Oxidative Oligomerization Of DBL Catechol, A Potential Cytotoxic Compound For Melanocytes, Reveals The Occurrence Of Novel Ionic Diels-Alder Type Additions Part 2

There were also compounds formed from the dimer with the loss of two protons. These compounds eluted at 17 min, 18 min, 20 min, and 21 min with a molecular mass of 353.1021, which is within 1.5 ppm of the theoretical mass for C20H16O6 (353.1013 amu). The CID spectrum of these compounds was significantly different, indicating multiple isomers are being formed in the reaction mixture (Figures 8–11).

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The peak eluting at 20 min showed only a water loss as the major production (m/z 335 ion in Figure 10). The peak eluting at 21 min showed the major peak with the loss of the COCH2 group (m/z 311 ions). This compound has to be the oxidized form of the DBL quinone dimer. On the other hand, the peak eluting at 18 min showed major decomposition ions at 335 (water loss), 311 (COCH2 loss), and a minor ion at m/z 293 (water and COCH2 loss). Note that the last decomposition ion is not possible for the DBL quinone dimer and is possible only for the oxidized form of the benzodioxan dimer. From these results it was inferred that two different kinds of dimers are formed in the reaction— a benzodioxan dimer and a DBL quinone dimer.

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These oxidized dimers will exhibit visible absorbance, much like any other simple unconjugated quinones. This is consistent with the absorbance maximum of 420 nm, which is due to the quinonoid compound accumulating in the DBL catechol–tyrosinase reaction mixture. The DBL quinone dimer could aromatize and further oxidize to the quinone methide. Whereas the benzodioxan dimer will undergo oxidation to quinone which will then isomerize to side chain desaturated compound. Thus, the initial dimers with m/z 355 are converted to the oxidized form of the dimers with m/z 353.

In addition to the dimeric products, trimeric compounds could also be observed in the mass spectrum of the reaction mixture. Again, two-parent ions at m/z 529.1486 are present, one eluting at 20 min and the other at 22 min (Figure 5 panel C). Their mass is within 3 ppm of the mass of the theoretical protonated trimeric compound (C30H26O9). Their CID spectra are shown in Figures 12 and 13. The CID of one isomer gave a major ion at 351, corresponding to the dimer's fully oxidized form. The other isomer gave considerably less amount of this production. It was not possible to distinguish the structure of the trimers based on the fragmentation pattern. Nevertheless, it was clear that different trimeric products are also formed in the reaction mixture. Thus, the results presented in this paper confirm that DBL catechol is extremely susceptible to oxidative polymerization as proposed in an earlier work from one of our groups [11].

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The formation of dimers and trimers can be explained by the reactivities of the quinonoid products formed in the reaction (Figure 14). Oxidation of DBL catechol produces its corresponding quinone, which is highly hydrophobic and can easily exhibit a cycloaddition reaction with the parent catechol. The ionic Diels-Alder addition of the DBL quinone to the parent catechol will produce two types of adducts as shown in Figure 14. The reaction of quinonoid carbonyl groups with the desaturated side chain will produce the benzodioxan dimer. In contrast, the dienone side chain addition with the desaturated side chain produces the pyran-type adduct simply designated as DBL quinone dimer. Both these compounds can undergo facile oxidation and further reaction to form trimeric compounds by similar Diels-Alder reactions. Although the biological occurrence of the Diels-Alder reaction is very rare, it has been reported to proceed in a few circumstances [20–23]. For example, one of our groups has recently shown that the quinone of N-acetyl dopa methyl ester is undergoing rapid cycloaddition, probably via ionic Diels-Alder reaction, generating a similar benzodioxan dimer [20]. The current studies also support the prevalence of such ionic Diels-Alder additions in the quinonoid chemistry of side-chain desaturated catechols. These cyclization reactions are all nonenzymatic and hence will be non-stereoselective, leading to the production of multiple isomeric products. The production of such multiple products during the nonenzymatic cyclization of enzymatically generated quinonoid species has been well documented in this laboratory for several dehydrodopa and dehydrodopamine derivatives [16–20].

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The potentmelanotoxicity of RK and its reduced product, rhododendron, is now well established [1–8,24]. While some of the reactions such as the depletion of thiols and addition to cellular nucleophiles are also common to other cytotoxic quinones, the unique genotoxicity of RK and rhododendron could be ascribed to their ability to exhibit multiple redox reactions that produce not only their corresponding quinonoid derivatives but also several side chain desaturated quinonoid species. In addition, a plethora of dimeric and trimeric compounds are produced, all with the ability to cause reactive oxygen species production, depletion of cellular thiols, and reaction with cellular macromolecules including proteins and DNA [11,24]. Compounds exhibiting such multiple redox reactions will therefore be more toxic than simple quinonoid compounds. It is rather difficult to pinpoint one or any other products of RK or rhododendron as a causative agent for inducing leukoderma and other myelotoxic effects. With these results in mind, we caution against the use of these compounds and other related catechols which have the potency to exhibit multiple redox reactions for the treatment of any melanin-related disorders.

3. Materials and Methods

Materials: DBL catechol was procured from Fujifilm-Wako Pure Chemicals (Osaka, Japan). Mushroom tyrosinase (specific activity 5771 units/mg of protein) was purchased from Sigma Chemical Co., St. Louis, MO. HPLC grade methanol and ammonium formate (99%) were obtained from Acros, Morris Plains NJ. Milli Q synthesis A10 Water purification system purchased from Millipore, Milford, MA was used to prepare HPLC grade water. Mobile phase solvents (formic acid, acetonitrile) for mass spectrometry were purchased from Fisher Chemical (Fair Lawn, NJ, USA) and were Optima LC/MS Grade. All other chemicals were of analytical grade and purchased from Fisher and/or VWR.

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Enzyme assays: A reaction mixture (1 mL) containing DBL catechol (usually 0.2 mM), about 5–10 µg of mushroom tyrosinase in 50 mM sodium phosphate buffer at specified pH was incubated at room temperature and the spectral changes associated with the oxidation was followed using a diode array spectrophotometer. Some reactions were conducted in acidic conditions. Chemical oxidation of DBL catechol with sodium periodate was conducted in a mole-to-mole ratio at specified pH values. Exact conditions are given under each figure legend.

Sample preparation for mass spectral studies: A reaction mixture containing 100 nmol of DBL catechol and 5 µg of tyrosinase was incubated in 1 mL of water at room temperature for two min and an aliquot of the reaction (100 mL) was quenched with (900 mL) 1% trifluoroacetic acid. This diluted mixture was subjected to mass spectrometric analysis. The diluted reaction was directly injected into the mass spectrometer. RP-nLC/ESI-MS conditions: An Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher, San Jose, CA, USA) coupled online to an EASY-nLC 1200 (Thermo Fisher, San Jose, CA, USA) was used to detect and characterize the products. The nLC system was operated at a fellow rate of 300 nL/min using a linear gradient of 0–70% B in 15 min. Mobile phase A was 96.1:3.9 0.1% formic acid in water/0.1% formic acid in acetonitrile. Mobile phase B was 80.0:20.0 0.1% formic acid in water/0.1% formic acid in acetonitrile. The sample was first desalted on a Thermo Fisher Scientific Acclaim PepMap 100 C18 HPLC column (3 µm particle size, 75 µm × 2 cm, 100 Å) before separation on a Thermo Fisher Scientific PepMap RSLC C18 EASY-Spray Column (3 µm particle size, 75 µm × 15 cm, 100 Å).

The Orbitrap Fusion Lumos mass spectrometer was operated in the small molecule mode. The global settings were as follows: ion source type NSI, a positive voltage of 1900 V, and an Ion Transfer Tube Temp of 275 ◦C. Ions for the MS scans were detected in the Orbitrap with a resolution of 30,000. The mass range was normal, and the scan range was set to 100–1000 m/z. The RF lens was set to 30% and the AGC target and maximum injection time were 4.0 × 105 and 50 ms, respectively. The data-dependent MS2 CID scans were run in conjunction with a targeted mass filter in which the targeted masses corresponded to the following protonated species: DBL (179.0708 m/z), DBL-quinone (177.0551 m/z), DBL-quinone dimer (355.1182 m/z), DBL-quinone trimer (529.1499 m/z), DBL-water adduct (197.0813 m/z), and DBL-dimer with a loss of 2H (353.1026 m/z). An intensity threshold of 2.0 × 103 was set on each mass with a mass tolerance of ± 10 ppm. Ions for the ddMS2 CID were isolated in the ion trap with a rapid scan rate and with an isolation window of 2 m/z. Ions were fragmented via CID with a fixed collision energy of 40%. The Q parameter for the CID activation was set to 0.25. The AGC target and maximum injection time were set to 1.0 × 104 and 500 ms. The cycle time for the data-dependent acquisition was set to 3 s.

Author Contributions: Conceptualization, M.S., S.I., and K.W.; methodology, M.S., J.E., R.M., and K.U.; formal analysis, M.S. and J.E.; investigation, M.S., J.E., R.M., and K.U.; resources, M.S., S.I., and K.W.; data curation, M.S., J.E., R.M., and K.U.; writing—original draft preparation, M.S.; writing—review and editing, M.S., S.I., K.W., and J.E.; visualization, M.S.; supervision, M.S. and J.E.; project administration, M.S. All authors have read and agreed to the published version of the manuscript.

Funding: This research received no external funding.

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

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Abbreviations

CID Collision induced decomposition

DBL 3,4-dihydroxybenzalacetone

LC/MS High-pressure liquid chromatography/mass spectrometry

RK Raspberry ketone

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