evidence of 1e― oxidations either when O2 or H2O2 were present, indicating 1) that (Fe = O)+2 reacted with H2O to form HO• radicals, or 2) with O2, creating O2• [16], which then reacted with (R-O-O-H) to generate HO• radicals [39]. On the other hand, Hage et al. (1995) found that in the conversion of benzene to phenol, if a small amount of H2O was added, the efficiency of conversion was increased, but other 1e― signature products were not detected [89].

Sawyer et al. (1996) surveyed the oxidizing abilities of Fe+2,+3, Cu+2, Co+2, and Mn+2 ions in anhydrous solvents with ROOH, with/out O2. Under an argon atmosphere, only the hydroxyl radical sources produced chain fusion events, none of the ions did. When air (20% O2) was substituted, all of the ions showed oxidation patterns consistent with HO• radicals. The authors concluded that the metal ions, activated by peroxide, reacted with solubilized O2, producing superoxide (O2 • or HO2 • ), which in turn reacted with H2O2 to generate reactive singlet oxygen (•O•) which then reacts with R-C-H to produce HO• radicals [41].

Barton et al. (1995, 1996) seconded the research of Sawyer's group, confirming that absent H2O, ferryl-oxo and perferryl-oxo ions perform distinct and distinctive 2- (and 4-) electron oxidations without mixing the unique chemistries of either ion [86, 90].

Mwebi (2005) also confirmed that when Fe+2 ions, H2O2, and H2O are reacted in aqueous conditions, all three secondary oxidants [(Fe = O)+2, (Fe = O)+3, and HO•] arise in that either (Fe = O)+2 and (Fe = O)+3 ions can abstract H• from H2O to create the HO• radical, the HO• radical can oxidize Fe+2 ions to Fe+3 ions, and H2O2 can reduce Fe+3 ions to Fe+2 ions [51].

### **5.2 Biological occurrence and utilization of the Fenton reagent**

Oceans covered Earth 4.4 billion years ago [91], evidence of bacteria dates back 3.5 billion years ago (92), and evidence of oxygenic photosynthesis 2.3 billion years ago [91, 92]. From at least that time living organisms have evolved to defend against, and/ or, utilize Fenton chemistry.

The use of the Fenton reagents to kill organisms or degrade biopolymers is widely distributed in the biosphere. Saprophytic fungi use Fenton reagents to degrade polysaccharides of woody plant tissues [93], including cellulose [93–96], callose [97], and hemicelluloses [98].

On the other side of the eukaryote kingdom, mammalian leukocytes and neutrophils pump Fe+2 ions [99, 100] and H2O2 into phagosomes to produce oxygen radicals [101] to effect pathogen killing [102–107]. For both nutrient mobilization and pathogen killing, these oxidants target external glycan including cell walls to cause cell lysis and/or internal glycans such DNA and RNA to facilitate death of bacteria and eukaryote parasites.

Moore and coworkers incubated *Saccharomyces cerevisiae* cells with an Fe+2-chelating anti-cancer medication. Treated and control cells were stained, fixed, and thinsectioned for electron microscopy. While studying chromosome damage the authors observed damage to the yeast cell walls by the anti-cancer drug [108–110].

Following Moore's lead, Lipke and coworkers treated 35S -labeled *S. cerevisiae* cells with the an Fe+2-binding anti-cancer medication, then compared protein levels release into growth media from treated and control cells [111], and cell lysis rates of treated and control cells after adding *Arthrobacter luteus* (Zymolyase) protease [112].

In Lim et al. (1995), the authors noted that pretreatment with yeast cells with an Fe+2-binding anti-cancer agent increased cell lysis rates by Zymolyase protease with: 1) Fe+2 + O2 or Fe+3 + O2, but not Ca+2, Co+2, Cu+2, Mn+2, Mg+2, and Zn+2 ions; 2) H2O2 could substitute for O2; and 3) Fe+2/H2O2 and Fe+3/H2O2 alone also accelerated yeast cell lysis; 4) H2O2 only controls did not accelerate Zymolyase lysis rates [112].

To understand the basis of cell wall weakening by Fe+2/H2O2, Ovalle et al. (2001) elected to separately test pure analogs of carbohydrates and proteins found in yeast wall [113]. Ovalle et al. (2001) assumed that partial oxidation of fungal wall monosaccharides would oxidize hydroxyl groups to aldehydes and/or carboxylic acids and developed a method for separating carbohydrates from 0 to 20 glucan units on polyacrylamide gels. Surveying the available literature of the time, the authors followed the method of Ahrgren et al. (1975) where dextran was preincubated with FeSO4 prior to addition of H2O2 [114].

#### *A History of the Fenton Reactions (Fenton Chemistry for Beginners) DOI: http://dx.doi.org/10.5772/intechopen.99846*

Ovalle et al. (2001) [113] labeled the aldehyde groups of glucose, maltose, maltotriose and enzymatically digested laminaran with 8- amino, 1-, 3-, 6-, naphthalene trisulfonate (ANTS) and NaCNBH3, by the method of Klock & Starr (1998) [115], to have glucan ladders to estimate degree of polymerization of carbohydrate chains separated by polyacrylamide gel electrophoresis. Ovalle et al. (2001) modified the method Klock & Starr (1998) to visualize carboxylic acids and by quenching aldehydes with NaBH4, then crosslinking ANTS to carboxylic acids with Nhydroxysuccinamide (NHS) and N-ethyl-N-(3-aminopropyl) carbodiimide (EDC) [116]. Ovalle et al. (2001) separately visualized de novo aldehydes and de novo carboxylic acids (after quenching aldehydes with NaBH4) on 10% stacking/ 20% running acrylamide gels.

Ovalle et al. (2020) [117] used the method of Ovalle et al. (2001) to determine if Fe+2/H2O2 would oxidize algal laminaran (d.p. ≈ 50–60 glucose units; 97–99% β1–3 glu / 1–3% β1–6 glu). To optimize metal ion-carbohydrate interactions, FeSO4 was incubated with carbohydrate for 1 min prior to addition of H2O2. The final ratio (glucose monomer: Fe+2: H2O2 = 10:1:1) was chosen to oxidize 10% of glucose monomers and reduce the likelihood of a secondary oxidation of glucose fragments to 1% maximum. Unoxidized laminaran did not enter that stacking gel. NaIO4 oxidized laminaran entered the stacking gel but stopped at the stacking gel/running gel interface. Fenton-oxidized laminaran produced smears, when labeled for either aldehyde or carboxylate groups. Enzyme- (Zymolyase) digested laminaran were used as glucan ladders when labeled for aldehydes or organic acids.

To label glucan fragments so as to be suitable for positive ion mass spectroscopy [118, 119], Ovalle et al. (2020) substituted tert-butyl ester of tyrosine (TBT) for ANTS with no other changes required. Ovalle et al. (2020) observed the elution of TBTlabeled glucan fragments with masses consistent with six classes of TBT-labeled molecules: aldoses, dialdoses, uronic acids, hexosuloses, aldonic acids (unlabeled), and hexulosonic acids (unlabeled) (**Figure 4**).

#### **Figure 4.**

*Comparison of particles of four molecule classes from Laminaran after Fenton oxidation.*
