molecules

Article Mulinane and Azorellane Diterpenoid Biomarkers by GC-MS from a Representative (Umbelliferae) Species of the

Bernd R.T. Simoneit 1,*, Daniel R. Oros 2, Rudolf Jaffé 3 , Alexandra Didyk-Peña 4,*, Carlos Areche 5, Beatriz Sepúlveda 6 and Borys M. Didyk 7

1 Department of Chemistry, College of Science, Oregon State University, Corvallis, OR 97331, USA 2 Consultant, 72 Marina Lakes Drive, Richmond, CA 94804, USA; [email protected] 3 Southeast Environmental Research Center and Department of Chemistry and Biochemistry, Florida International University, 3000 NE 151st Street, North Miami, FL 33181, USA; jaffer@fiu.edu 4 Escuela de Química y Farmacia, Facultad de Medicina, Universidad Andres Bello, Viña del Mar 2520000, 5 Departamento de Química, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago 8320000, Chile; [email protected] 6 Departamento de Ciencias Químicas, Universidad Andres Bello, Viña del Mar 2520000, Chile; [email protected] 7 Consultant, Casilla 942, Viña del Mar 2520000, Chile; [email protected] * Correspondence: [email protected] (B.R.T.S.); [email protected] (A.D.-P.); Tel.: +1-541-737-2081 (B.R.T.S.)  Academic Editor: Motoo Tori  Received: 16 January 2019; Accepted: 13 February 2019; Published: 14 February 2019

Abstract: Extracts of bled resin from compacta, of the Azorelloideae family from the Andes (>4000 m), were analyzed by gas chromatography-mass spectrometry. The mass spectra of the dominant compounds of the resin and its hydrogenation products were documented. The most abundant compounds were oxygenated diterpenoids, namely mulinadien-20-oic (∆11,13 and ∆11,14) acids, azorell-13-en-20-oic acid, 13α,14β-dihydroxymulin-11-en-20-oic acid, and azorellanol, with a group of azorellenes and mulinadienes. The mass spectra of the novel diterpenoid hydrocarbons with the azorellane and mulinane skeletons were also presented. This study documents the molecular diversity of these diterpenoid classes, and could be of great utility for future organic geochemical, environmental, archeological, pharmaceutical, and forensic chemistry studies.

Keywords: Azorella compacta; diterpenoids; GC-MS; standards

1. Introduction Natural products or their derivatives in the ambient environment or geological record are used by organic geochemists as tracers for sources, transport and alteration processes of organic matter, e.g., [1–7]. Remote locales, for example, the high Andes region in , are ideal sites as pristine references for background studies of such processes. An early report on the lipids and biomarkers in sediment from Lake Lejia in a caldera in Antofagasta, Chile, found traces of diterpenoids typical of resinous vegetation [8]. The region is barren of trees, especially gymnosperms that produce resin, and potential sources such as the Araucariaceae grow far to the south [9]. Subsequently, we reported on the lipid and resin composition of a dried sample of Laretia compacta (common name Llareta) from San Pedro de Atacama [10]. The dried material had a strong resinous odor and thus was implied as a possible source of diterpenoids in the Altiplano environment. The extract yield of the whole based on dry weight comprised 4.2 mg/g (0.2%) lipids and 2.5 mg/g (0.12%)

Molecules 2019, 24, 684; doi:10.3390/molecules24040684 www.mdpi.com/journal/molecules Molecules 2019, 24, x 684 FOR PEER REVIEW 22 of of 15 extract yield of the whole plant based on dry weight comprised 4.2 mg/g (0.2%) lipids and 2.5 mg/g (0.12%)terpenoids terpenoids [10]. The [10]. terpenoid The terpenoid distribution distribution consisted consisted of mono-, of sesqui- mono‐ and, sesqui diterpenoids,‐ and diterpenoids, and the latter and theare latter of interest are of here interest because here tri- because and tetracyclic tri‐ and tetracyclic diterpenoids diterpenoids were the were dominant the dominant compounds. compounds. Laretia compacta is now called called AzorellaAzorella compacta compacta ofof the the Apiaceae:Azorelloideae Apiaceae:Azorelloideae family, family, also known as Umbelliferae [11]. [11]. These grow slowly at high altitudes (>4000 m) in in the northern Andes of Chile and as dense dense pillow pillow-like‐like structures structures close close to to the the ground ground with with subterranean subterranean stems stems and and roots. roots. The Azorelloideae family evolved from thethe firstfirst UmbelliferaeUmbelliferae duringduring thethe Cretaceous,Cretaceous e.g., [12,13]. [12,13]. Azorella sp. have historically been used on the Altiplano as firewood firewood and cooking fuel by local cultures, andand laterlater in in early early industrial industrial and and mining mining activities activities as as fuel, fuel, which which contributed contributed to its to scarcity its scarcity and andcurrent current protection. protection. The speciesThe species is now is now more more common, common, and represents and represents the dominant the dominant biomass biomass in these in Andeanthese Andean environments. environments. The plants The andplants resin and exudates resin exudates have been have used been by Andeanused by cultures Andean for cultures medicinal for medicinalpurposes where, purposes for example,where, for hot example, water infusions hot water serve infusions as herbal serve remedies as herbal for various remedies ailments for various [14,15]. ailmentsConsequently, [14,15]. natural Consequently, product characterizationnatural product andcharacterization pharmacological and pharmacological studies have been studies carried have out beenon A. carried compacta outand on related A. compacta species. and Numerous related species. investigations Numerous reported investigations structure determinations reported structure and determinationspharmacological and potential pharmacological studies of potential the resin studies compounds, of the thatresin were compounds, isolated that and were purified isolated from and the purifiedmajor Andean from the Apiaceae major Andean species, Apiaceae namely Mulinum species, namelyand Azorella Mulinum[16– and35]. Azorella This has [16–35]. led to a This description has led toof a mainly description two novel of mainly diterpenoid two novel skeletons diterpenoid among skeletons the natural among products, the natural specifically products, the specifically mulinanes theand mulinanes azorellanes and (Figure azorellanes1), with the(Figure assignment 1), with of theirthe assignment skeletal numbering of their conventionsskeletal numbering [ 16,21]. conventionsThe biosynthetic [16,21]. pathway The biosynthetic for the mulinane pathway and for azorellane the mulinane diterpenoids and azorellane has been diterpenoids proposed as has starting been proposedfrom a phytatetraene as starting from [24]. a Laboratory phytatetraene syntheses [24]. Laboratory of polar mulinane syntheses diterpenoids of polar mulinane have diterpenoids also recently havebeen reportedalso recently [36, 37been]. reported [36,37].

Skeleton numbering convention

16 16 16 16

12 12 12 12 13 13 13 13 11 11 14 14 11 11 17 17 17 14 17 14 1 9 1 9 9 1 9 10 10 1 10 15 15 10 15 15 8 8 8 2 2 8 5 5 2 2 3 3 5 5 7 7 3 7 3 7 6 6 4 4 4 6 4 6 18 20 18 20 18 20 18 20

19 19 19 19 13(H)-Mulinane 13(H)-Mulinane 13(H)-Azorellane 13(H)-Azorellane Figure 1. CarbonCarbon numbering numbering convention for the mulinane and azorellane skeletons.

Here, we characterize the molecular composition of Azorella compacta resin based on mass spectrometric interpretation, interpretation, and and correlation correlation and and comparison comparison with with known known standards. standards. We Wereport report the massthe mass spectra spectra and and gas gas chromatography chromatography-mass‐mass spectrometry spectrometry (GC (GC-MS)‐MS) characteristics characteristics of of the the dominant dominant compounds of of the the resin resin and and of ofthe the hydrogenated hydrogenated resin resin as the as thefree freeand andderivatized derivatized products. products. This informationThis information is unique, is unique, because because prior prior reports reports in inthe the natural natural product product literature literature only only provided provided mass spectrometric data on some compounds. Our data further documents the molecular diversity of this novel diterpenoidditerpenoid class class (mulinane (mulinane and and azorellane), azorellane), information information which which could could be of be great of great utility utility for future for futureorganic organic geochemical, geochemical, environmental, environmental, archeological, archeological, pharmaceutical, pharmaceutical, and forensic and chemistryforensic chemistry studies. studies. 2. Results and Discussion 2. ResultsThe relative and Discussion composition of the fresh resin was 1% sesquiterpenoid and 99% diterpenoids, excludingThe relative a low amountcomposition of bornyl of the acetate fresh resin (<0.5%). was No1% lipidsesquiterpenoid components and from 99% plant diterpenoids, wax were excludingdetected. Ita islow of amount interest toof notebornyl the acetate breakdown (<0.5%). of theNo diterpenoidslipid components into 19% from hydrocarbons plant wax were and detected.80% polar It oxygenated is of interest compounds, to note the breakdown and the same of the proportions diterpenoids were into retained 19% hydrocarbons upon hydrogenation. and 80% polarThis observationoxygenated wascompounds, unexpected and becausethe same most proportions bled conifer were resinsretained are upon characterized hydrogenation. by higher This observationoxygenated was diterpenoid unexpected contents because (Simoneit, most bled unpublished conifer resins data). are characterized by higher oxygenated diterpenoid contents (Simoneit, unpublished data).

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TypicalTypical examples examples ofof GC GC-MS‐MS total total ion ion current current traces traces for forA. compactaA. compacta resinresin and hydrogenated and hydrogenated resin resinindicated indicated the molecular the molecular complexity complexity of the of compound the compound mixtures mixtures (Figure (Figure 2). The2 ).poor The resolution poor resolution of the ofunderivatized the underivatized resin acids resin (cf. acids Figure (cf. Figure2a) was2 a)due was to their due polarity. to their polarity. Sample components Sample components were classified were classifiedinto three into groups three groups of compounds, of compounds, sesquiterpenoid, sesquiterpenoid, diterpenoid diterpenoid hydrocarbons hydrocarbons and and oxygenated oxygenated diterpenoids,diterpenoids, and and are are discussed discussed as such. as such. Only oneOnly sesquiterpenoid, one sesquiterpenoid, namely namely spathulenol spathulenol (I, all structures (I, all arestructures shown in are Appendix shown inA ),Appendix which hydrogenates I), which hydrogenates to two isomers to two of isomers spathulanol of spathulanol (II), was observed. (II), was Theobserved. relative The concentrations relative concentrations of all identified of all compounds identified compounds are listed in are Table listed1. in Table 1.

FigureFigure 2. 2.Examples Examples of of GC-MS GC‐MS total total ion ion currentcurrent tracestraces for A. compacta resinresin and and hydrogenated hydrogenated resin resin analyzedanalyzed as: as: ( a(,ac,)c) total total extracts, extracts, and and ( b(b,d,d)) silylatedsilylated extracts.extracts. Roman numerals numerals refer refer to to structures structures in in TableTable1 and 1 and Appendix AppendixA. A.

Table 1. Relative concentrations of the major compounds identified in resin and hydrogenated resin of Table 1. Relative concentrations of the major compounds identified in resin and hydrogenated resin Azorella compacta. of Azorella compacta.

Structure b c Hydrogenated Structurea Compound Composition MW ID ResinResin (n = 7) Hydrogenated Number Compound Composition b MW ID c Resin (n = 4) a NumberI Spathulenol C H O 220 S 1.4(n = 7) Resin (n = 4) 15 24 I IISpathulenol Spathulanol C15H26OC15H24O 222220 SS 1.4 3 III 20-Normulina-11,13-diene C H 258 T 0.5 II Spathulanol 19 30 C15H26O 222 S 3 IV 20-Norazorell-13-ene C19H30 258 T 1 IIIV 20 20-Norazorellane,‐Normulina‐11,13α + β‐diene C H C19H30 260258 TT 0.5 1 19 32 IVVI 20 20-Normulinane,‐Norazorell‐13α +‐eneβ C19H34 C19H30 262258 TT 1 0.3 VII 13β(H)-Azorell-14-ene C20H32 272 T 9 V 20‐Norazorellane, α + β C19H32 260 T 1 VIII Azorell-13-ene C20H32 272 I 2 19 34 VIIX 20 13‐αNormulinane,(H)-Azorell-14-ene α + β C20H32 C H 272262 TT 2.5 0.3

VIIX 13 13ββ(H)(H)-Mulina-11,14-diene‐Azorell‐14‐ene C20H32 C20H32 272272 IT 16 9 XI Mulina-11,13-diene C20H32 272 L 7 VIII Azorellα ‐13‐ene C20H32 272 I 2 XII 13 (H)-Mulina-11,14-diene C20H32 272 I 4 IXXIII 13 13αβ(H)(H)-Azorellane‐Azorell‐14‐ene C20H34 C20H32 274272 IT 2.5 26 α X XIV13 13β(H)(H)-Azorellane‐Mulina‐11,14‐diene C20H34 C20H32 274272 II 16 15 XV 13β(H)-Mulinane C20H36 276 I 11 XIXVI Mulina 13α(H)-Mulinane‐11,13‐diene C H C20H32 276272 IL 7 8 20 36 XIIXVII 13 13-Hydroxyazorellanesα(H)‐Mulina‐11,14‐diene C20H34OC20H32 290272 LI 4 14 XVIII 13α-Hydroxymulinane C H O 292 L 22 XIII 13β(H)‐Azorellane 20 36 C20H34 274 I 26 XIX 13β(H)-Mulina-11,14-dien-20-oic acid C20H30O2 302 S 10 XIVXX 13 Mulina-11,13-dien-20-oicα(H)‐Azorellane acid C H O C20H34 302274 SI 100 15 20 30 2 XVXXI 13 Azorell-13-en-20-oicβ(H)‐Mulinane acid C20H30O2 C20H36 302276 SI 17 11 XXII 13β(H)-Azorellan-20-oic acid C H O 304 S 5 XVI 13α(H)‐Mulinane 20 32 2 C20H36 276 I 8 XXIII 13α(H)-Azorellan-20-oic acid C20H32O2 304 S 30 XVII 13‐Hydroxyazorellanes C20H34O 290 L 14

XVIII 13α‐Hydroxymulinane C20H36O 292 L 22

XIX 13β(H)‐Mulina‐11,14‐dien‐20‐oic acid C20H30O2 302 S 10

XX Mulina‐11,13‐dien‐20‐oic acid C20H30O2 302 S 100

XXI Azorell‐13‐en‐20‐oic acid C20H30O2 302 S 17

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XXII 13β(H)‐Azorellan‐20‐oic acid C20H32O2 304 S 5

XXIII 13α(H)‐Azorellan‐20‐oic acid Table 1.C20ContH32O.2 304 S 30

XXIV 13β(H)‐Mulinan‐20‐oic acid C20H34O2 306 S 44

StructureXXV 13α(H)‐Mulinan‐20‐oic acid C20bH34O2 306 c S Hydrogenated100 a Compound Composition MW ID Resin (n = 7) NumberXXVI Mulinol C20H34O2 306 L 7 Resin (n = 4)

XXIVXXVII 13Dihydromulinolβ(H)-Mulinan-20-oic acid C20H34O2 C20H36O2 306308 SI 446 XXV 13α(H)-Mulinan-20-oic acid C H O 306 S 100 XXVIII 13α‐Hydroxymulin‐11‐en‐20‐oic acid 20 34 2 C20H32O3 320 S 8 XXVI Mulinol C20H34O2 306 L 7 XXVIIXXIX Dihydromulinol13α‐Hydroxymulinan‐20‐oic acid C H O C20H34O3 308322 IS 25 6 20 36 2 XXVIIIXXX 1320α-Hydroxymulin-11-en-20-oic‐Acetoxymulina‐11,13‐diene acid C20H32O3 C22H34O2 320330 SL 15 8 α XXIXXXXI 1313-Hydroxymulinan-20-oicα,14β‐Dihydroxymulin acid‐11‐en‐20‐oic C acid20H34 O3 C20H30O4 322336 SL 11 25 XXX 20-Acetoxymulina-11,13-diene C22H34O2 330 L 15 XXXII 13α,14β‐Dihydroxymulinan‐20‐oic acid C20H34O4 338 I 32 XXXI 13α,14β-Dihydroxymulin-11-en-20-oic acid C20H30O4 336 L 11 XXXIIXXXIII 13Azorellanolα,14β-Dihydroxymulinan-20-oic acid C20H34O4 C22H36O3 338348 IL 16 12 32 XXXIIIa Structures Azorellanol are shown in Appendix A. b Listed C22H36 asO 3the natural 348products, polar L compounds 16 analyzed 12 as a b Structuresmethyl areesters shown and/or in Appendix trimethylsilylA. Listed derivatives. as the natural c Identification: products, polar I = interpretation compounds analyzed and correlation as methyl of esters and/or trimethylsilyl derivatives. c Identification: I = interpretation and correlation of MS fragmentation pattern andMS GC retentionfragmentation time as pattern derivative and from GC standard, retention T =time suggested as derivative interpretation from basedstandard, on MS T and= suggested GC retention timeinterpretation only, S = standard, based L =on literature MS and citation, GC retention n = number time of only, analyses. S = standard, L = literature citation, n = number of analyses. 2.1. Diterpenoid Hydrocarbons 2.1. Diterpenoid Hydrocarbons There were two norditerpenes, 20-normulina-11,13-diene (III) and 20-norazorell-13-ene (IV), There were two norditerpenes, 20‐normulina‐11,13‐diene (III) and 20‐norazorell‐13‐ene (IV), which upon hydrogenation yielded four isomers, namely 20-normulinane (VI) and 20-norazorellane (V) which upon hydrogenation yielded four isomers, namely 20‐normulinane (VI) and 20‐norazorellane both as the 13β(H) and 13α(H)-epimers (Figure3a–f, also see Figure SM-2b). We inferred the absence (V) both as the 13β(H) and 13α(H)‐epimers (Figure 3a–f, also see Figure SM‐2b). We inferred the of C-20absence for theseof C‐ novel20 for norditerpenes, these novel norditerpenes, rather than C-16 rather or C-17,than basedC‐16 or on C the‐17, facile based elimination on the facile of the functionalizedelimination C-20of the group functionalized in the mass C‐ spectra20 group of thein the acid mass standards spectra discussed of the acid below. standards The configuration discussed at C-5below. is most The likelyconfiguration 5β(H), butat C‐ based5 is most on likely the preferential 5β(H), but based loss or on absence the preferential of a C-20 loss functional or absence group, of coulda C also‐20 functional be represented group, as could 5α(H). also be represented as 5α(H).

Figure 3. Cont.

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Figure 3. Cont.

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FigureFigure 3. 3.Mass Mass spectra spectra of of the the diterpenoid diterpenoid hydrocarbons hydrocarbons in A. incompactaA. compacta resins. Theresins. KI values The relative KI values relativeto n‐alkanes to n-alkanes on a DB on‐5 column a DB-5 are column shown in are parentheses shown inon each parentheses mass spectrum: on each (a) 20 mass‐normulina spectrum:‐ (a) 20-normulina-11,13-diene11,13‐diene (III), (b) 20‐norazorell (III), (b‐13) 20-norazorell-13-ene‐ene (IV), (c) 13β(H)‐20 (IV),‐normulinane (c) 13β(H)-20-normulinane (VI), (d) 13α(H)‐20 (VI),‐ (d)normulinane 13α(H)-20-normulinane (VI), (e) 13β(H) (VI),‐20‐norazorellane (e) 13β(H)-20-norazorellane (V), (f) 13α(H)‐20‐norazorellane (V), (f) 13 (V),α(H)-20-norazorellane (g) azorell‐13‐ene (V),(VIII), (g) azorell-13-ene(h) 13β(H)‐azorell (VIII),‐14‐ene ( h(VII),) 13 (βi)(H)-azorell-14-ene 13α(H)‐azorell‐14‐ene (VII), (IX), (ji) mulina 13α(H)-azorell-14-ene‐11,13‐diene (XI), (k (IX),) (j) mulina-11,13-diene13β(H)‐mulina‐11,14 (XI),‐diene (k (X),) 13 (βl)(H)-mulina-11,14-diene 13α(H)‐mulina‐11,14‐diene (X), (XII), (l) 13(mα) (H)-mulina-11,14-diene13β(H)‐azorellane (XIII), (n (XII),) (m)13 13αβ(H)(H)-azorellane‐azorellane (XIV), (XIII), (o) 13 (nβ)(H) 13‐mulinaneα(H)-azorellane (XV), and (XIV), (p) 13α ((H)o)‐ 13mulinaneβ(H)-mulinane (XVI). (XV), and (p) 13α(H)-mulinane (XVI). There were six isomers of C20 diterpene hydrocarbons, which after hydrogenation yielded four isomers of the saturated diterpanes. The interpretations of the mass spectra as mulina‐11,13‐diene There were six isomers of C20 diterpene hydrocarbons, which after hydrogenation yielded four isomers(XI), 13 ofβ the(H)‐ saturatedmulina‐11,14 diterpanes.‐diene (X) Theand 13 interpretationsα(H)‐mulina‐11,14 of the‐diene mass (XII) spectra (Figure as 3j–l) mulina-11,13-diene were inferred (XI),from 13β (H)-mulina-11,14-dienethe literature [32], although (X) the and match 13α(H)-mulina-11,14-diene was poor. The identifications (XII) (Figure of azorell3j–l)‐13 were‐ene (VIII), inferred 13β(H)‐azorell‐14‐ene (VII) and 13α(H)‐azorell‐14‐ene (IX) were based on the GC elution order and from the literature [32], although the match was poor. The identifications of azorell-13-ene (VIII), interpretations of the mass spectrometric fragmentation patterns (Figure 3g–i; details are given in 13β(H)-azorell-14-ene (VII) and 13α(H)-azorell-14-ene (IX) were based on the GC elution order and Figure SM‐2b) when compared to the C‐20 functionalized standards. interpretations of the mass spectrometric fragmentation patterns (Figure3g–i; details are given in Upon hydrogenation to the mulinane and azorellane skeletons (Figure 1), we obtained two Figureisomers, SM-2b) i.e., when 13α(H) compared and 13β(H), to thefor the C-20 position functionalized of the methyl standards. group at C‐13. We assigned the elution orderUpon as hydrogenationwas observed for to the the α and mulinane β kaurane and and azorellane phyllocladane skeletons standards (Figure [38,39],1), that we is obtained the 13β(H) two isomers,isomer i.e., eluted 13α prior(H) and to the 13 13β(H),α(H) for epimer. the position The diterpanes, of the namely methyl 13 groupβ(H)‐azorellane at C-13. We(XIII), assigned 13α(H)‐ the elutionazorellane order as(XIV), was 13 observedβ(H)‐mulinane for the (XV),α and andβ kaurane 13α(H)‐mulinane and phyllocladane (XVI), have standards similar mass [38, 39spectra], that is the(Figure 13β(H) 3m–p), isomer but eluted different prior molecular to the 13 weights.α(H) epimer. The key Theion for diterpanes, all is m/z 191, namely with M 13‐Cβ3(H)-azorellaneH7 (m/z 231) (XIII),for 13 theα(H)-azorellane azorellanes and (XIV), M‐C5H 1311β ((H)-mulinanem/z 205) for the (XV), mulinanes and 13 (cf.α(H)-mulinane Supplementary (XVI), material have Figure similar SM mass‐ spectra2b). (FigureThese four3m–p), compounds but different may be molecular potential weights. biomarkers The for key the ion geologic for all record is m/ zand191, of with interest M-C to3 H7 organic geochemistry. The mass spectra of both C20 hydrocarbon groups are the first report of these (m/z 231) for the azorellanes and M-C5H11 (m/z 205) for the mulinanes (cf. Figure SM-2b). These four compoundsnovel parent may diterpenoid be potential skeletons, biomarkers and for they the do geologic not match record with and any of mass interest spectra to organic of other geochemistry. reported tri‐ and tetracyclic diterpanes (e.g., abietanes, atisanes, pimaranes, kauranes, phyllocladanes, etc.). The mass spectra of both C20 hydrocarbon groups are the first report of these novel parent diterpenoid skeletons, and they do not match with any mass spectra of other reported tri- and tetracyclic diterpanes 2.2. Oxygenated Diterpenoids (e.g., abietanes, atisanes, pimaranes, kauranes, phyllocladanes, etc.). The oxygenated diterpenoids comprised mainly mulina‐11,13‐dien‐20‐oic acid (XX) with the 2.2.isomers Oxygenated 13β Diterpenoids(H)‐mulina‐11,14‐dien‐20‐oic (XIX) and azorell‐13‐en‐20‐oic (XXI) acids. Their identification was based on comparison of the mass spectra of standard natural products as the free The oxygenated diterpenoids comprised mainly mulina-11,13-dien-20-oic acid (XX) and derivatized compounds (Figure 4d–f,k–o and Supplementary material Figure SM‐1o–p, β withrespectively), the isomers and 13 their(H)-mulina-11,14-dien-20-oic respective GC retention indices. (XIX) One characteristic and azorell-13-en-20-oic of the mass spectra (XXI) of acids.all TheirC‐ identification20 carboxylic acids was and based C‐20 on methyl comparison or TMS ofcarboxylates the mass spectrais the facile of standard loss of that natural moiety products from the as the freerespective and derivatized molecular ion compounds (cf. Supplementary (Figure4 materiald–f,k–o Figure and Figure SM‐2a). SM-1o–p, respectively), and their respective GC retention indices. One characteristic of the mass spectra of all C-20 carboxylic acids and C-20 methyl or TMS carboxylates is the facile loss of that moiety from the respective molecular ion (cf. Figure SM-2a).

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Figure 4. Cont.

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Figure 4. Cont.

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FigureFigure 4. Mass4. Mass spectra spectra of of the the oxygenated oxygenated diterpenoids diterpenoids in A.A. compacta compacta resins,resins, including including KI KIvalues values shownshown in in parentheses: parentheses: (a ()a 13)β‐ 13hydroxyazorellaneβ-hydroxyazorellane (XVII), (XVII), (b) 13α‐ (bhydroxyazorellane) 13α-hydroxyazorellane (XVII), (c) 13 (XVII),α‐ (c)hydroxymulinane 13α-hydroxymulinane (XVIII), ( (XVIII),d) 13β(H) (‐dmulina) 13β‐11,14(H)-mulina-11,14-dien-20-oic‐dien‐20‐oic acid (XIX, standard), acid (e (XIX,) mulina standard),‐11,13‐ (e) mulina-11,13-dien-20-oicdien‐20‐oic acid (XX, standard), acid (f) (XX, azorell standard),‐13‐en‐20‐oic (f )acid azorell-13-en-20-oic (XXI, standard), (g) 13 acidβ(H) (XXI,‐azorellan standard),‐20‐ (g) 13oicβ (H)-azorellan-20-oicacid (XXII, standard), acid(h) 13 (XXII,α(H)‐azorellan standard),‐20 (‐hoic) 13 acidα(H)-azorellan-20-oic (XXIII, standard), (i) acid13β(H) (XXIII,‐mulinan standard),‐20‐ (i) 13oicβ (H)-mulinan-20-oicacid (XXIV, standard), acid (j) 13 (XXIV,α(H)‐mulinan standard),‐20‐oic (j) acid 13α (XXV,(H)-mulinan-20-oic standard), (k) methyl acid (XXV, mulina standard),‐11,13‐ (k) methyldien‐20 mulina-11,13-dien-20-oate‐oate (XX, standard), (l) methyl (XX, 13 standard),β(H)‐mulina (l)‐11,14 methyl‐dien 13‐β20(H)-mulina-11,14-dien-20-oate‐oate (XIX, standard), (m) methyl (XIX, standard),13β(H)‐ (mulinanm) methyl‐20‐ 13oateβ(H)-mulinan-20-oate (XXIV, standard), (n) (XXIV,methyl standard), 13α(H)‐mulinan (n) methyl‐20‐oate 13 (XXV,α(H)-mulinan-20-oate standard), (o) (XXV,methyl standard), azorell (‐o13)‐ methylen‐20‐oate azorell-13-en-20-oate (XXI, standard), (p) (XXI,20‐acetoxymulina standard), (‐p11,13) 20-acetoxymulina-11,13-diene‐diene (XXX), (q) 13α,14β‐ (XXX),dihydroxymulinan (q) 13α,14β-dihydroxymulinan-20-oic‐20‐oic acid (XXXII), (r) azorellanol acid (XXXII),(XXXIII, standard), (r) azorellanol (s) mulinol (XXXIII,‐diTMS standard),(XXVI), (s) mulinol-diTMS(t) dihydomulinol (XXVI),‐diTMS (XXVII), (t) dihydomulinol-diTMS (u) 13α‐hydroxymulin (XXVII),‐11‐en‐20 (u‐oic) 13 acidα-hydroxymulin-11-en-20-oic‐diTMS (XXVIII, mulinolic acid-diTMSacid‐diTMS, (XXVIII, standard), mulinolic (v) 13α‐hydroxymulinan acid-diTMS,‐20‐ standard),oic acid‐diTMS (v (XXIX,) 13α standard),-hydroxymulinan-20-oic (w) 13α,14β‐ dihydroxymulin‐11‐en‐20‐oic acid‐triTMS (XXXI), and (x) 13α,14β‐dihydroxymulinan‐20‐oic acid‐ acid-diTMS (XXIX, standard), (w) 13α,14β-dihydroxymulin-11-en-20-oic acid-triTMS (XXXI), triTMS (XXXII). and (x) 13α,14β-dihydroxymulinan-20-oic acid-triTMS (XXXII). Significant amounts of 20‐acetoxymulina‐11,13‐diene (XXX), azorellanol (7β‐acetoxy‐13α‐ Significant amounts of 20-acetoxymulina-11,13-diene (XXX), azorellanol hydroxyazorellane, XXXIII), and 13α,14β‐dihydroxymulin‐11‐en‐20‐oic acid (XXXI) were also (7β-acetoxy-13α-hydroxyazorellane, XXXIII), and 13α,14β-dihydroxymulin-11-en-20-oic acid (XXXI) identified. The major fragment ions of the mass spectrum of 20‐acetoxymulina‐11,13‐diene (Figure were4p) also matched identified. with Thethose major listed fragment in the literature ions of for the its mass direct spectrum insertion ofprobe 20-acetoxymulina-11,13-diene mass spectrum [26]. The (Figuremass4p) spectrum matched of the with azorellanol those listedstandard in (Figure the literature 4r and Supplementary for its direct material insertion Figure probe SM‐1v mass as spectrumthe TMS [26 derivative)]. The mass matched spectrum with its ofliterature the azorellanol listing [21]. standard There was (Figure also a trace4r and of the Figure tentatively SM-1v as theidentified TMS derivative)13‐epi‐azorellanol matched (Supplementary with its literature material listingFigure [21SM].‐1k), There which was eluted also prior a trace to of theazorellanol. tentatively The identified structure 13- ofepi 13-azorellanolα,14β‐dihydroxymulin (Figure SM-1k),‐11‐en‐20‐ whichoic acid eluted(Figure prior4w) was to based azorellanol. on Thecorrelation structure ofwith 13 αthe,14 standardβ-dihydroxymulin-11-en-20-oic 13α‐hydroxy‐14‐oxomulin acid‐11‐en (Figure‐20‐oic 4acidw) was(XXXIV, based Supplementary on correlation withmaterial the standard Figure 13SMα‐-hydroxy-14-oxomulin-11-en-20-oic1j,w,z). Mulinol (13α,20‐dihydroxymulin acid‐11‐ (XXXIV,ene, XXVI) Figure and 13 SM-1j,w,z).α‐hydroxymulin Mulinol‐ (13α11,20-dihydroxymulin-11-ene,‐en‐20‐oic acid (mulinolic acid, XXVI) XXVIII) and were 13 αobserved-hydroxymulin-11-en-20-oic as minor components. The acid mass (mulinolic spectrum acid, XXVIII)of mulinol were observed (Figure 4s) as minorwas correlated components. with Thethat mass of its spectrum hydrogenation of mulinol product (Figure (Figure4s) was4v) and correlated the withpublished that of its MS hydrogenation listing [20]. The product mass (Figurespectra4 ofv) mulinolic and the published acid matched MS listingwith those [ 20]. of The the mass standard spectra of mulinolic(Figure 4u acid and matched Supplementary with those material of the Figure standard SM‐1d,h). (Figure 4u and Figure SM-1d,h). The hydrogenated resin sample contained the corresponding saturated diterpenoids (Figure 2c,d). As discussed above for the hydrocarbons, the elution order assumed for the dominant 13β(H)‐ and 13α(H)‐mulinan‐20‐oic acids (XXIV and XXV, respectively) was beta before alpha (Figure 4i,j,m,n

Molecules 2019, 24, 684 10 of 15

The hydrogenated resin sample contained the corresponding saturated diterpenoids (Figure2c,d). As discussed above for the hydrocarbons, the elution order assumed for the dominant 13β(H)- and 13α(H)-mulinan-20-oic acids (XXIV and XXV, respectively) was beta before alpha (Figure4i,j,m,n and Figure SM-1r,s, respectively). The same was the case for the 13β(H)- and 13α(H)-azorellan-20-oic acids (XXII and XXIII, respectively) and their mass spectra are shown in Figure4g,h and Figure SM-1r,s, respectively (refer to Figure SM-2a). These diterpanoid acids may be potential diagenetic products in the geologic record and thus are of interest to organic geochemistry. The hydrogenation products of mulinol and dihydroxymulin-11-en-20-oic acid were also detectable as dihydromulinol (XXVII, Figure4t) and 13 α,14β-dihydroxymulinan-20-oic acid (XXXII, Figure4x).

3. Samples and Experimental Methods A sample of A. compacta resin was obtained by cutting to bleed an Azorella compacta plant growing off the road B245 from El Tatio to San Pedro de Atacama in the Paso la Vizcacha (Chile, Region II, 22◦22034”S, 68◦100”W, altitude 4556 m). The fresh resin was split into two parts, one dissolved completely in dichloromethane/methanol (DCM/MeOH, 3:1 v/v), and the duplicate sample was dissolved in n-hexane. Aliquots (50 µL) of these total extracts were converted to trimethylsilyl derivatives by reaction with N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA, Sigma-Aldrich, St. Louis, MO, USA) and pyridine for 3 h at 70 ◦C, e.g., [40]. Prior to analysis, the excess silylating reagent was evaporated by nitrogen blow down and the sample mixture dissolved in an equivalent volume of n-hexane. Other aliquots of sample solutions were treated with trimethylsilyldiazomethane (2M in hexane, Sigma-Aldrich) at room temperature for 30 min to methylate carboxylic acids [41]. The excess reagent was reacted with concentrated acetic acid, the solution dried by nitrogen blow down, and the products dissolved in DCM/MeOH (3:1) for analysis. The following reference standards were also derivatized and analyzed by these methods: spathulenol (I), mulina-11,13-dien-20-oic acid (XX), azorell-13-en-20-oic acid (XXI), 13α-hydroxymulin-11-en-20-oic acid (XXVIII), azorellanol (XXXIII), 13α-hydroxy-14-oxomulin-11-en-20-oic acid (XXXIV), 7β,13α-dihydroxymulin-11-ene (XXXV), 15α-acetoxymulina-11,13-dien-20-oic acid (XXXVI), and 18-acetoxymulina-11,13-diene-16,20-dioic acid (XXXVII). Aliquots of the total extracts and mulina-11,13-dien-20-oic acid were hydrogenated. The samples were diluted with DCM/MeOH (3:1) in a round bottom flask (50 mL) with a magnetic stir bar and PtO2 (Arcos Organics, Fisher, Pittsburgh, PA, USA, 83% Pt) as catalyst. H2 at atmospheric pressure was installed with a latex balloon (500 mL) over the neck of the flask and hydrogenation allowed to proceed at room temperature for 8 h. The catalyst was filtered off and the sample concentrated by nitrogen blow down for analysis. Molecules 2019, 24, 684 11 of 15

The gas chromatography-mass spectrometry (GC-MS) analyses of the total extracts and aliquots of the derivatized and hydrogenated total extracts (typical injection volume 1 µL) were performed on a Hewlett-Packard model 6890 GC coupled to a Hewlett-Packard model 5973 MSD (Palo Alto, CA, USA)). Separation of compounds was achieved on a fused silica capillary column coated with DB-5MS (Agilent, Santa Clara, CA, USA) 30 m × 0.25 mm i.d., 0.25 µm film thickness). The GC operating conditions were as follows: temperature held at 65 ◦C for 2 min, increased from 65 to 300 ◦C at a rate of 6 ◦C min−1, and final isothermal held at 300 ◦C for 20 min. Helium was used as carrier gas. The samples were injected splitless with the injector temperature at 280 ◦C. The mass spectrometer was operated in the electron impact mode at 70 eV and scanned from 50 to 650 da. Data were acquired and processed with the Chemstation software (Hewlett-Packard, Palo Alto, CA, USA). Compounds were identified by comparison of mass spectra and retention times with those of authentic standards, with literature data, and interpretation of mass spectrometric fragmentation patterns of unknowns. The Kovats [42] GC retention indices (KI) relative to n-alkanes are shown on each mass spectrum and are determined for elution on a DB-5 capillary column. The extract of the dried plant (prior report by [10]) was reanalyzed. The dominant diterpenoids were the same as found in the new resin sample and are not discussed further.

4. Conclusions In this study, we document the molecular characteristics and diversity of novel diterpenoid classes of natural products, namely mulinanes and azorellanes, from resin of the Andean Apiaceae plant species Azorella compacta. The dominant compounds were oxygenated diterpenoids, mainly mulinadien-20-oic (∆11,13 and ∆11,14) acids, azorell-13-en-20-oic acid, 13α,14β-dihydroxymulin-11-en-20-oic acid, and azorellanol, as confirmed by standards. The diterpenoid hydrocarbons included mulinadienes and azorellenes, which are novel compounds of potential interest to organic geochemistry. The mulinane and azorellane diterpenoid hydrocarbons, resulting from the hydrogenation of the resin, may be potential biomarkers for the geologic record, while the corresponding oxygenated diterpenoids may be applied for environmental studies.

Supplementary Materials: Additional mass spectra of related and derivatized natural products are collected in the Supplemental Materials section available with this article. Author Contributions: B.R.T.S. and B.M.D. conceived and designed the work; B.R.T.S., R.J., C.A. and B.S. performed the experiments; B.R.T.S. and D.R.O. analyzed and interpreted the data; B.R.T.S. wrote the paper, and all coauthors reviewed and edited the paper. Funding: This research received no external funding. Acknowledgments: We thank Ljubomir Tomasevic for the bled resin sample, and Leszek Marynowski and S. Kurkiewicz for some GC-MS data. This is publication number 897 from the Southeast Environmental Research Center and Institute of Water and Environment at Florida International University. Conflicts of Interest: The authors declare no conflicts of interest. Molecules 2019, 24, 684 12 of 15

Appendix A Molecules 2019, 24, x FOR PEER REVIEW 12 of 15

Appendix I

OH OH

I. Spathulenol II. Spathulanols III. 20-Normulina IV. 20-Norazorell V. 20-Norazorellanes -11,13-diene -13-ene

VI. 20-Normullinanes VII. 13(H)-azorell VIII. Azorell-13-ene IX. 13(H)-azorell X. 13(H)-mulina -14-ene -14-ene -11,14-diene

XI. Mulina-11,13 XII. 13(H)-mulina XIII. 13(H)- XIV. 13(H)- XV. 13(H)- -diene -11,14-diene azorellane azorellane mulinane

OH OH

CO2H CO2H

XVI. 13(H)- XVII. 13-Hydroxy- XVIII. 13-Hydroxy- XIX. 13(H)-Mulina-11,14- XX. Mulina-11,13- mulinane azorellane mulinane dien-20-oic acid dien-20-oic acid

CO H CO H CO H CO H 2 CO2H 2 2 2

XXI. Azorell-13- XXII. 13(H)-azorell- XXIII. 13(H)-azorell- XXIV. 13(H)-muli- XXV. 13(H)-muli- en-20-oic acid an-20-oic acid an-20-oic acid nan-20-oic acid nan-20-oic acid

Figure A1. Cont.

Molecules 2019, 24, 684 13 of 15 Molecules 2019, 24, x FOR PEER REVIEW 13 of 15

OH OH OH OH

CO H CO H OH OH 2 2

XXVI. Mulinol XXVII. Dihydromulinol XXVIII. 13-Hydroxymulin- XXIX. 13-Hydroxy- 11-en-20-oic acid mulinan-20-oic acid

OH OH OH OH OH

O

O O CO2H CO2H O XXX. 20-Acetoxymulina- XXXI. 13,14-Dihydroxy- XXXII. 13,14-Dihydroxy- XXXIII. Azorellanol 11,13-diene mulin-11-en-20-oic acid mulinan-20-oic acid CO2H OH OH O OAc AcO OH CO H CO2H 2 CO2H

XXXIV. 13-Hydroxy-14- XXXV. 7,13-Dihydroxy- XXXVI. 15-Acetoxymulina XXXVII. 18-Acetoxymulina oxomulin-11-en-20-oic acid mulin-11-ene -11,13-dien-20-oic acid -11,13-dien-16,20-dioic acid Figure A1. Chemical structures cited and listed in Table1. References

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