Unprecedented deoxygenation at C-7 of the ansamitocin core during mutasynthetic biotransformations.

861 Unp eceden ed deoxygena ion a C-7 o he ansami ocin co e du ing mu asyn he ic bio ans o ma ions Tobias Knobloch1, Ge ald D äge 1, We a Collisi2, Flo enz Sasse2 and And eas Ki schning*1 Full Resea ch Pape Open Access Add ess: 1Ins i u e o O ganic Chemis y and Cen e o Biomolecula D ug Resea ch (BMWZ), Leibniz Uni e si y Hanno e , Schneide be g 1b, 30167 Hanno e , Ge many and 2Depa men o Chemical Biology, Helmhol z Cen e o In ec ious Resea ch (HZI), Inho ens aße 7, D-38124 B aunschweig, Ge many Email: And eas Ki schning* - [email p o ec ed] * Co esponding au ho Keywo ds: ansami ocins; an ibio ics; an i umo agen s; mu asyn hesis; na u al p oduc s Beils ein J. O g. Chem. 2012, 8, 861–869. doi:10.3762/bjoc.8.96 Recei ed: 23 Ap il 2012 Accep ed: 16 May 2012 Published: 11 June 2012 This a icle is pa o he Thema ic Se ies "Biosyn hesis and unc ion o seconda y me aboli es". Gues Edi o : J. S. Dickscha © 2012 Knobloch e al; licensee Beils ein-Ins i u . License and e ms: see end o documen . Abs ac We desc ibe he unp eceden ed o ma ion o six ansami ocin de i a i es ha a e deoxygena ed a C-7 o he ansami ocin co e, ob ained du ing e men a ion expe imen s by employing a a ie y o Ac inosynnema p e iosum mu an s and mu asyn he ic app oaches. We sugges ha he o ma ion o hese de i a i es is based on elimina ion a C-7/C-8 ollowed by educ ion(s) o he in e media e enone. In bioac i i y es s, only ansami ocin de i a i es bea ing an es e side chain a C-3 showed s ong an ip oli e a i e ac i i y. 861 In oduc ion Na u al p oduc s s ill play an impo an ole as lead s uc u es o he ea men o in ec ious diseases and cance . Howe e , na u al p oduc s ha e los some o hei a ac ion o he de el- opmen o pha maceu icals because o hei s uc u al complexi y and he di icul ies associa ed wi h accessing analogues o s uc u e–ac i i y ela ionship s udies. To al syn- hesis app oaches a e s ill a ou de o ce and a e ha dly employed, while commonly semisyn hesis as well as bio echno- logical app oaches a e widely pu sued in indus ial esea ch [1-3]. In es iga ions in o he biosyn hesis o na u al p oduc s ha e no only allowed us o unde s and he syn he ic p inciples ha na u e pu sues, bu ha e also p o ided ools, mainly based on gene ic enginee ing, which can be exploi ed in na u al p od- uc syn hesis [4]. P oduce s ains gene ically blocked in he biosyn hesis o impo an and complex na u al p oduc s can se e as such new ools. The syn he ic concep based on hese blocked mu an s is Beils ein J. O g. Chem. 2012, 8, 861–869. 862 Scheme 1: Summa y o ansami ocin biosyn hesis and s uc u e o he ela ed ansamycin an ibio ic geldanamycin (6). e med “mu a ional biosyn hesis”, o in sho mu asyn hesis, and i elies on he cellula up ake o modi ied biosyn he ic in e media es, some imes e med mu asyn hons, and hei inco po a ion in o complex seconda y me aboli es [5-7]. When making use o mu an s ha a e blocked in ea ly s ages o a gi en biosyn hesis pa hway, he concep o mu asyn hesis may be compa ed o a (pa ial) na u al p oduc o al syn hesis. When u he modi ica ion o an ad anced biosyn he ic in e - media e wi h an es ablished co e s uc u e owa ds bioac i e na u al p oduc s and analogues is conduc ed, mu asyn hesis may be ega ded as he “endgame” o a o al syn hesis [4]. The ansami ocins (may ansinoids) 3–5 a e ideally sui ed o mu asyn he ic modi ica ions and he c ea ion o new analogues because hey a e highly po en an i umo ac i e compounds ha inhibi he g ow h o di e en leukemia cell lines as well as human solid umo s a e y low concen a ions (10−3 o 10−7 µg/mL) [8]. In con as o colchicine, may ansinoids such as ansami ocins bind o β- ubulin monome s a a si e o e lap- ping he inca alkaloid binding si e [9]. Recen ly, we disclosed se e al mu asyn he ic s udies aimed a he p oduc ion o de i a i es o ansami ocins 3–5 [10-12] as well as o geldanamycin (6), u ilizing mu an s ains o Ac inosynnema p e iosum, he ansami ocin p oduce [13-17], and S ep omyces hyg oscopicus, he geldanamycin p oduce [18,19]. These enginee ed s ains a e unable o biosyn hesize 3-amino-5-hyd oxybenzoic acid (1) [20], he common s a e uni o bo h polyke ide syn hases (PKS) (Scheme 1). These assembly-line- ype mul ienzymes a e esponsible o se ing up he comple e ca bon backbone o bo h ansamycin an ibio ics [21-24]. Mo e p ecisely, he biosyn hesis o ansami ocins elies on a ype I modula polyke ide syn hase (PKS), wi h 3-amino-5- hyd oxybenzoic acid (1, AHBA) [20] as he s a e uni ol- lowed by chain ex ension by one “glycola e”, h ee p opiona e and h ee ace a e uni s. The las PKS module holds seco- p oansami ocin, which is eleased and cyclized, p esumably by an ansamycin amide syn hase (Asm9) [21-24], o yield he 19-membe ed mac ocyclic lac am p oansami ocin (2). P oansami ocin (2) is ans o med in o bioac i e compounds 3–5 by a se o pos -PKS ailo ing s eps, ollowing a p ede e - mined, only pa ly lexible logic (Scheme 1) [16]. Complemen ing ou s udies wi h mu an s ain A. p e iosum HGF073, blocked in he biosyn hesis o he PKS s a e uni AHBA 1 [13-16], we ecen ly epo ed he use o a mu an o A. p e iosum blocked in Asm12 (chlo ina ion) and Asm21 Beils ein J. O g. Chem. 2012, 8, 861–869. 863 Figu e 1: Fe men a ion p oduc s, p oansami ocin (2) and de i a i es 7–9, o he Asm12 and Asm21-blocked (chlo ina ion, ca bamoyla ion) mu an s ain A. p e iosum Δasm12/21 (yields gi en as isola ed p oduc pe olume o e men a ion b o h) [17]. (ca bamoyla ion) and he e o e p oducing p oansami ocin (2) in good yield (up o 106 mg/L o e men a ion b o h) [17]. Addi- ionally, we isola ed small amoun s o O-me hyl p oansami- ocin 7 (2.3 mg/L), 10-epi-p oansami ocin 8 (3.5 mg/L) and wo dias e eome ic byp oduc s 9a and 9b (7.6 mg/L; 1:1 a io) om he e men a ion b o h o A. p e iosum Δasm12/21 (Figu e 1). We also showed ha none o hese p oansami ocin de i a i es exhibi an ip oli e a i e ac i i y. He ein, we desc ibe he unp eceden ed o ma ion o ansami- ocin de i a i es ha a e deoxygena ed a C-7 o he ansami- ocin co e, ob ained by us du ing e men a ion expe imen s using a a ie y o A. p e iosum mu an s and mu asyn he ic app oaches. Resul s and Discussion In he cou se o ou well-es ablished mu asyn hesis expe i- men s wi h A. p e iosum HGF073, a mu an ha is unable o p oduce he essen ial s a e uni AHBA (1) by i sel , we achie ed he gene a ion o se e al no el ansami ocin de i a- i es, among o he hings, based on he simple mu asyn hon 3-amino-5-chlo obenzoic acid (10, Scheme 2). Wi h he excep ion o p oansami ocin analogue 11a, all new me aboli es we e isola ed on a p epa a i e scale and ully cha - ac e ized. These compounds co espond o he known s epwise sequence o pos ke ide syn hase ailo ing ans o ma ions, and hose p oduc s and simila compounds ha e been epo ed by us be o e [16]. As e iden om he a ie y o compounds isola ed, p oansami ocin analogues esul ing om supplemen a ion o AHBA analogue 10 o A. p e iosum HGF073 we e no e i- cien ly p ocessed by he enzymes in ol ed in pos -PKS ailo ing. In addi ion o he o dina y compounds 11a–e, he expe imen yielded h ee compounds o an unp eceden ed ype (11 –h) whose appea ance could no be a ibu ed o he known ailo ing ans o ma ions. He ein, we now desc ibe o he i s ime he isola ion and cha ac e iza ion o compounds 11 –h sha ing, in con as o all o he (p o)ansami ocin de i a i es known so a , he common ea u e o deoxygena ion a C-7. In addi ion, p oansami ocin de i a i es 11g–h a e no able o hei C-9 alcohol, while 11h shows addi ional ca bamoyla ion o he unusual alcohol moie y. The ex ao dina y p oansami ocin de i a i es 11 –h we e ully cha ac e ized, excep o he con- igu a ion a C-9 in 11g and 11h (single dias e eome ). O e lap wi h o he signals in he 1H NMR spec a hampe ed comple e assignmen o all coupling cons an s a C-9 excep o J9,10 = 7.2 Hz, which, howe e , is no diagnos ic. In con inua ion o ou expe imen s wi h ad anced biosyn he ic in e media es, such as p oansami ocin (2) [25] and seco-acid de i a i es [26,27] se ing as mu asyn hons in expe imen s wi h ea ly-s age-blocked mu an s, we also es ed he unusual me aboli es 9a and 9b [17] as p ecu so s o u he p ocessing by he AHBA(−)-mu an o he ansami ocin p oduce (Scheme 3). O iginally ob ained by e men a ion o a mu an blocked o he g ea es ex en in he pos -PKS ans o ma ion sequence (A. p e iosum Δasm12/21) [17], i was ques ionable whe he hese compounds, di e ing subs an ially om p oansami ocin (2) bo h by a ea anged diene sys em and an alcohol moie y a C-14, would be accep ed by he ailo ing enzymes. Su p isingly, when he ea anged oxida ion p oduc s 9a and 9b we e each supplemen ed o cul u es o A. p e iosum HGF073, new p od- uc s we e o med. While he ca bamoyla ed de i a i e 12 esul ing om bio ans o ma ion o alcohol 9b can be a ib- u ed o he ac i i y o he ca bamoyl ans e ase Asm21, he eby p o iding an indica ion pe aining o i s subs a e lexibili y, p oduc s 13a and 13b ob ained om bo h expe imen s a e mo e unusual and di e om he s a ing ma e ial by being deoxy- gena ed a C-7. Howe e , he s uc u e o compound 12 s ongly con as s wi h all o he o he 7-O-ca bamoyla ed (p o)ansami ocin de i a- i es ha we ha e ob ained wi h his kind o eeding expe i- men so a [17]. In he case o compound 12, he ca binol- Beils ein J. O g. Chem. 2012, 8, 861–869. 864 Scheme 2: Mu asyn he ic p epa a ion o ansami ocin de i a i es 11a–h by using 3-amino-5-chlo obenzoic acid (10) as a mu asyn hon (yields in % a e calcula ed wi h e e ence o he amoun o mu asyn hon 10 ed). amide moie y is no p esen in a cyclic hal aminal o m (δC-9 ~197 ppm ins ead o ~82 ppm), despi e he ac ha he ke o g oup a C-9 is s ill p esen . This esul may be a ibu ed o he ke o g oup a C-9 being in conjuga ion wi h he diene sys em, esul ing in a educed ac i i y o he ca bonyl g oup and an al- e a ion o he mac olac am ing con o ma ion. We based he de e mina ion o deoxygena ion a C-7 in 13a and 13b on MS da a and he appea ance o a seconda y ca bon a om in exchange o he e ia y ca binol a C-7 on phase-sensi i e 1H–13C-co ela ion NMR spec a (HSQC). I needs o be no ed ha NOE-analysis combined wi h molecula modeling did no allow elucida ion o he absolu e con igu a ion a C-14 o he new de i a i es 13a and 13b, as i did no allow o he s a ing compounds 9a and 9b [17]. In o de o shed ligh on he sequence o e en s leading o deoxygena ion, he ca bamoyla ed de i a i e 12 was again ed o a cul u e o A. p e iosum HGF073, and he o ma ion o new p oduc s was analyzed by UPLC-HRMS o he pa ially pu i- ied c ude ex ac (Scheme 3). Indeed, he expec ed deoxy- gena ed p oduc 13b could be de ec ed, bu was also accompa- nied by he o ma ion o alcohol 9b. Compound 9b may ei he ha e esul ed om Michael addi ion o wa e o he suspec ed Beils ein J. O g. Chem. 2012, 8, 861–869. 865 Scheme 3: Mu asyn he ic bio ans o ma ion o p oansami ocin de i a i es 9a and 9b wi h AHBA(−) mu an A. p e iosum HGF073 (s a ing ma e ials could be eisola ed: 9a: 77% eisola ed, 9b: 23% eisola ed). Scheme 4: Fe men a ion p oduc s 14–16 o acyl ans e ase Asm19-blocked mu an A. p e iosum HGF059 (Δasm19) (yields gi en as isola ed p od- uc pe olume o e men a ion b o h). in e media e enone 17 (see la e in Scheme 5) o om hyd oly ic clea age o he ca binolamide 12. A blocked mu an o A. p e iosum ha is unable o ca y ou acyla ion o he C-3 alcohol o he ca bamoyla ed p oansami- ocin de i a i e p ecu so due o gene ic inac i a ion o he acyl ans e ase Asm19, was epo ed by Floss e al. [21,28]. I was known ha mu an s ain A. p e iosum HGF059 p oduces he expec ed ansami ocin de i a i e 14 in good yield (Scheme 4) [28]. Besides he es e side chain, compound 14 also lacks he Beils ein J. O g. Chem. 2012, 8, 861–869. 866 Scheme 5: Possible mechanism o deoxygena ion a C-7 o p oansami ocin de i a i es. epoxy unc ionali y and he N-me hyl g oup. These wo ailo ing s eps inalize he biosyn hesis o ansami ocin P-3 (AP-3, 4) and occu only a e acyla ion has aken place (Scheme 1). While examining he e men a ion ex ac o byp oduc s we we e able o iden i y wo new me aboli es, 15 and 16. Fo ma- ion o compound 15 can be aced back o ine icien chlo ina- ion, a phenomenon ha we ha e encoun e ed be o e in o he eeding expe imen s wi h p oansami ocin [25]. Mo e unusual is me aboli e 16, which is ye ano he example o a case in which deoxygena ion a C-7 has aken place. In summa y, deoxygena ion may occu when he AHBA- blocked mu an A. p e iosum HGF073 o he acyl ans e ase Asm19-blocked mu an A. p e iosum HGF059 a e employed, whe eas he educ ion p ocess does no occu wi h he blocked mu an A. p e iosum ∆asm12/21. The majo di e ence be ween hese h ee mu an s is he p esence (HGF073, HGF059) o absence (Δasm12/21) o he ca bamoyl ans e ase Asm21. Ca bamoyla ion o p oansami ocin de i a i es a C-7 (e.g. 11a, 9a/b and 7) in oduces a ai ly good lea ing g oup β-posi ioned o he ke o g oup, he eby acili a ing elimina ion o enones 17 (Scheme 5). In he case o compound 12, which p edominan ly exis s in a o m lacking he ypical cyclized ca binolamide moie y, he C-9 ke o g oup can p ese e i s elec onic p ope ies, hus acili- a ing he elimina ion s ep. Finally, he ac i i y o a ailo ing educ ase, which in all likelihood is no pa o he PKS, ca alyzes he educ ion o he α,β-unsa u a ed bond, yielding he deoxygena ed de i a i es (11 , 13a/b, 16). Dias e eoselec i e educ ion o he C-9 ke o g oup and ca bamoyla ion o in e - media e 11 would hen esul in compounds 11g and 11h, es- pec i ely. A ac o con ibu ing o he o ma ion o deoxygena ed p oansami ocin de i a i es is likely he usage o he ca bamoyl- a ed p ecu so s by he Asm19 acyl ans e ase. Acyla ion is a c ucial bo leneck s ep in he o he wise pa ly lexible sequence o pos -PKS ans o ma ions. When his s ep canno occu due o he absence o an ac i e acyl ans e ase (as in A. p e iosum HGF059), o i he ans o ma ion is ine icien (e.g., com- pound 11b) o e en nonexis en due o an unusual subs a e (e.g., compound 12), ca bamoyla ed in e media es accumula e. Indeed, he ca bamoyla ed p oduc 11b was he majo p oduc o he mu asyn hesis expe imen wi h mu asyn hon 10. The ca bamoyla ed compounds may hen be channeled in o he pa hway leading o deoxygena ed p oduc s. Depending on he subs a e, his p ocess may be qui e e icien . Fo ins ance, no ca bamoyla ed p oduc could be isola ed a e he bio ans o - ma ion o compound 9a, indica ing an e icien ans o ma ion o he suspec ed ca bamoyla ed in e media e o he inal p od- uc 13a. All (p o)ansami ocin de i a i es ully cha ac e ized by NMR we e also subjec ed o in i o biological es ing wi h di e en human cell lines de i ed om umo s o he umbilical ein. The esul s om hese es s a e gi en as alues o he hal -maximal inhibi o y concen a ion o he espec i e ansami ocin de i a- Beils ein J. O g. Chem. 2012, 8, 861–869. 867 Table 1: An ip oli e a i e ac i i y IC50 [nmol/L] o 11c–e in compa ison o AP-3 (4).a compound cell line U-937 A-431 SK- OV-3 PC-3 MCF-7 HUVEC AP-3 (4) 0.01 0.08 0.05 0.06 n.d. 0.02 11c 0.5 1.58 0.66 0.30 0.90 0.32 11d 0.05 0.10 0.05 0.16 0.11 0.08 11e 0.18 0.35 0.21 0.53 0.41 0.21 aValues shown a e means o wo de e mina ions in pa allel; human cell lines: U-937 (his iocy ic lymphoma), A-431 (epide moid ca cinoma), SK-OV-3 (o a y adenoca cinoma), PC-3 (p os a e adenoca cinoma), MCF-7 (b eas adenoca cinoma), HUVEC (umbilical ein endo helial cells); n.d. = no de e mined. i es in compa ison o he “gold s anda d” ansami ocin P-3 (4, Table 1). As expec ed [14], all (p o)ansami ocin de i a i es lacking he es e side chain a C-3 (11b, 11g–h, 12–16) do no show any an ip oli e a i e ac i i y (IC50 > 800 nM) o a leas wo o he cell lines lis ed in Table 1. Compounds 11c–e, bea ing he es e side chain a C-3, p edomi- nan ly showed ac i i ies in he pM ange. As seen also wi h AP-3 (4), he e is no signi ican di e ence be ween cance ous and heal hy cells. The mos ac i e compound was he N-me hyl de i a i e 11d, which eached he ac i i y o he s anda d AP-3 (4) o selec ed cell lines. Conclusion In conclusion, we disclose he isola ion and chemical and an ip oli e a i e ac i i y cha ac e iza ion o se e al no el ansami ocin de i a i es ha a e deoxygena ed a C-7. We used h ee di e en A. p e iosum mu an s and a a ie y o mu asyn- he ic app oaches. These p elimina y s udies on he deoxygena- ion a C-7 sugges ha i occu s by elimina ion a C-7/C-8 a e ca bamoyla ion has aken place, ollowed by educ ion medi- a ed by an unknown educ ase, which is no pa o he main assembly line PKS. As expec ed, all ansami ocin compounds bea ing he es e side chain a C-3 p edominan ly showed ac i - i ies in he pM ange. Expe imen al Analy ical de ails a e gi en in he Suppo ing In o ma ion File 1. Cul i a ion In gene al, cul i a ion o mic obial s ains on aga pla es was conduc ed in a He aeus incuba o a 30 °C, while cul i a ion in a shake lask was pe o med in a mul ile el New B unswick Scien i ic Inno a 4900 gy a o y mul i-shake a 200 pm a 29 °C. Unless o he wise no ed, he cul i a ion media we e p epa ed using dis illed wa e and s e ilized by au ocla ing: YMG medium – 10 g/L mal ex ac (Sigma), 4 g/L yeas ex ac (Bac o), 4 g/L D(+)- glucose∙H2O; YMG aga – YMG medium plus 22 g/L aga (Bac o); K-medium [29], basal composi ion ( inal s a concen a ion in he main cul u e co esponds o 5/6 o he alues gi en o his medium due o dilu ion) – 60 g/L dex in om maize s a ch (Fluka), 30 g/L D(+)-mal ose∙H2O (Fluka), 5.25 g/L co onseed lou (P o lo), 5 g/L CaCO3, 4.5 g/L yeas ex ac (Bac o), 300 mg/L K2HPO4 (Fluka, T ace- Selec ), 2 mg/L FeSO4∙7H2O. K-medium, addi i e – au o- cla ed and added sepa a ely: 3 g/L L- aline ( inal s a concen- a ion in he main cul u e, om a 3% (w/ ) s ock solu ion). A. p e iosum HGF073 is a eplica e o s ain HGF056 epo ed in [22]. Fe men a ion o A. p e iosum s ains A. p e iosum s ains (HGF073, HGF059, Δasm12/21) we e s o ed as spo e suspensions in 40% ( / ) glyce ol/wa e a −80 °C, and used o he inocula ion o YMG aga pla es. Following incuba ion o he pla es o 4 d a 30 °C, 5–8 well- spo ula ed colonies we e ans e ed o a 1.5 mL ube cha ged wi h 1 mL o s e ile dis illed wa e and illed o app ox. 50% heigh wi h s e ile glass beads (Ø = 2 mm, washed wi h dilu e hyd ochlo ic acid). A e o ex-mixing, he esul ing suspen- sion was used o he inocula ion o p ecul u es in bo om- ba led 250 mL E lenmeye lasks cha ged wi h YMG medium (50 mL pe lask, wi h addi ional s eel sp ing). P ecul u es we e shaken o 2 d a 29 °C be o e inocula ion o main p oduc ion cul u es (1/15 dilu ion). Cul i a ions we e pe o med in K-medium wi h addi i es – 42 mL K-medium wi h L- aline, 3 mL p ecul u e and one d op o SAG 471 an i- oam (GE Baye Silicones) [30] – by using nonba led 250 mL E lenmeye lasks ( inal olume: 35–60 mL pe lask, wi h addi ional s eel sp ing). Shaking was con inued a 29 °C o a o al cul i a ion ime o 7–10 days. Beils ein J. O g. Chem. 2012, 8, 861–869. 868 Fo de ec ion o no el p oduc s om es cul u es, samples o he cul u e b o h (200 μL) we e mixed wi h e hanol (200 μL), cen i uged (20800g, 3 min, 4 °C) and he clea supe na an subjec ed o UPLC-ESIMS analysis. Failing de ec ion o no el p oduc s, he cul u e b o h was ex ac ed h ee imes wi h e hyl ace a e, d ied o e MgSO4, concen a ed in acuo, and il e ed o e silica gel wi h e hyl ace a e, and he sol en was emo ed in acuo. The esidue was dissol ed in me hanol (1 mL) and subjec ed o UPLC-ESIMS analysis. Fo isola ion o no el p oduc s om (la ge-scale) e men a ions, he combined e men a ion b o h was ex ac ed wi h e hyl ace a e as desc ibed abo e, and he c ude ex ac subjec ed o a sequence o ch oma og aphic pu i ica ions (Suppo ing In o - ma ion File 1). Mu asyn hesis wi h A. p e iosum HGF073 In mu asyn hesis expe imen s wi h A. p e iosum HGF073, p oduc ion cul u es we e shaken o 2 d a e inocula ion (see abo e) be o e mu asyn hons we e added (9a, 9b, 10 o 12). Fo no el mu asyn hons, p oduc i i y o he s ain was i s moni- o ed by pa allel eeding o mu asyn hons o which accep ance was known (e.g., he na u al s a e building block: 3-Amino-5- hyd oxybenzoic acid, hyd ochlo ide sal (1)). Mu asyn hons we e dissol ed in DMSO/wa e [p e e ably 1:1; olume o eeding solu ion no exceeding 10% ( / ) wi h espec o he ecipien cul u e] and s e ilized by il a ion. Mu asyn hon 10 (1.25 mmol/L o e men a ion b o h) was added o p oduc ion cul u es wi h a combined olume o 945 mL con inuously (d op-wise) o e he ime-cou se o 3.5 d, by using au ocla able, sy inge pump-d i en eeding capilla ies – B ain ee Scien i ic BS-9000-8 sy inge pump wi h Upchu ch Scien i ic high-pu i y Te lon® PFA ubing (1/16” OD, 0.1” ID) and Te zel® connec o s. Bio ans o ma ion o he p oansami ocin de i a i es 9a, 9b and 12 was ca ied ou by supplemen ing a p oduc ion cul u e o A. p e iosum HGF073 (45 mL inal olume; K-medium; see abo e) wi h he espec i e de i a i e [9a/b (each: 4.5 mg, 9.8 µmol, dissol ed in 2 mL DMSO:H2O = 1:1); 12 (0.1 mg, 0.2 µmol, dissol ed in 1 mL DMSO)] in a single po ion a e 2.5 ( o 12) o 3.5 d ( o 9a/b) o shaking. Following bio ans o ma ion ( e e men a ion) o p oansami- ocin de i a i e 12, only UPLC-ESIMS analysis o he silica- gel- il e ed e hyl ace a e ex ac aken up in me hanol (see abo e) was ca ied ou . The e en ion imes and mass spec a o he de ec ed p oduc de i a i es 9b and 13b we e iden ical o hose o he p e iously isola ed ma e ials (Suppo ing In o ma- ion File 1). Suppo ing In o ma ion The suppo ing in o ma ion p o ides pu i ica ion p o ocols o e men a ions, a sho desc ip ion o he cell p oli e a ion assay, analy ical desc ip ions o new me aboli es and copies o 1H and 13C NMR spec a. Suppo ing In o ma ion File 1 Analy ical de ails and compound spec a. [h p://www.beils ein-jou nals.o g/bjoc/con en / supplemen a y/1860-5397-8-96-S1.pd ] Acknowledgemen s This wo k was suppo ed by he Deu sche Fo schungsgemein- scha (G an Ki-397, 13-1) and he Fonds de Chemischen Indus ie. Re e ences 1. C agg, G. M.; G o haus, P. G.; Newman, D. J. Chem. Re . 2009, 109, 3012–3043. doi:10.1021/c 900019j 2. Newman, D. J.; C agg, G. M. J. Na . P od. 2007, 70, 461–477. doi:10.1021/np068054 3. on Nussbaum, F.; B ands, M.; Hinzen, B.; Weigand, S.; Häbich, D. Angew. 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Use o SAG 471 an i- oam o e men a ion o A. p e iosum was sugges ed. License and Te ms This is an Open Access a icle unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. The license is subjec o he Beils ein Jou nal o O ganic Chemis y e ms and condi ions: (h p://www.beils ein-jou nals.o g/bjoc) The de ini i e e sion o his a icle is he elec onic one which can be ound a : doi:10.3762/bjoc.8.96