*4.3. Oxidation of 9,10-Dihydroxystearic Acid* (**9**) *to 9,10-Dioxostearic Acid* (**10**)

A mixture of diol **10** (1.55 g, 4.9 mmol), Fe(NO3)3·9 H2O (20 mg, 0.049 mmol), TEMPO (8.0 mg, 0.049 mmol), and NaCl (3 mg, 0.049 mmol) in toluene (45 mL) was stirred at 100 ◦C for 4–5 h. The reaction mixture was poured into water and extracted with ethyl acetate. The organic phase was dried and concentrated under reduced pressure to give the crude dioxo derivative **11** (1.83 g, 75% purity by GC/MS analysis, estimated content of compound **11** 1.37 g) which was employed in the successive step without further purification. 1H NMR (CDCl3, 400 MHz) [43]: δ = 2.72 (4H, t with *J* = 7.3 Hz, 2CH2CO), 2.35 (2H, t with *J* = 7.4 Hz, CH2COOH), 1.69–1.49 (6H, m, 3 CH2), 1.40–1.20 (16H, m, 8 CH2), 0.92–0.84 (3H, m, CH3). 13C NMR (CDCl3, 100.6 MHz) [43]: δ 200.3, 200.2, 180.0, 36.2, 36.1, 34.2, 31.9, 29.4, 29.3, 29.2, 29.1, 29.04, 28.96, 24.2, 23.2, 23.1, 22.8, 14.2. GC/MS (EI) as a methyl ester, obtained by treatment with MeOH and trimethylsilyldiazomethane 10% in hexane, tr = 22.48 min: *m*/*z* (%) = 326 (M+, 1), 295 (5), 185 (100), 141 (54).

#### *4.4. Oxidative Cleavage of 9,10-Dioxostearic Acid* (**11**) *to Azelaic* (**2**) *and Pelargonic Acid* (**3**)

A mixture of crude dioxo derivative **11** (1.75 g, 75% purity, estimated content of compound **11** 4.21 mmol) and 35% H2O2 w/w (579 μL, 6.73 mmol) in toluene (35 mL) was stirred at 30 ◦C for 3 h. A saturated solution of NaHSO3 (750 μL) was added, followed by the addition of H2SO4 2 M till pH = 2. The reaction mixture was then extracted with ethyl acetate. The organic phase was dried and concentrated under reduced pressure to give a crude mixture containing 93% (GC/MS) of acids **2** and **3**, which was heated at 50 ◦C for 1 h in a 1:1 mixture of EtOAc and water. Water was separated, and diacid **2** crystallized upon cooling. Other two extractions of the organic phase with hot water allowed the isolation of diacid **2** as a pure compound. Pelargonic acid **3** was isolated from the organic phase showing 91% chemical purity (GC/MS).

Azelaic acid (**2**): 578 mg (73%, >99% chemical purity by GC/MS and NMR); 1H NMR (CDCl3, 400 MHz) [44]: δ = 2.35 (4H, t with *J* = 7.4 Hz, 2CH2COOH), 1.75–1.55 (4H, m, 2CH2), 1.4–1.2 (6H, m, 3CH2). 13C NMR (CDCl3, 100.6 MHz) [44]: δ = 180.2, 34.2, 29.0, 28.9, 24.3. GC/MS (EI) as a methyl ester, obtained by treatment with MeOH and trimethylsilyldiazomethane 10% in hexane, tr = 13.9 min: *m*/*z* (%) = 185 (M<sup>+</sup> - 31, 55), 152 (100), 143 (47), 111 (63).

Pelargonic acid (**3**): 512 mg (77%, 91% chemical purity by GC/MS); 1H NMR (CDCl3, 400 MHz) [45]: δ = 2.35 (2H, t with *J* = 7.5Hz, CH2COOH), 1.75–1.55 (2H, m, CH2), 1.4–1.2 (10H, m, 5CH2), 0.80–0.95 (3H, m, CH3). 13C NMR (CDCl3, 100.6 MHz) [45]: δ = 180.5, 34.2, 31.9, 29.3, 29.20, 29.18, 24.8, 22.7, 14.2. GC/MS (EI) as a methyl ester, obtained by treatment with MeOH and trimethylsilyldiazomethane 10% in hexane, tr = 9.33 min: *m*/*z* (%) = 172 (M+, 0.5), 141 (15), 129 (18), 87 (45), 74 (100).

#### **5. Conclusions**

The chemo-enzymatic conversion of oleic acid into azelaic and pelargonic acids herein described represents a sustainable alternative to ozonolysis, currently employed at the industrial scale. Azelaic acid can be produced in high chemical purity in 44% isolation yield after three steps, avoiding column chromatography purifications. Intermediate diol **10** and final azelaic acid **2** are purified by crystallization from acetonitrile and water, respectively. The procedure shows some valuable aspects, even if it is not a one-pot process, as those using H2O2 and tungsten derivatives already known in the literature [13–18].

The reagents of the three steps are: (i) H2O2 35% for the epoxidation of acid **4** and the oxidative cleavage of diketone **11**, and atmospheric oxygen for diol **10** oxidation, both producing H2O as a by-product; (ii) H2O for the acid-catalyzed hydrolysis of both epoxide **9** and anhydride **12,** generating no side-product, being fully incorporated in the reacting products. The organic solvents used during the reactions are limited to acetonitrile and toluene; water and ethyl acetate are employed for quenching and separation procedures. The final oxidative cleavage of dioxo derivative **11** occurs in mild conditions and generates a very tiny quantity of oxidized impurities, thus increasing the economic value of the process, and reducing the complexity and cost of final azelaic acid purification. Hydrogen peroxide is itself very effective in promoting the cleavage with no need for catalysts or harsh acidic or alkaline conditions and generating water as a side-product. The reaction medium can be either toluene or acetonitrile. The use of an enzymatic method to produce in situ H2O2 will be considered for further development of the process.

Studies are now in progress to apply this synthetic procedure to soapstock recovered from the neutralization step during vegetable seed oil refining at Oleificio Zucchi. A pre-treatment step has to be added where the lipase-mediated hydrolysis of the triglycerides, which are inevitably present in this by-product, is carried out.

A further development of the process will also be the optimization of the entire sequence in continuous flow mode, taking advantage of the fact the enzyme employed for the generation of the key peroxy species is already marketed in immobilized form. Besides an expected higher productivity value and an advantage for the process scalability study, the use of a continuous flow reactor will most likely increase the stability of lipase, and allow for a more reliable evaluation of lipase reusability.

**Supplementary Materials:** The following are available online, Table S1: Effect of H2O2/oleic acid molar ratio and temperature on the chemo-enzymatic epoxidation of oleic acid, Table S2: Effect of the amount of lipase and solvent on the chemo-enzymatic epoxidation of oleic acid, Table S3: Effect of the amount of aqueous 2M sulfuric acid on the ring-opening of epoxystearic acid **9**, Characterization of 8-((2*SR*,3*RS*)-3-octyloxiran-2-yl)octanoic acid (**9**), Recovery and re-use of Novozyme 435, Procedure for the oxidative cleavage of 9,10-dioxostearic acid (**11**) to give a mixture of 9-(nonanoyloxy)-9-oxononanoic acid (**12**), azelaic (**2**), and pelargonic acid (**3**), Column chromatography separation of azelaic (**2**) and pelargonic acid (**3**).

**Author Contributions:** Conceptualization, E.B. and D.T.; methodology, E.B., D.T., and G.D.L.; experimental procedure, M.V., M.C.G., F.T., and D.C.; characterization, F.G.G., M.V., M.C.G., and G.D.L.; data analysis, F.T., D.C., E.B.; writing—original draft preparation, E.B., F.T., D.C.; writing—review and editing, E.B., D.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Fondazione Cariplo—INNOVHUB, project SOAVE (Seed and vegetable Oils Active Valorization through Enzymes), grant number 2017-1015.

**Conflicts of Interest:** The authors declare no conflict of interest.
