Identification and Total Synthesis of Two Previously Unreported Odd-Chain Bis-Methylene-Interrupted Fatty Acids with a Terminal Olefin that Activate Protein Phosphatase, Mg2+/Mn2+-Dependent 1A (PPM1A) in Ovaries of the Limpet Cellana toreuma

Diverse non-methylene-interrupted (NMI) fatty acids (FAs) with odd-chain lengths have been recognized in triacylglycerols and polar lipids from the ovaries of the limpet Cellana toreuma, however their biological properties remain unclear. In this study, two previously unreported odd-chain NMI FAs, (12Z)-12,16-heptadecadienoic (1) and (14Z)-14,18-nonadecadienoic (2) acids, from the ovary lipids of C. toreuma were identified by a combination of equivalent chain length (ECL) values of their methyl esters and capillary gas chromatography-mass spectrometry (GC-MS) of their 3-pyridylcarbinol derivatives. On the basis of the experimental results, both 1 and 2 were synthesized to prove their structural assignments and to test their biological activity. The ECL values and electron impact-mass (EI-MS) spectra of naturally occurring 1 and 2 were in agreement with those of the synthesized 1 and 2. In an in vitro assay, both 1 and 2 activated protein phosphatase, Mg2+/Mn2+-dependent 1A (PPM1A) up to 100 μM in a dose-dependent manner.


Introduction
Non-methylene-interrupted (NMI) fatty acids (FAs) with two or more methylene groups between the double bonds are frequently distributed in marine invertebrates [1,2], including mollusks and sponges, as well as in some terrestrial plant seeds [3,4], however their biological role and function are poorly understood. Among representative marine NMI FAs in sponges, some typical ∆5, 9-dienoic FAs have inhibitory activities against key enzymes targeted for DNA uncoiling and cleavage and FA synthesis of some bacterial cells [1,2], as well as cytotoxicity against some bacteria and some cancer cell lines [1,2]. Among several ∆5,9-dienoic FAs and their related derivatives which are targeted enzyme inhibitors, (5Z,9Z)-5,9-heptacosadienoic acid from the marine sponge Amphimedon sp. shows the most potent inhibitory activity against topoisomerase [1,2]. In addition, a mixture of (5Z,9Z)-5,9-tricosadienoic and (5Z,9Z)-5,9-tetracosadienoic acids from the marine sponge Agelas oroides In Figure 2, both 1 and 2, as minor and uncommon NMI FA components, were detected along with previously described odd-chain isomers, three NMI heptadecadienoic, and two NMI nonadecadienoic acids [12,13], although, 9,16-heptadecadienoic acid (17:2Δ9,16), which is a structural analogue of 19:2Δ11,18, has been recognized in biological samples for the first time in this study. Generally, most NMI FAs are found as minor lipid components as compared with methylene-interrupted FAs, i.e., with the presence of one methylene group between the double bonds. In Figure 2, both 1 and 2, as minor and uncommon NMI FA components, were detected along with previously described odd-chain isomers, three NMI heptadecadienoic, and two NMI nonadecadienoic acids [12,13], although, 9,16-heptadecadienoic acid (17:2∆9,16), which is a structural analogue of 19:2∆11,18, has been recognized in biological samples for the first time in this study. Generally, most NMI FAs are found as minor lipid components as compared with methylene-interrupted FAs, i.e., with the presence of one methylene group between the double bonds.
Mar. Drugs 2019, 17, x FOR PEER REVIEW 3 of 13 chromatography (TLC), however, they were not isolated due to their minute quantity. Despite their minute amounts, 1 and 2 in TAG and polar lipids were detected by gas chromatography-mass spectrometry (GC-MS). In Figure 2, both 1 and 2, as minor and uncommon NMI FA components, were detected along with previously described odd-chain isomers, three NMI heptadecadienoic, and two NMI nonadecadienoic acids [12,13], although, 9,16-heptadecadienoic acid (17:2Δ9,16), which is a structural analogue of 19:2Δ11,18, has been recognized in biological samples for the first time in this study. Generally, most NMI FAs are found as minor lipid components as compared with methylene-interrupted FAs, i.e., with the presence of one methylene group between the double bonds. Figure 2. Partial GC-MS total ion current chromatograms of the methyl esters of the dienoic FAs with 1 (A) and 2 (B) fractionated by using 5% (w/v) argentation TLC, from TAG in ovaries of C. toreuma. To determine the elution order of the methyl esters of 1 and 2, authentic methyl octadecanoate (18:0) and methyl eicosanoate (20:0), as saturated straight-chain FA methyl ester markers, were added into this fraction. Figure 2. Partial GC-MS total ion current chromatograms of the methyl esters of the dienoic FAs with 1 (A) and 2 (B) fractionated by using 5% (w/v) argentation TLC, from TAG in ovaries of C. toreuma.

A B
To determine the elution order of the methyl esters of 1 and 2, authentic methyl octadecanoate (18:0) and methyl eicosanoate (20:0), as saturated straight-chain FA methyl ester markers, were added into this fraction. suggested that there was a bis-methylene-interrupted dienoic FA with a terminal olefin. To further confirm the presence of these key diagnostic fragment ions as described above, the EI-MS spectrum of the 3-pyridylcarbinol derivative of 1 was analyzed. Consequently, all the diagnostic ions of 1, obtained by synthesis ( Figure 5B), agreed well with those of naturally occurring 1. On the basis of these characteristic fragment ions, naturally occurring 1 was finally confirmed as 12, 16-heptadecadienoic acid, namely, bis-methylene-interrupted heptadecadienoic acid with a terminal olefin. The enriched fraction including the target methyl esters of 1 and 2 ( Figure 2) was submitted to GC-MS analysis and chemical derivatization reaction with 3-pyridylcarbinol.      To further confirm the presence of these key diagnostic fragment ions as described above, the EI-MS spectrum of the 3-pyridylcarbinol derivative of 1 was analyzed. Consequently, all the diagnostic ions of 1, obtained by synthesis ( Figure 5B), agreed well with those of naturally occurring 1. On the basis of these characteristic fragment ions, naturally occurring 1 was finally confirmed as 12, 16-heptadecadienoic acid, namely, bis-methylene-interrupted heptadecadienoic acid with a terminal olefin. ECL values are practically used to identify a dienoic FA (as its methyl ester), because the ECL of a dienoic FA depends, in principle, on the contribution of the two constitutive ethylenic bonds between the double bonds [22].  ECL values are practically used to identify a dienoic FA (as its methyl ester), because the ECL of a dienoic FA depends, in principle, on the contribution of the two constitutive ethylenic bonds between the double bonds [22]. In this study, the ECL values for methyl esters and 3-pyridylcarbinol derivatives of 1 and 2, obtained by synthesis, agreed well with those of naturally occurring 1 and 2, indicating that the double bond in these compounds has a Z configuration. Thus, the structures of 1 and 2 were finally established as (12Z)-12,16-heptadecadienoic and (14Z)-14,18-nonadecadienoic acids, respectively. With the EI-MS spectra for the 3-pyridylcabiol derivatives of bis-methylene-interrupted dienoic FAs with a terminal olefin, predominant characteristic ions for m/z [M − 41] + and m/z [M − 1] + of both 1 and 2, obtained by synthesis, were unequivocally confirmed in this study. This new knowledge will be very helpful in the future to clarify the structural assignment for undescribed bis-methylene-interrupted FA analogues having a terminal olefin.

Total Synthesis of 1 and 2
The total synthesis of 1 and 2 started with commercially available diols 3a and 3b, respectively. Monoprotection of 3a and 3b, with chloromethyl methyl ether (MOMCl), provided the mono-MOM ethers 4a and 4b, which were converted to the aldehydes 5a and 5b by pyridinium chlorochromate (PCC) oxidation. The Wittig reaction of 5a and 5b with the Wittig reagent under salt-free conditions [23] provided the dienes 6a and 6b in 84% and 94% yield, respectively. Deprotection of the MOM protecting group in 6a and 6b, followed the Jones oxidation of the resulting alcohols, gave rise to 1 and 2 (Scheme 1).
ECL values, the double bond of both bis-methylene-interrupted structures was tentatively proposed to have a Z configuration. (Therefore, to further determine biological properties of naturally occurring 1 and 2, as well as to elucidate their precise structures, we have synthesized these compounds.) In this study, the ECL values for methyl esters and 3-pyridylcarbinol derivatives of 1 and 2, obtained by synthesis, agreed well with those of naturally occurring 1 and 2, indicating that the double bond in these compounds has a Z configuration. Thus, the structures of 1 and 2 were finally established as (12Z)-12,16-heptadecadienoic and (14Z)-14,18-nonadecadienoic acids, respectively. With the EI-MS spectra for the 3-pyridylcabiol derivatives of bis-methylene-interrupted dienoic FAs with a terminal olefin, predominant characteristic ions for m/z [M − 41] + and m/z [M − 1] + of both 1 and 2, obtained by synthesis, were unequivocally confirmed in this study. This new knowledge will be very helpful in the future to clarify the structural assignment for undescribed bis-methylene-interrupted FA analogues having a terminal olefin.

Biological Activity of 1 and 2
Unfortunately, little is known about the structural diversity and biological properties of NMI FAs in marine mollusks. Our continuing work with highly diverse uncommon NMI FAs, especially their odd-chain analogues and isomers in minute amounts in germ cells of Japanese limpets, has resulted in the discovery of structurally interesting and biologically active 1 and 2, along with 23:2Δ9Z,20Z, as novel PPM1A activators, although their functions are still to be elucidated. We expect that studies on the isolation and identification of marine undescribed NMI FAs will provide a good starting point for further investigations of their biological functions, as well as to explore small Unfortunately, little is known about the structural diversity and biological properties of NMI FAs in marine mollusks. Our continuing work with highly diverse uncommon NMI FAs, especially their odd-chain analogues and isomers in minute amounts in germ cells of Japanese limpets, has resulted in the discovery of structurally interesting and biologically active 1 and 2, along with 23:2∆9Z,20Z, as novel PPM1A activators, although their functions are still to be elucidated. We expect that studies on the isolation and identification of marine undescribed NMI FAs will provide a good starting point for further investigations of their biological functions, as well as to explore small molecules to increase or improve targeting health effects. Furthermore, a combination of chemically synthesis and structural studies on previously undescribed NMI FAs could yield important findings for their unreported biological properties.

General Experimental Procedures
General experimental procedures for all chemical transformations and methods for the determination of the structure of synthesized compounds were performed as described in our former paper including the apparatus for the measurement of 1 H and 13 C NMR, IR, and low-and high-resolution electron impact (EI) mass spectra [24]. The copies of 1 H and 13 C NMR spectra for all new synthetic compounds (4b, 6a, 6b, 1, and 2)  Fresh ovaries of C. toreuma were dissected. Polled ovaries (15 g wet weight) were suspended in 50 mL of CHCl 3 -MeOH (2:1, v/v) and were homogenized for 1 min at 16,000 rpm by using IKA Ultra-Turrax T25 basic (IKA Japan, Nara, Japan). Lipids were extracted from homogenized ovaries by the Bligh and Dyer method [25]. The fractionation and identification of TAG and polar lipids by TLC were performed according to our previous experimental procedure [8]. The amounts of TAG and polar lipids (mainly phosphatidylcholine and phosphatidylethanolamine) obtained were 104 mg and 56 mg, respectively.
FA methyl esters were prepared as previously described [13]. FA methyl esters, depended on the degree of FA unsaturation, were obtained by using 5% (w/v) argentation TLC, as described previously [13]. The fractionated FA methyl esters, including methyl esters of 1 and 2, in TAG and polar lipids were approximately 3 mg and 1 mg, respectively. These FA methyl esters were subjected to gas-liquid chromatography (GLC) and GC-MS analyses and chemical derivatization reaction with 3-pyridylcarbinol [13].
GC-MS analyses of FA methyl esters and 3-pyridylcarbinol derivatives, as well as GLC analyses of FA methyl esters, were performed according to our previous works [12,13]. To a stirred solution of 3a or 3b (4.30 mmol) in THF (12 mL) was added NaH (60%, 181 mg, 4.52 mmol) at 0 • C, and the reaction mixture was stirred at 0 • C for 15 min. To the reaction mixture was added MOMCl (0.35 mL, 4.52 mmol), and the resulting mixture was stirred for 16 h at room temperature. The reaction was quenched with saturated NH 4 Cl aqueous solution, and the aqueous mixture was extracted with CH 2 Cl 2 (3 × 3 mL). The organic extracts were combined, dried, and evaporated to give a colorless oil, which was chromatographed on a SiO 2 column (15 g, EtOAc/n-hexane = 1/10-1/5) to give 4a [26] or 4b. To a stirred solution of 4a or 4b (2.16 mmol) in CH 2 Cl 2 (10 mL) were added PCC (837 mg, 3.88 mmol) and NaOAc (424 mg, 5.18 mmol), and the resulting mixture was stirred at room temperature for 16 h. The reaction mixture was filtered through a celite pad and washed with CH 2 Cl 2 (3 × 3 mL). The filtrate and washings were combined and evaporated to give a black oil, which was chromatographed on a SiO 2 column (10 g, EtOAc/n-hexane = 1/10) to give 5a or 5b, which were immediately used for the next Wittig reaction.

Conclusions
The structure determination of the previously unreported 1 and 2 in ovaries of the limpet C. toreuma was archived by comparison of their ECL values and EI-MS analyses of the synthetic counterparts 1 and 2. A total synthesis of compounds 1 and 2 was achieved in five-step linear synthetic sequence starting from commercially available diols 3a and 3b, respectively. Selective formation of internal Z-double bonds was performed by the Wittig reaction under salt-free conditions. Naturally occurring 1 and 2 were finally identified as (12Z)-12,16-heptadecadienoic and (14Z)-14,18-nonadecadienoic acids, respectively. In addition, both 1 and 2, as well as structurally related NMI FAs, produced similar PPM1A activation in vitro assay to previously reported 18:1n-9 and 18:2n-6. In the future, to facilitate understanding of their structural diversity and biological properties, additional studies are needed to clarify of the biological functions of 1 and 2, along with other structurally related NMI FAs, in living organisms.