Belizentrin, a Highly Bioactive Macrocycle from the DinoflagellateProrocentrum belizeanumHumberto J. Domínguez,† Jose G. Napolitano,† M. Teresa Fernandez-Sanchez,‡ David Cabrera-García,‡
Antonello Novelli,§ Manuel Norte,† Jose J. Fernandez,*,† and Antonio Hernandez Daranas*,†,∥
†Institute for Bio-Organic Chemistry “Antonio Gonzalez”, Center for Biomedical Research of the Canary Islands, and ∥Department ofChemical Engineering and Pharmaceutical Technology, Faculty of Pharmacy, University of La Laguna, 38206 La Laguna, Tenerife,Spain‡Department of Biochemistry and Molecular Biology, and §Department of Psychology, Institute of Biotechnology of Asturias,Campus “El Cristo”, University of Oviedo, Oviedo 33006, Spain
*S Supporting Information
ABSTRACT: Belizentrin (1), a novel 25-membered polyketide-derived macrocycle,was isolated from cultures of the marine dinoflagellate Prorocentrum belizeanum. Thismetabolite is the first member of an unprecedented class of polyunsaturated andpolyhydroxylated macrolactams. The structure of 1 was primarily determined byNMR and computational methods. Pharmacological assays with cerebellar cellsshowed that 1 produces important changes in neuronal network integrity atnanomolar concentrations.
Marine organisms are an important source of bioactivemolecules, with a significant number of them currently
in clinical trials. In fact, due to their novel structural features,many of these marine-derived products are considered as “first-in-class” drugs.1 Marine dinoflagellates, in particular the genusProrocentrum, produce some of the most active and complexsecondary metabolites found in nature.2−7 Many of thesecompounds exhibited unparalleled antiproliferative or immu-nosuppressive bioactivities, making them potential drugcandidates or valuable pharmacological tools in drug discoveryand development.8
In this report, we describe the isolation and structureelucidation of belizentrin (1), a chemically unique and highlybioactive macrocycle obtained from large-scale cultures ofProrocentrum belizeanum. This new polyketide-derived com-pound has a highly oxidized side chain and is, to the best of ourknowledge, the first macrolactam isolated from a marinedinoflagellate.9
Belizentrin was isolated from the methanol extract of a cellpellet obtained from a 1000 L culture of P. belizeanum (strainPBMA01). Chromatographic fractionation of the crude extractwas carried out using gel permeation and reversed-phasechromatography, which led to the isolation of 3.1 mg of 1 as awhite amorphous solid. After a preliminary purity assessmentwas performed by LC−MS (obtaining a single-peak total ioncurrent chromatogram), samples of 1 were subjected to bothNMR analysis and biological testing.The molecular formula of 1, C48H73NO17 (with a total of 13
degrees of unsaturation), was established by HRMS analysis([M + Na]+ C48H73NO17Na, calcd m/z 958.4776, obsd m/z
958.4794). The UV spectrum of 1 showed an absorptionmaximum at 270 nm, which is consistent with the presence of apolyconjugated carbonyl group. Analysis of the HSQCexperiment showed the presence of 9 olefinic carbons(accounting for 10 olefinic protons as one carbon is an exo-type methylene), 4 methyl groups, 13 aliphatic methylenes, and14 aliphatic methines (including 13 oxymethynes). In addition,8 quaternary carbons were identified in the HMBC experiment,including 2 carboxamides, 1 carboxy carbon, 3 olefinic carbons,and 2 aliphatic carbons directly attached to oxygen atoms asdeduced by their chemical shifts (72.2 and 97.9 ppm).A comprehensive analysis of COSY, TOCSY, HSQC, and
HSQC-TOCSY experiments led to the identification of six1H−1H spin systems (Figure 1). These fragments wereconnected through the analysis of long-range 1H,13Ccorrelations extracted from the HMBC experiment (Figure1). An interesting structural feature of 1 is the presence of bothan ester and an amide group on its macrocycle backbone. Thelocation of the amide group was determined by the distinctivechemical shifts of C11′ (41.3 ppm) and the methylene protonsH11′ (3.41/3.84 ppm) as measured in CD3OD. On the otherhand, considering the downfield shifts of both C19 and H19(73.0 and 5.36 ppm, respectively), the ester functionalityconnected this position with C1′, closing a 25-memberedmacrocycle. Further analysis of the HMBC experiment enabledthe identification of three ether linkages (Figure 1), thereby
forming one oxolane (B) and two oxane rings (A/C), andaccounting for the remaining unsaturations.Once the planar structure of 1 was established, the
determination of key stereochemical features of this complexmolecule was undertaken. The configuration of the doublebonds Δ17,18, Δ2′,3′, and Δ9′,10′ was assigned as E on the basis ofthe observed 3JH,H values (>15 Hz in all cases) and ROE cross-correlations (Figure 1). Because the remaining double bondsincluded quaternary carbons, the determination of theirgeometry relied on the analysis of dipolar correlations. As aresult, the configuration of the Δ4′,5′ olefin was assigned as Ebased on the ROE cross-peaks between Me12′ and one of theH6′ methylene protons, as well as by the ROE between H3′and H5′. Similarly, the Z configuration of the Δ21,22 doublebond was assigned by the observation of an intense ROEcorrelation between Me34 and H22.The relative configuration of all the stereogenic centers in the
six-membered ring A was unambiguously determined using acombination of scalar coupling measurements and a thoroughanalysis of the ROESY experiment. A trans-diaxial relationshipbetween protons H3, H4, H5, and H6 was established on thebasis of their large vicinal coupling constants (3JH3,H4 = 8.9 Hz,3JH4,H5 = 8.9 Hz, and 3JH5,H6 = 10.5 Hz). The smaller J-couplingvalue between H6 and H7 (3JH6,H7 = 4.2 Hz) was diagnostic of agauche relationship between these neighboring protons. Thecross-peak network observed in the ROESY experimentbetween H3, H5, and H8 confirmed that these protons werelocated on the same ring face, whereas the dipolar correlationsbetween H2, H4, H6, and H7 positioned these protons on theopposite side (Figure 2A).Even though the configurational analysis of saturated five-
membered rings represents a substantial challenge due to theirinherent conformational flexibility,10 the relative configurationof the oxolane ring B could be established through the analysisof dipolar correlations between cis-oriented protons in 1,3-relative positions.11 The ROE cross-peak between H12 andH14, as well as the one between H14 and one of the H16methylene protons, indicated that these nuclei were located onthe same ring face. Additional dipolar correlations between oneof the H13 methylene protons and H11, Me33, and H15 wereobserved, thereby confirming that these protons werepositioned on the opposite ring face (Figure 2B).In the case of ring C, the relative configuration of all four
chiral centers was established by ROE analysis due to theexistence of two quaternary carbons (Figure 2C). Thus, the
dipolar correlation between H25 and Me35 indicated that bothgroups are located on the same ring face, whereas the dipolarcorrelation between H26ax and H28 positioned these nuclei onthe opposite ring face. In addition, the ROE cross-peaksbetween H28, and both H30 and H31 were crucial to establishthe relative configuration of the quaternary carbon C29 (Figure2C).Next, we approached the relative configuration within the 25-
membered macrocycle, where four out of the five chiral carbonswithin this moiety are located in the oxane ring C.Consequently, the difficulty was to connect their relativeconfigurations with the remote position C19. Taking intoaccount that all of the involved stereocenters are confinedwithin a conformationally restricted macrocycle, this taskshould be accessible from ROE and 3JH−H measurementsfollowed by computational simulations.12−14 Therefore, ino rde r to ana l y z e the NMR da t a , we fi x ed a25R*,27S*,28R*,29S* relative configuration for ring C, andmolecular modeling simulations were undertaken using a C1−C15 truncated model of the two possible C19 epimers (Figure3 and Figure S15, Supporting Information).15 For this task, an
Figure 1. Planar structure of belizentrin (1), including key NMRcorrelations. The 1H,1H spin systems are numbered I−VI.
Figure 2. Selected dipolar correlations observed in the five- and six-membered rings of belizentrin (1).
Figure 3. Selected dipolar correlations observed for the macrocyclicmoiety of belizentrin (1). Interatomic distances are shown inangstroms.
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unrestricted hybrid search method was used.16 First, 5000cycles of molecular dynamics simulated annealing at 1000 Kwere carried out, followed by 5000 cycles of large-scale lowmode search steps. These cycles resulted in 24 and 8 stableconformers for the 19S* and 19R* epimers within a 10 kJ/molthreshold of the global minimum. Afterward, geometricaloptimizations at the DFT level using the B3LYP functionalwith the 6-31G**(+) basis set were undertaken. Once theresulting Boltzmann populations were calculated, four relevantconformers (70, 20, 5, and 4%) were found for the 19S*epimer, while only one major conformer (98%) resulted for the19R* epimer (Figure S15, Supporting Information). Thecalculated structures were examined looking for concordanceswith the available NMR data (ROE and 3JH−H). Thus, it turnedo u t t h a t t h e c a l c u l a t e d s t r u c t u r e s f o r t h e19S*,25R*,27S*,28R*,29S* stereoisomer explained the dipolarcorrelations observed between the pairs H2′−H312′, H312′−H6′, H6′−H8′, and H8′−H10′ as well as the transannular ROEbetween H2′ and H24 (Figure 3). Moreover, on the other faceof the macrocycle an analogous NOE network could beobserved, reinforcing the previous observations. Particularlydiagnostic were the correlations between H19 and both Me34and H17 (Figure 3 and Table S2, Supporting Information).Moreover, the measured 3JH,H values for H19 (ddd, 9.2, 8.9, 4.8Hz) perfectly matched the selected structure. On the otherhand, the structure of the 19R* epimer showed discrepancieswith the experimental data.We also used quantum mechanical calculations of chemical
shifts as an alternative approach of analyzing the NMR data.This methodology has proved to be an excellent tool foraddressing the stereochemistry of complex natural prod-ucts.17,18 Thus, we calculated 1H and 13C chemical shifts forboth C19 epimers of the macrocyclic portion of 1 andestimated their DP4 probabilities.19 Particularly informativewere the calculated chemical shifts for both H20 (Figure S14,Supporting Information). Using the DP4 parameter, it ispossible to assign stereochemical relationships with quantifiablec o n fi d e n c e . 2 0 T h e r e s u l t w a s t h a t , t h e19S*,25R*,27S*,28R*,29S* configuration (also selected usingthe NOE and 3JH,H data) was predicted with >99% probability,reinforcing our previous conclusions.The C1−C18 side chain of 1 is stereochemically more
complex than the macrocyclic portion as includes 11asymmetric carbons. Nevertheless, considering that the relativeconfiguration within the C3−C7 and C12−C15 cyclicstereoclusters was previously determined, the remaining taskwas to connect both rings via a four-carbon acyclic tether (C8−C11) that included three stereogenic centers. In the absence oflong-range heteronuclear coupling constants (decomposition of1 occurred before we could do such measurements, Figure 11,Supporting Information), we made use of Kishi’s universalNMR database concept.21 Thus, we measured 1H−1H couplingconstants within the C9−C12 portion of 1 (3JH9,H10 = 3.9 Hz,3JH10,H11 = 2.8 Hz, and 3JH11,H12 = 5.8 Hz) from 1H and COSY-DQF experiments (Table S1, Supporting Information). Theobserved 3JH,H profile corresponds either to an all-anti or all-synrelationship between the oxygen substituents of this portion butdiscards all other possibilities.21 In order to solve thisuncertainty, we also made use of chemical shift calculations.Thus, we built models of the 16 possible diasteroisomers of theC1−C18 fragment and performed conformational searches foreach one (Table S5, Supporting Information). Afterward, allconformers within a 10 kJ/mol threshold of the global
minimum found for each stereoisomer were geometricallyoptimized at the HF/3-21G level. Finally, single-point energyand NMR shielding constants calculations were calculated usingDFT at the mpw1pw91/6-31G**(+) level for all thoseconformers (201 structures) using a Poisson−Boltzmannmethanol solvation model. Subsequently, for each diaster-oisomer, a Boltzmann weighted average of their NMR chemicalshifts and the corresponding DP4 probabilities were estimated.This calculation resulted in the prediction of a singles t e r e o i s o m e r o f r e l a t i v e c o n fi g u r a t i o n ,3R*,4S*,5R*,6R*,7R*,9S*,10R*,11S*,12S*,14S*,15R*, with a90.1% probability when both 1H and 13C chemical shifts weretaken into account (Tables S6 and S7, Supporting Informa-tion). Remarkably, this stereoisomer shows an all synrelationship between the oxygen substituents of the C9−C12portion in correspondence with Kishi’s NMR database results.In summary, the relative configurations within the macrolactamand the polyhydroxilated side chain had been assigned,although the relationship between their configurations isarbitrary as they are connected by a double bond at C17−C18.From our point of view, belizentrin shows structural
similarities with macrolide immunosuppressants.22 Recently,several lines of evidence have demonstrated unexpectedactivities of immunossuppresant drug targets in the centralnervous system including the modulation of tubulin polymer-ization and τ protein function,23 axon regeneration andsprouting,24 neurite outgrowth,25 and neuronal apoptosis.26
Consequently, to study the possible biological actions of 1 onneuronal survival and function we used primary cultures ofcerebellar cells.26,27 Exposure of cultured neurons to 1 resultedin strong changes in neuronal network integrity followed by celldeath. Neurite weakness and fragmentation was evident startingat concentrations of 100 nM, while exposure to concentrationsof 1 over 300 nM caused complete degeneration of neuronalsomas (Figure 4). These effects required 24 h exposure to 1,while no visible morphological signs of toxicity could be
Figure 4. Effect of various concentrations of belizentrin (1) oncultures of cerebellar cells. (Top) Fluorescence photomicrographs ofneurons before and after exposure to 1 for 24 h. Live neurons showeda bright green color in the cell body, whereas neurites and deadneurons did not retain any fluorescein and their nuclei appearedstained in red by ethidium bromide. Complete disintegration ofneurites was observed in cells exposed to 200 nM of 1. (Bottom)Dose−response curve (mean ± SD).
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observed with shorter exposures. Dose−response experimentswere performed taking advantage of the capability of viable cellsto retain fluorescein. The concentration of 1 that produced a50% reduction in maximum neuronal survival after 24 h(EC5024) was estimated at approximately 193 ± 7 nM. Thesebiological effects on neuronal cultures were consistentlyobserved over time. This bioactivity is therefore a stablecharacteristic of the compound, suggesting the involvement ofstructural elements not affected by the observed decompositionof the molecule.In summary, this study described the structural character-
ization of belizentrin (1), a new and structurally uniquemacrolide that exhibited potent bioactivity in neuronal survivalassays in vitro. Interestingly, 1 affected the strength andintegrity of neurites long before any reduction in neuronalviability could be observed. From a structural point of view, therelatively low stability of 1 precluded the acquisition of acomplete set of spectroscopic data, in particular heteronuclearnJC,H measurements, thus adding a new degree of difficulty to analready challenging elucidation task. Nevertheless, a thoroughanalysis of the available NMR data, combined with molecularmodeling simulations and quantum mechanical calculations,enabled the evaluation of potential diastereomeric structuresand ultimately made possible a full proposal for the relativeconfiguration of this complex natural product.
■ ASSOCIATED CONTENT*S Supporting Information
Experimental procedures, spectral data, and computationalresults. This material is available free of charge via the Internetat http://pubs.acs.org.
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTSThis research work was funded by EU Grant Nos. FP7-KBBE-3-245137-MAREX and FP7-REGPOT-2012-CT2012-31637-IMBRAIN as well as by SAF2011-28883-C03-01 and 03 fromMINECO and CEI10/00018 from MECD, Spain. H.D.acknowledges MINECO for a Ph.D. scholarship.
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