Revision of the Structure of Acremine P from a Marine-Derived Strain Of

molecules

Communication Revision of of the the Structure Structure of of Acremine Acremine P P from from a a Marine-Derived Strain of Acremonium persicinum Marine-Derived Strain of Acremonium persicinum Mary J. Garson 1,*, Warren Hehre 2, Gregory K. Pierens 3 and Suciati 4 Mary J. Garson 1,*, Warren Hehre 2, Gregory K. Pierens 3 and Suciati 4 1 School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD 4072, Australia 1 2 SchoolWavefunction of Chemistry Inc., and Irvine, Molecular CA 92612, Biosciences, USA; [email protected] University of Queensland, Brisbane, QLD 4072, Australia 2 3 WavefunctionCentre for Advanced Inc., Irvine, Imaging, CA 92612, University USA; [email protected] of Queensland, Brisbane, QLD 4072, Australia; 3 [email protected] for Advanced Imaging, University of Queensland, Brisbane, QLD 4072, Australia; 4 [email protected] of Pharmacy, Airlangga University, Surabaya, East Java 60286, Indonesia; [email protected] 4 * FacultyCorrespondence: of Pharmacy, [email protected]; Airlangga University,Tel.: Surabaya, +61-3365-3605 East Java 60286, Indonesia; [email protected] * Correspondence: [email protected]; Tel.: +61-3365-3605 Academic Editor: Margaret A. Brimble AcademicReceived: Editor: 28 February Margaret 2017; A. Accepted: Brimble 23 March 2017; Published: 24 March 2017 Received: 28 February 2017; Accepted: 23 March 2017; Published: 24 March 2017 Abstract: The previously published structure of the fungal metabolite acremine P is revised by Abstract:re-evaluation The ofpreviously chemical shiftpublished values structure and NOESY of the data, fungal and bymetabolite DFT calculations. acremine P is revised by re-evaluation of chemical shift values and NOESY data, and by DFT calculations. Keywords: Acremonium; fungi; NOESY; DFT calculations; biosynthesis Keywords: Acremonium; fungi; NOESY; DFT calculations; biosynthesis

1. Introduction 1. Introduction Fungi from the genus Acremonium isolated from both terrestrial or marine sources have been reportedFungi to from produce the meroterpenoids,genus Acremonium alkaloids, isolated peptidesfrom both or terrestrial oxygenated or metabolitesmarine sources [1–6 ].have Recently, been wereported reported to produce the structure meroterpenoids, elucidation, alkaloids, including pept a stereochemicalides or oxygenated investigation, metabolites of the[1–6]. meroterpenoid Recently, we reportedacremine the P ( 1structure) (Chart1 )elucidation, from a strain including of Acremonium a stereochemical persicinum investigation,isolated from of thethe marinemeroterpenoid sponge acremineAnomoianthella P (1) rubra(Chart, obtained1) from offshorea strain of from Acremonium Mooloolaba persicinum in southeast isolated Queensland. from the marine The chemical sponge Anomoianthellacorrelation of acreminerubra, obtained P with itsoffshore co-metabolite from Mooloolaba acremine A (in2) bysoutheast catalytic Queensland. hydrogenation The under chemical mild correlationconditions of (H acremine2, Pd/C, 24P with h) was its instrumentalco-metabolite inacremine defining A the (2) carbonby catalytic framework hydrogenation and partial under relative mild conditionsconfiguration (H2 of, Pd/C, acremine 24 h) P, was while instrumental the absolute in configuration defining the at carbon C-6 was framework determined and by partial NMR analysisrelative configurationof O-methylmandelate of acremine (MPA) P, while ester the derivatives. absolute configuration The remaining at C-6 stereochemical was determined elements by NMR were analysis resolved of O-bymethylmandelate evaluation of NOESY (MPA)data, ester molecularderivatives. modeling The remaining and selected stereochemical heteronuclear elements coupling were resolved constant by evaluationvalues [7]. of NOESY data, molecular modeling and selected heteronuclear coupling constant values [7].

ChartChart 1. 1. SelectedSelected meroterpenoids meroterpenoids fromfromA. A. persicinum persicinum. . In this communication, we revise the carbon framework of acremine P based on a comparison of calculatedIn this and communication, experimental weNMR revise chemical the carbon shift da frameworkta. The predictions of acremine of Pchemical based on shift a comparison values by quantumof calculated chemical and experimental methods have NMR provided chemical valuable shift in data.sights The into predictions natural product of chemical structures, shift valuesincluding by guidingquantum the chemical choice of methods diastereomer have provided for structure valuable confirmation insights intoby total natural synthesis product [8–10]. structures, Recent includingexamples which highlight the role of computed chemical shift values in the stereochemical evaluation of natural Molecules 2017, 22, 521; doi:10.3390/molecules22040521 www.mdpi.com/journal/moleculeswww.mdpi.com/journal/molecules Molecules 2017, 22, 521 2 of 5 guiding the choice of diastereomer for structure conﬁrmation by total synthesis [8–10]. Recent examples which highlight the role of computed chemical shift values in the stereochemical evaluation of natural products include vannusal B [11] and nobilisitine [12]. The structural revision of acremine A likewise necessitated a review of its overall relative stereochemistry, and this was informed by nOe data as well as molecular modeling. Finally, a biosynthetic pathway based on oxidative cleavage of a didehydro analogue of acremine Q is proposed.

2. Results and Discussions The structures of acremines P and Q were examined using a combination quantum chemical/empirical approach (Spartan, Wavefunction Inc., Irvine, CA, USA) [13] that provides 13C-NMR shifts with a mean absolute error (MAE) of ~1.6 ppm. The calculations involve Boltzmann averaging from a ωB97X-V/6-311+G(2df,2p) model using conformer geometries from the ωB97X-D/6-31G* model and chemical shifts from ωB97X-D/6-31G*, but are also empirically corrected to account for the local environment. There was close agreement between the calculated vs. experimental 13C-NMR shift values for acremine Q (3), thereby corroborating the structure and stereochemistry of the metabolite as previously published. For acremine P, originally assigned structure 2, the preliminary computational search for conformers revealed a single dominant conformer, with the closest alternative being upwards of 10 kJ/mol higher in energy and thus not contributing signiﬁcantly to the equilibrium mixture. As shown in Table1, there was a signiﬁcant mismatching of the calculated vs. experimental 13C-NMR shift values for acremine P, with deviations of 20.4 ppm for the alkene carbon (C-2) and 23.0 ppm for the hydroxymethine carbon (C-7). Consequently, the published structure for acremine P could not be correct. While deviations of 3 ppm between the experimental and calculated values are considered acceptable, even up to 5–6 ppm for carbonyl groups, a deviation of >10 ppm between experimental and calculated values is diagnostic of an incorrect structure [8,14].

Table 1. Comparison of experimental vs. calculated 13C-NMR chemical shifts for acremine P (4). 1

Carbon Exptl. Calc. (1) Calc. (4a) Calc. (4b) Calc. (4c) Calc. (4d) 1 192.3, C 197.6 193.3 193.5 193.0 192.3 2 102.4, CH 122.8 108.7 104.0 108.7 102.4 3 162.5, C 159.0 164.6 163.3 165.5 162.5 4 99.0, C 108.0 101.3 116.5 101.0 99.0 5 59.1, CH 55.5 60.3 58.7 60.4 59.1 6 57.4, C 57.2 57.3 57.5 57.7 57.4 7 95.0, CH 72.0 95.9 99.4 98.8 95.0 8 86.2, CH 89.0 88.7 82.2 82.0 86.2 9 78.2, C 81.5 80.4 80.8 83.4 78.2 10 25.8, CH3 21.9 23.9 27.6 26.2 25.8 11 23.4, CH3 21.9 23.2 23.1 22.9 23.4 12 14.4, CH3 13.4 15.9 15.8 15.9 14.5 1 Carbon numbering selected so that carbon chemical shift values align with those provided in Reference [7].

A re-evaluation of the 13C-NMR shift values suggested that the signal at 95.0 ppm (C-7), previously assigned to a secondary alcohol center, was instead associated with an acetal or lactol center. Furthermore, the alkene carbon signals (102.4 and 162.5 ppm) indicated a polarized double bond, likely enolized given the number of oxygen atoms in the molecule. With this information, three candidate structures (4)–(6) (Chart2), each of which contained a lactol functionality, were assessed against the previously reported HMBC data. In acremine P, the lactol proton at δH 5.83 (d) and the signal at ™H 4.15 (s) for the hydroxymethine proton (H-8) each showed HMBC bond correlations to the acetal carbon at 99.0 ppm. In isomer 4, these correspond to three bond correlations, whereas in isomers 5 and 6, the lactol proton and the hydroxymethine proton, respectively, were each four bonds distant from the acetal center. Furthermore, in planar structure 5, an HMBC between the lactol proton and the alkene C-3 at 162.5 ppm would be anticipated. Molecules 2017, 22, 521 2 of 5

products include vannusal B [11] and nobilisitine [12]. The structural revision of acremine A likewise necessitated a review of its overall relative stereochemistry, and this was informed by nOe data as well as molecular modeling. Finally, a biosynthetic pathway based on oxidative cleavage of a didehydro analogue of acremine Q is proposed.

2. Results and Discussions The structures of acremines P and Q were examined using a combination quantum chemical/ empirical approach (Spartan, Wavefunction Inc., Irvine, CA, USA) [13] that provides 13C-NMR shifts with a mean absolute error (MAE) of ~1.6 ppm. The calculations involve Boltzmann averaging from a ωB97X-V/6-311+G(2df,2p) model using conformer geometries from the ωB97X-D/6-31G* model and chemical shifts from ωB97X-D/6-31G*, but are also empirically corrected to account for the local environment. There was close agreement between the calculated vs. experimental 13C-NMR shift values for acremine Q (3), thereby corroborating the structure and stereochemistry of the metabolite as previously published. For acremine P, originally assigned structure 2, the preliminary computational search for conformers revealed a single dominant conformer, with the closest alternative being upwards of 10 kJ/mol higher in energy and thus not contributing significantly to the equilibrium mixture. As shown in Table 1, there was a significant mismatching of the calculated vs. experimental 13C-NMR shift values for acremine P, with deviations of 20.4 ppm for the alkene carbon (C-2) and 23.0 ppm for the hydroxymethine carbon (C-7). Consequently, the published structure for acremine P could not be correct. While deviations of 3 ppm between the experimental and calculated values are considered acceptable, even up to 5–6 ppm for carbonyl groups, a deviation of >10 ppm between experimental and calculated values is diagnostic of an incorrect structure [8,14].

Table 1. Comparison of experimental vs. calculated 13C-NMR chemical shifts for acremine P (4). 1

Carbon Exptl. Calc. (1) Calc. (4a) Calc. (4b) Calc. (4c) Calc. (4d) 1 192.3, C 197.6 193.3 193.5 193.0 192.3 2 102.4, CH 122.8 108.7 104.0 108.7 102.4 3 162.5, C 159.0 164.6 163.3 165.5 162.5 4 99.0, C 108.0 101.3 116.5 101.0 99.0 5 59.1, CH 55.5 60.3 58.7 60.4 59.1 6 57.4, C 57.2 57.3 57.5 57.7 57.4 7 95.0, CH 72.0 95.9 99.4 98.8 95.0 8 86.2, CH 89.0 88.7 82.2 82.0 86.2 9 78.2, C 81.5 80.4 80.8 83.4 78.2 10 25.8, CH3 21.9 23.9 27.6 26.2 25.8 11 23.4, CH3 21.9 23.2 23.1 22.9 23.4 Molecules 2017, 22, 521 12 14.4, CH3 13.4 15.9 15.8 15.9 14.5 3 of 5 1 Carbon numbering selected so that carbon chemical shift values align with those provided in Reference [7].

Molecules 2017, 22, 521 3 of 5

structures (4)–(6) (Chart 2), each of which contained a lactol functionality, were assessed against the Chart 2. Candidate structures for acremine P. previously reported HMBCChart data. 2. InCandidate acremine P, structuresthe lactol proton for at acremine δH 5.83 (d) P. and the signal at ™H 4.15 Molecules(s) for 2017 the, 22hydroxymethine, 521 proton (H-8) each showed HMBC bond correlations to the acetal carbon at3 of 5 A re-evaluation of the 13C-NMR shift values suggested that the signal at 95.0 ppm (C-7), previously 99.0 ppm. In isomer 4, these correspond to three bond correlations, whereas in isomers 5 and 6, the lactol assigned to a secondary alcohol center, was instead associated with an acetal or lactol center. Therefore,structuresproton planar and(4)–( the6 structure) (Charthydroxymethine 2), 3eachwas of prot consideredwhichon, respectively, contained further a were lactol witheach functionality, four DFT bonds computations weredistant assessed from the undertakenagainst acetal the on four Furthermore, the alkene carbon signals (102.4 and 162.5 ppm) indicated a polarized double bond, likely diastereomerspreviouslycenter. (4a Furthermore,– reported4d) (Chart HMBC in 3planar) data. following structure In acremine 5 a, an conformational P, HMBC the lactol between proton the at search lactol δH 5.83 proton on(d) and eachand the the of signal alkene the at C-3 four ™ Hat 4.15 candidates. (s) enolizedfor the hydroxymethine given the number proton of oxygen (H-8) atoms each showedin the molecule. HMBC bondWith thiscorrelations information, to the three acetal candidate carbon at Diastereomers162.54a ppmand would4d had be anticipated. two contributing conformers, while 4b and 4c each had one contributing 99.0 ppm.Therefore, In isomer planar 4, these structure correspond 3 was toconsidered three bond further correlations, with DFT whereas computations in isomers undertaken 5 and 6 on, the four lactol 13 conformer.proton Threediastereomers and of the the hydroxymethine( four4a–4d) candidates(Chart 3) protfollowing wereon, respectively, a reasonable conformational were matches eachsearch four on to eachbonds the of distanttheC data,four from candidates. suggesting the acetal that the correct planarcenter.Diastereomers structure Furthermore, 4a of and acreminein 4dplanar had twostructure P contributing had 5 been, an HMBC conformers, elucidated; between while the further, 4b lactoland 4c proton these each had and data one the enabledcontributing alkene C-3 diastereomer at 4b, for which162.5conformer. the ppm C-4 would Three chemical be of anticipated. the shiftfour candidates was calculated were reasonable at 116.5 matches ppm to compared the 13C data, tosuggesting an experimental that the value of correctTherefore, planar planar structure structure of acremine 3 was P hadconsidered been elucidated; further withfurther, DFT these computations data enabled undertaken diastereomer on 4b four, 99.0 ppm, to be eliminated. It was not feasible to use the computational data to distinguish between diastereomersfor which the (4a C-4–4d chemical) (Chart shift3) following was calculated a conformational at 116.5 ppm searchcompared on toeach an experimentalof the four valuecandidates. of 13 the three remainingDiastereomers99.0 ppm, to diastereomers 4abe andeliminated. 4d had It two was owing contributing not feasible to the to conformers, similarityuse the computational while of their 4b and data 4c C-NMRto eachdistinguish had shift one between contributing values, the and also to three remaining diastereomers owing to the similarity of their 13C-NMR shift values, and also to the the errors inevitablyconformer. Three inherent of the tofour the candidates quantum were mechanical reasonable matches calculations. to the 13C Itdata, was suggesting noted that that thethe quantum errors inevitably inherent to the quantum mechanical calculations. It was noted that the quantum correct planar structure of acremine P had been elucidated; further, these data enabled diastereomer 4b, mechanical calculationsmechanical calculations revealed revealed stereoisomers stereoisomers 4a4a andand 4d to4d be tothe belowest the in lowest energy in energy. for which the C-4 chemical shift was calculated at 116.5 ppm compared to an experimental value of 99.0 ppm, to be eliminated. It was not feasible to use the computational data to distinguish between the three remaining diastereomers owing to the similarity of their 13C-NMR shift values, and also to the errors inevitably inherent to the quantum mechanical calculations. It was noted that the quantum mechanical calculations revealed stereoisomers 4a and 4d to be the lowest in energy

ChartChart 3. Candidate 3. Candidate diastereomers diastereomers of acremine of acremine P. P. The three remaining stereoisomers could be further distinguished by the zero coupling between the The threevicinal remaining lactol and stereoisomershydroxymethine protons, could bewhich further necessitated distinguished a bond angle byclose the to zero90°. Selected coupling between homonuclear (H7–H8) couplings were calculated using the methods of Kutateladze et al. [15], yielding the vicinal lactol and hydroxymethine protons, which necessitated a bond angle close to 90◦. Selected values of 0.2, 4.1, 3.9 and 0.3 Hz for JH7–H8 in stereoisomers 4a–4b, respectively. The two stereoisomers homonuclear(4a (H7–H8), 4d) thus fitted couplings the couplingChart were data.3. calculatedCandidate It was noted diastereomers using that all the four of methods stereoisomersacremine ofP. Kutateladzeshowed a heteronuclear et al. [ 15], yielding (C4-H8) J value close to 5 Hz, and so these data did not distinguish 4a from 4d. NOe data also supported values of 0.2, 4.1, 3.9 and 0.3 Hz for JH7–H8 in stereoisomers 4a–4b, respectively. The two stereoisomers theThe choice three of remaining stereoisomer, stereoisomers in that from couldtheir 3D be stereostructuresfurther distinguished both isomers by the 4azero and coupling 4d were betweenexpected the (4a, 4d) thusvicinalto ﬁtted show lactol thean andnOe coupling betweenhydroxymethine the data. lactol It protonprotons, was notedand which one thaton necessitatedly of all the four two amethyl stereoisomers bond groups;angle closein contrast, showed to 90°. isomers Selected a heteronuclear (C4-H8) J homonuclearvalue4b and close 4c (H7–H8)were to each 5 Hz,couplings expected and were soto show these calculated nOes data between using did the the not methods lactol distinguish proton of Kutateladze and 4abothfrom etmethyl al. [15],4d groups.. yielding NOe data also supportedvalues theIn acremine choice of 0.2, 4.1, P, of the 3.9 stereoisomer, lactol and proton0.3 Hz atfor δH inJ 5.83H7–H8 that shows in fromstereoisomers an nOe their to the 3D4a methyl–4b stereostructures, respectively. signal at δH 1.43The for two both H-11, stereoisomersisomers but not 4a and 4d (4a,to 4d the) thus methyl fitted signal the at coupling δH 1.47 (Figure data. It1). was The notedcalculated that chemical all four shiftsstereoisomers were further showed examined a heteronuclear using the were expected(C4-H8)DP4+ to Jcomputational showvalue close an nOeto approach 5 Hz, between and developed so these the databy lactol Sarotti did not protonet al distinguish. to assign and the 4a one mostfrom only probable4d. NOe of the diastereomerdata two also methylsupported [16]. groups; in contrast, isomerstheUsing choice the 4bof 13 stereoisomer,C-NMRand 4c datawere alone, in that each the from probability expected their 3D was stereostructures to99.7% show that 4d nOes was both the betweenisomers correct diastereomer.4a and the 4d lactol were protonexpected and both to show an nOe between the lactol proton and one onδly of the two methyl groups; in contrast, isomers δ methyl groups. In acremine P, the lactol protonH-7 at H 5.83 shows an nOe to the methyl signal at H 4b and 4c were each expected to show nOes between the lactol proton and both methyl groups. 1.43 for H-11, but not to the methylMe-11 signal at δH 1.47 (Figure1). The calculated chemical shifts were In acremine P, the lactol proton at δH 5.83 shows an nOe to the methyl signal at δH 1.43 for H-11, but not further examined using the DP4+ computational approach developed by Sarotti et al. to assign the to the methyl signal at δH 1.47 (Figure 1). The calculatedE-10 chemical shifts were further examined using the 13 most probableDP4+ computational diastereomer approach [16]. UsingdevelopedH-8 the by SarottiC-NMR et al. datato assign alone, the most the probable probability diastereomer was 99.7%[16]. that 4d was the correctUsing the diastereomer. 13C-NMR data alone, the probability was 99.7% that 4d was the correct diastereomer.

Me-10 H-7 Me-11 Figure 1. Three-dimensional image of stereoisomer 4d showing nOe to Me-11.

E-10

H-8

Me-10

Figure 1. FigureThree-dimensional 1. Three-dimensional image image of of stereoisomerstereoisomer 4d showing4d showing nOe to Me-11. nOe to Me-11.

Molecules 2017, 22, 521 4 of 5 Molecules 2017, 22, 521 4 of 5

In our earlier publication publication [7], [7], we we reported reported that that the the hydrogenation hydrogenation of of acremine acremine P Pyielded yielded acremine acremine A A((2) 2as) asthe the sole sole product, product, and andused used this information this information to specify to specify the absolute the absolute configur conﬁgurationation at C-6 of atacremine C-6 of acremineP; clearly P;this clearly information this information must be must in error be in since error sincethe dioxolane the dioxolane ring ringof the of therevised revised structure structure is isincompatible incompatible with with the the tetrahydrofuran tetrahydrofuran ring ring previously previously ascribed ascribed to to acremine acremine P. P. Nevertheless,Nevertheless, wewe anticipated that acremine P would have the samesame 6R6R conﬁgurationconfiguration as itsits co-metaboliteco-metabolite acremine A. Scheme 11 shows shows aa plausibleplausible biosyntheticbiosynthetic routeroute toto thethe revisedrevised structurestructure ofof acremineacremine P.P. AA didehydro didehydro derivative, generated by the action of a P450P450 enzymeenzyme onon thethe co-metaboliteco-metabolite acremine Q, couldcould undergoundergo oxidative ring cleavage of thethe C-3/C7C-3/C7 double bond [17], [17], thereby generating an aldehyde group at thethe original C-7 position. position. The The 1,3-dioxolane 1,3-dioxolane ring ring is isform formeded by by cyclization cyclization of the of the C-4 C-4 hydroxy hydroxy group group onto onto the thealdehyde, aldehyde, and and would would be beanticipated anticipated to toprovide provide the the thermodynamically thermodynamically most most stable stable lactol lactol product. Finally, nucleophilicnucleophilic attack of the C-9C-9 hydroxyhydroxy groupgroup ontoonto thethe C-3C-3 carbonylcarbonyl generatesgenerates aa hemiacetalhemiacetal intermediate;intermediate; the the subsequent dehydration dehydration step step which which generates generates an an enol enol ether is facilitatedfacilitated by thethe associated formation formation of of an an αα,β,β-unsaturated-unsaturated carbonyl. carbonyl.

SchemeScheme 1. 1.Putative Putative biosynthetic biosynthetic pathwaypathway toto acremine P ( 4d)) from from a a dehydro dehydro derivative derivative of of acremine acremine Q. Q.

3. Materials and and Methods Methods The isolationisolation and characterizationcharacterization of acremine P hashas beenbeen previouslypreviously reportedreported [[7].7]. DetailsDetails ofof computational calculations calculations are are given given in in the the supplementary supplementary materials. materials.

4. Conclusions 4. Conclusions In conclusion, our revision of the structure of acremine P illustrates the valuable role of In conclusion, our revision of the structure of acremine P illustrates the valuable role of computational studies in evaluating the structures and stereochemistry of stereochemically complex computational studies in evaluating the structures and stereochemistry of stereochemically complex natural products. At the same time, a single piece of data in the original study, i.e., the suggested natural products. At the same time, a single piece of data in the original study, i.e., the suggested conversion of acremine P into acremine A by hydrogenation, compromised the structural study and conversion of acremine P into acremine A by hydrogenation, compromised the structural study and thereby incorrectly informed the structure determination. thereby incorrectly informed the structure determination. Supplementary Materials: Supplementary materials are available online. Supplementary Materials: Supplementary materials are available online. Acknowledgments: We thank the University of Queensland for financial support. Acknowledgments: We thank the University of Queensland for financial support. Author Contributions: M.J.G., W.H. and G.K.P. analyzed the data; S. contributed the sample of acremine P; M.J.G. andAuthor G.K.P. Contributions: wrote the paper. M.J.G., W.H. and G.K.P. analyzed the data; S. contributed the sample of acremine P; ConflictsM.J.G. and of G.K.P. Interest: wroteThe the authors paper. declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References

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Sample Availability: A sample of acremine P is available from the authors.

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