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Natalamycin A, a

aDepartment of Biological Chemistry and M

School, 240 Longwood Avenue, Boston,

jon_clardy@hms.harvard.edubDepartment of Bacteriology, University of W

Building, 1550 Linden Drive, Madison, Wisc

† Electronic supplementary informationcomputational procedures, bioassay dataX-ray crystal data, Cartesian coordinate994285. For ESI and crystallographic dataDOI: 10.1039/c4sc01136h

‡ These authors contributed equally.

§ Current address: Natural Product ReseSungkyunkwan University, 300 CheonchRepublic of Korea.

{ Current address: Leibniz Institute for NBiology e.V., Hans Knoll Institute (HKIGermany.

k Current address: Natural Products & ExHawaii Cancer Center, 701 Ilalo Street, H

** Current address: Centre for SocialEvolution, Department of BiologUniversitetsparken 15, 2100 Copenhagen

Cite this: Chem. Sci., 2014, 5, 4333

Received 17th April 2014Accepted 14th July 2014

DOI: 10.1039/c4sc01136h

www.rsc.org/chemicalscience

This journal is © The Royal Society of C

n ansamycin from a termite-associated Streptomyces sp.†

Ki Hyun Kim,‡§a Timothy R. Ramadhar,‡a Christine Beemelmanns,‡{a Shugeng Cao,kaMichael Poulsen,**b Cameron R. Currieb and Jon Clardy*a

We report a preliminary functional and complete structural characterization of a highly unusual

geldanamycin analog, natalamycin A, that was isolated from Streptomyces strain M56 recovered from a

South African nest of Macrotermes natalensis termites. Bioassay-guided fractionation based on

antifungal activity led to the isolation of natalamycin A, and a combination of NMR spectroscopy and X-

ray crystallographic analysis, including highly-accurate quantum-chemical NMR calculations on the

largest and most conformationally-flexible system to date, revealed natalamycin A's three-dimensional

solid- and solution-state structure. This structure along with the structures of related compounds

isolated from the same source suggest a geldanamycin-like biosynthetic pathway with unusual post-PKS

modifications.

Introduction

Bacterially-produced small molecules modulate interactionsbetween the microbial producers and their relatives, allies,competitors, and hosts. These molecules have long interestedchemists because of their potential therapeutic value, theirremarkable molecular structures, and the challenges posed bytheir synthesis and biosynthesis. In a systematic effort todiscover new members of this family, we have been exploringthe bacterial symbionts of insect agricultural systems.

Agriculture in insects is ancient, originating millions of yearsago when insect farmers – ants, beetles, and termites – formed

olecular Pharmacology, Harvard Medical

Massachusetts, 02115, USA. E-mail:

isconsin-Madison, 6145 Microbial Science

onsin, 53706, USA

(ESI) available: Experimental and, characterization data, NMR spectra,s, and calculated NMR shis. CCDCin CIF or other electronic format see

arch Laboratory, School of Pharmacy,eon-dong, Jangan-gu, Suwon 440-746,

atural Product Research and Infection), Beutenbergstrasse 11a, 07745 Jena,

perimental Therapeutics, University ofonolulu, Hawaii, 96813, USA.

Evolution, Section for Ecology andy, University of Copenhagen,East, Denmark.

hemistry 2014

obligate mutualisms with specialized food fungi.1 The insectbenets by feeding on the fungus, and the fungus benets bythe insect providing plant material for the fungus to consume inprotected gardens. A third party to these symbioses, bacteria,fend off pathogens by producing antibacterial and antifungalagents that suppress specialist and generalist crop pathogens.The secondary metabolites produced by the bacteria in theseagricultural systems are expected to undergo multiple rounds ofalteration and selection, which likely makes them promisingtherapeutic candidates.1,2

In an effort to extend earlier studies, we investigated termite-associated Actinobacteria and their potential for producingbiologically active small molecules.3 Screening bacterial isolatesagainst the cultivar (Termitomyces spp.) and pathogen (Pseu-doxylaria spp.) revealed that while most of the Actinobacteriaproducedmolecules with antifungal activity, Streptomyces strainM56, isolated from the fungal comb material of a MacrotermesnatalensisMn802 colony, showed exceptionally high and broad-range antifungal activity. Further investigation of Streptomycessp. M56 led to the isolation of natalamycin A (1) and relatedansa macrolides (Fig. 1).

Results and discussion

Fungal comb material was collected from a South African M.natalensis Mn802 colony (Fig. 2) and bacteria from the combwere grown and subcultured to yield a Streptomyces isolatecalled M56. M56 is most closely related to Streptomyces malay-siensis 1160 as judged by 100% 16S rRNA sequence identity. Ona preparative scale, M56 was grown on ISP-2 agar for 14 daysand was subsequently extracted using iPrOH. The concentratedcrude extract was subjected to bioassay-guided fractionation

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Fig. 1 Natalamycin A, geldanamycin and other analogs isolated fromStreptomyces sp. M56.

Fig. 2 South AfricanMacrotermes natalensis colony and fungal comb.The white nodules are produced by Termitomyces and are eaten bythe termites.

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against Pseudoxylaria X802 using a disk assay. Fractionationbegan with C18 solid phase extraction followed by MeOH/H2Oelution. Two fractions (80% MeOH in H2O and 100% MeOH)exhibited antifungal activity, and they were combined andsubjected to preparative-scale C18 reverse-phase HPLC. 1HNMR analysis of a contiguous set of bioactive fractions indi-cated that geldanamycin (2),4 a known antifungal agent, waspresent. However, additional compounds were observed inthese fractions. Through a careful series of semi-preparativereverse-phase HPLC experiments with a phenyl-hexyl column,reblastatin (3),5 17-O-demethyl-geldanamycin (4),6 19-S-methyl-geldanamycin (5),7 17-amino-17-demethoxy-geldanamycin (6),8

methyl geldanamycinate derivative 7,9 and recently reportedgeldanamycin analog 9 were resolved and isolated.10 There were

4334 | Chem. Sci., 2014, 5, 4333–4338

also two previously unreported metabolites that were isolated.The rst was 17-amino-17-demethoxy-methyl geldanamycinate(8), which is a plausible biosynthetic precursor to 6,11 and wascharacterized through MS and 2D NMR analysis. The secondand more structurally challenging metabolite was a fusedbicyclic [6.4.0] ansa macrolide, which we named natalamycin A(1) aer its association with M. natalensis. The yield of 1 whengrown on 50 ISP-2 agar plates (14 cm diameter) was 0.7 mg. Wewere unable to observe natalamycin A production if the bacteriawere grown in ISP-2 liquid media.

While the fractions containing mixtures of natalamycin A,geldanamycin and its analogs exhibited antifungal activityagainst the Pseudoxylaria X802 fungus, activity was alsoobserved against the Termitomyces T112 cultivar. This differsfrom the selectivities seen in beetle and ant systems, but itagrees with recent competition studies by Visser and co-workersin which Actinobacteria from termite nests were found toinhibit both cultivar and pathogenic fungi.12 They concludedthat a mutualistic symbiosis would still be possible if the anti-biotics were applied with spatial localization. It should also benoted that M56 produces not fully characterized metaboliteswith antifungal activity that are not geldanamycin analogs.Furthermore, natalamycin A exhibited weak or no activityagainst the fungal cultivar, S. cerevisiae, B. subtilis, and otherisolated fungi. Studies are underway to further evaluate nata-lamycin A's bioactivity in a larger set of assays.

In regard to the structural elucidation of natalamycin A (1),analysis through HR-ESI-MS provided the unfamiliar molecularformula of C35H50N2O11 (m/z: 697.3310 [M + Na]+), and subse-quent analysis relied heavily on NMR spectroscopy. NMRanalysis revealed that it contains the macrolactam core of gel-danamycin (2). Comparison of all NMR data for 1 and 3 revealedthat the six-membered carbocycle is a substituted phenol, notthe substituted benzoquinone of 2. NMR analysis also revealedan additional spin-system which appeared to form, inconjunction with the phenolic core, an unprecedented fusedbicyclic [6.4.0] fragment that had not been found in any previ-ously reported ansamycins. COSY and HMBC analysis allowedfor the determination of bond connectivities for the 8-membered ring. It is interesting to note that while 1 was origi-nally analyzed in CD3OD, the crucial HMBC correlation estab-lishing the connectivity of the acetal moiety to the aromatic corewas only observed by NMR analysis in CDCl3. This allowed forthe assignment of C20 and C21 as the bridgehead carbonsversus fusion through C19 and C20. Altogether, NMR analysisprovided bonding arrangements for 1, but it was unable toclearly dene the relative stereochemistries of the methoxy andmethyl groups on the eight-membered heterocyclic ring or toconnect those relative stereochemistries with those of themacrolactam.

In order to fully elucidate the relative and absolute structureof 1, X-ray diffraction analysis was used. Aer multipleattempts, small crystals of 1 were grown through slow evapo-ration from CHCl3 with minimal MeOH. Achieving sufficientresolution and data quality for the measurement of these crys-tals virtually precluded the use of in-house diffractometers.Therefore, data collection was performed using high-ux

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synchrotron radiation with a helium cold stream (Chem-MatCARS, Advanced Photon Source, Argonne National Labora-tory), and the crystal structure was successfully solved andrened with sufficient resolution and quality (Fig. 3).13 Fortu-itous inclusion of three CHCl3 solvent molecules within theasymmetric unit allowed for the unambiguous determination ofabsolute stereochemistry at a shorter typical synchrotronwavelength (0.41 A) with a Hoo y parameter of 0.00(4).14 Themethoxy and methyl groups on the eight-membered ringwere both determined as 10R and 40R and the stereocenterson the macrolactam ring matched those of geldanamycin.Natalamycin A crystallizes in a closed C-clamp conformation in asimilar fashion to 19-(2-furyl)geldanamycin synthesized byMoody and co-workers with its cis-conguredmacrolactam,15 andin a fashion different from the solid-state conformation of 17-azetidinyl-17-demethoxygeldanamycin, which has an openextended conformation with its trans-congured macrolactam.16

Next, we wanted to predict 1H and 13C NMR chemical shisfor 1 with sufficient accuracy to understand its predominantsolution conformation, to verify the assignment of experimentalchemical shis, and to establish reliable procedures for struc-tural elucidation of other natural products in the laboratory.The conformation of geldanamycin analogs also has specialsignicance as it affects binding to Hsp90, geldanamycin'stherapeutic target. Previous conformational studies of 2,17 17-azetidinyl-17-demethoxygeldanamycin,16 and 19-(2-furyl)gelda-namycin15 used NMR and/or in silico analysis, and Moody andco-workers outlined the importance of geldanamycin analogsadopting a C-clamp conformation for target binding.15

We used a recent quantum-chemical NMR calculationmethod developed by Tantillo and co-workers18 that success-fully revised the structures of aquatolide18a and nobilistine A18b

as it promised the sensitivity we needed. The method dependson nding relevant conformers, which is not trivial for 1 due toits size and exibility. Natalamycin A contains 98 atoms and has294 degrees of freedom. This poses a particular challenge inregards to nding conformers, energetically rankingconformers and eliminating irrelevant ones through verycomputationally intensive DFT optimization and frequencycalculations, and performing high-level NMR calculations, all ina high-throughput fashion. Through the use of multipleconformational search tools along with the aid of the X-raycrystal structure and subsequent DFT geometry optimizations

Fig. 3 Wall-eye stereoview of the crystal structure of 1 exhibiting a C-clamp conformation, with CHCl3 solvent hidden for clarity. Ball-and-stick representation displayed; see ESI† for ORTEP plots.

This journal is © The Royal Society of Chemistry 2014

using cluster hardware, two relevant conformers of 1 – C-clampand extended –were afforded, with the C-clamp being the globalminimum structure (DG298.15 K ¼ 1.08 kcal mol�1, 86% pop-ulation). NMR calculations with implicit CHCl3 solvation,empirical scaling of the isotropic shis, averaging of equivalentproton shis, and Boltzmann averaging afforded the calculated1H and 13C shis relative to TMS (Fig. 4).

The calculated 1H and 13C NMR chemical shis correspondvery well to experimental values. The respective corrected meanabsolute deviation (CMAD) for 1H and 13C shis are 0.13 ppmand 2.1 ppm, and the respective maximum shi deviation foreach is 0.57 ppm and 4.2 ppm. These error statistics values arelow and closely correspond to those obtained by Tantillo and co-workers in their NMR calculation studies.18 The data stronglysuggest that 1 adopts a C-clamp solution conformation due tosignicant differences in the error statistics for each individualconformer arising from 1H and 13C shi discrepancies. For the

Fig. 4 Comparison of Boltzmann-averaged computed NMR chemicalshifts (bolded) and experimental values (underlined and italicized) for 1shown on the major C-clamp conformer with error statistics for (a) 1Hand (b) 13C. Computed values were obtained using the SCRF (chlo-roform)-mPW1PW91/6-311+G(2d,p)//B3LYP/6-31+G(d,p) level oftheory, and experimental values were obtained in CDCl3.

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Fig. 5 Biosynthetic hypothesis for natalamycin A (1) and 9 from Streptomyces sp. M56.

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C-clamp conformer, the CMAD values for 1H and 13C shis are0.12 ppm and 2.2 ppm, respectively, whereas for the extendedconformer it is 0.38 ppm and 3.2 ppm, respectively. Theextended conformer has larger shi discrepancies overall. TheC-clamp conformer geometry also aligns with the experimentalROESY correlations. These results show that given properlyidentied relevant conformers, highly accurate chemical shiscan be computed for large and exible secondary metabolites,which further validates the power and versatility of the methodby Tantillo and co-workers for natural product structuralstudies. To the best of our knowledge, this is the largest andmost conformationally exible molecule in which thisquantum-mechanical NMR calculation method has beengenerally used, and is the rst time that it has been employedfor the purposes of resolving conformation versus bondconnectivities.

Geldanamycin was isolated from Streptomyces hygroscopicusin 1970, and since then the ansamycin family of structurallycomplex and therapeutically promising molecules has grownconsiderably,4,19 serving as an impetus for biosynthetic studies.These studies have revealed that ansamycins are assembledfrom a 3-amino-5-hydroxybenzoic acid (AHBA) starter unit fol-lowed by the addition of extender units – malonate, methyl-malonate, and methoxymalonate – to form their polyketidebackbones.20 Aer chain extension, the resulting seco-pro-geldanamycin is released with concomitant macrolactamformation to release progeldanamycin.21 Progeldanamycinundergoes a variety of post-PKS modication – C-17 hydroxyl-ation, C-17-O-methylation, C-21 oxidation, C-7 carbamoylation,and a late stage C-4/C-5 dehydrogenation – to form familymembers. The recent isolation of several new metabolitesfunctionalized at C-19 highlight the possibilities of other familymembers from alternative post-PKS modications.7,9,22 Anotherapproach to ansamycin structural diversication using chainextender units has recently been reported.23

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The pathway for ansamycin biosynthesis in S. hygroscopicuscoupled with the identication of compounds 1–9 in this studyon M56 allows a plausible biosynthetic hypothesis for 1 and 9 tobe drawn (Fig. 5). In this formulation the PKS ansamycinpathway could lead to 10 through early release of seco-pro-geldanamycin followed by the known modications listedabove. Alternatively progeldanamycin could undergo the samemodications to give 11, the macrolactam version of 10. A ve-carbon fragment with the sort of functionality shown in 12could react with either 10 or 11 to eventually give 1 or 9. It ishypothetically possible that a ve-carbon fragment of thisnature can be derived from 4-hydroxy-2-oxopentanoate, which isan intermediate in 3-phenylpropionate catabolism. This cata-bolic pathway is known in Escherichia coli and other microbes.24

For actinomycetes, an enzyme within this pathway that forms 4-hydroxy-2-oxopentanoate from 2-oxopenta-4-enoate (2-oxopent-4-enoate hydratase) is predicted in an annotated gene sequencebelonging to Streptomyces sp. SirexAA-E (GenBank accessionnumber for hydratase: AEN11956.1), which is a member of abacterial/fungal symbiotic community associated with Sirexnoctilio, an invasive pinewood-boring wasp.25

Conclusions

This study explored the biosynthetic potential of Streptomycessp. M56 isolated from the fungal comb of a Macrotermes nata-lensis Mn802 nest with an initial focus on natalamycin A (1).Natalamycin A was structurally characterized with NMR spec-troscopy and X-ray crystallography. Additionally, quantum-chemical NMR calculations were successfully used on thelargest and most conformationally-exible system to date topredict its solution conformation, as opposed to previousapplications of this technique to solely determine bondconnectivities. The identication of structurally related metab-olites that are likely biosynthetic precursors coupled with the

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known geldanamycin biosynthetic pathway allowed a plausiblebiosynthetic pathway to compounds 1 and 9 to be constructedas a basis for further studies. Efforts to sequence and assemblethe G + C rich/highly redundant Streptomyces sp. M56 genomeand efforts to maximize production of 1 for biological testingare currently underway in our laboratory.

Acknowledgements

We are grateful for nancial support through the NationalInstitutes of Health (R01-GM086258 to J.C., U19-AI109673 andRC4-GM096347 to J.C. and C.R.C, and F32-GM108415 to T.R.R.)and through the German National Academy of Sciences Leo-poldina for a postdoctoral fellowship to C.B. We thank Dr Yu-Sheng Chen (ChemMatCARS, Advanced Photon Source,Argonne National Laboratory), for assistance with single-crystaldata collection. ChemMatCARS Sector 15 is principally sup-ported by the Divisions of Chemistry (CHE) and MaterialsResearch (DMR), the National Science Foundation, under grantnumber NSF/CHE-1346572. Use of the Advanced Photon Source,an Office of Science User Facility operated for the U.S. Depart-ment of Energy (DOE) Office of Science by Argonne NationalLaboratory, was supported by the U.S. DOE under Contract No.DE-AC02-06CH11357. We also thank Dr Shao-Liang Zheng(Department of Chemistry and Chemical Biology, HarvardUniversity) for assistance with single-crystal data collection andhelpful discussions on X-ray crystallography. Quantum-chem-ical calculations were run on the Odyssey cluster supported bythe FAS Division of Science, Research Computing Group atHarvard University. We thank Dr Duur K. Aanen for providingT112 and Mr Saria Otani for providing the termite/fungal combimages for Fig. 2 and the graphical abstract.

Notes and references

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