initially , growth of the fungus on 
 semisolid media led to the robust 
 production of 1 at approximately 50 mg l. the compound was purified on the basis of promising activity against m. tuberculosis .
the methanol extract from nrrl 50135 was 
 purified using a preparative c18 column to isolate 1 .
the c nmr spectra ( table 1 ) showed 27 carbon signals , 
 while its h nmr spectra presented five olefinic protons , 
 five methyls , four methylenes , and six methines .
the carbon connectivities 
 in the decalin ring and the olefinic side chain were established by 
 cosy , hmbc , and hsqc .
their association with equisetin - type tetramic 
 acid was evident from a review of literature data that showed that 1 is related to phomasetin , with the addition of two carbons 
 in the olefinic side chain and with the presence of an n - methyl group . in order to establish the relative and absolute configuration 
 of 1 and evaluate its antimycobacterial activity , we 
 fermented nrrl 50135 for additional material . unfortunately ,
as is 
 often observed in secondary metabolites isolated from cultivated microorganisms , 
 nrrl 50135 ceased production of 1 before completion of 
 this work . in order to achieve production of 1
, we identified 
 the biosynthetic locus for its production and moved it into the expression 
 host , fusarium heterosporum , as reported elsewhere . in this context , the major compounds produced 
 were not those found in nrrl 50135 , but instead comprised approximately 
 800 mg of desmethyl pyrrolocin analogues b ( 2 ) and c 
 ( 3 ) , along with a smaller amount of 1 , per 
 kilogram of fermentation .
compound 2 , which was isolated as an off - white , amorphous 
 solid , was the major metabolite found in the extract .
high - resolution 
 esims determined its molecular formula to be c26h38no5 . likewise 3 , which was also isolated 
 as an off - white , amorphous solid , has a molecular formula of c26h38no5 , as established by high - resolution 
 esims .
comparison of the uv spectra and h , c , cosy , hsqc , and hmbc data of 2 and 3 revealed that the two have the same carbon structure and that they 
 differ only in their stereochemistry .
furthermore , the hresims and 
 nmr data showed that 2 and 3 are the n - desmethyl analogues of 1 .
however , the proton 
 and carbon chemical shift values of 1 were more similar 
 to 3 than to 2 ( table 1 ) .
nmr 
 data showed that compound 2 has a cis - fused decalin structure . in the h nmr spectrum of 2 ,
the decalin ring junction signals h-3 (  2.72 ppm ) 
 and h-8 (  2.09 ppm ) were deshielded compared to the same protons 
 in 1 and 3 .
this is consistent with other cis - decalin tetramic acid analogues when compared to their trans - fused counterparts .
the nmr data also indicated 
 that the fused cyclohexyl ring assumes a chair conformation where 
 h-3 is axial while h-8 is equatorial ( figure 1 ) .
the absence of a diaxial coupling between these two protons caused 
 h-8 to be seen as a broad singlet in the h nmr spectrum , 
 while h-3 was seen as a broad doublet with j = 10.2 
 hz due to its coupling with h-4ax (  1.38 ppm ) .
the 
 roesy spectrum of 2 also showed a correlation between 
 h-3 and h-8 , which confirmed that the two are not in a diaxial configuration 
 ( figure 1 ) .
h-3 was further observed to correlate 
 with h-5ax (  0.87 ppm ) and h-7ax (  
 1.11 ppm ) , while h-8 was also seen to correlate with both h-7ax and h-7eq (  1.56 ppm ) .
this was also the 
 case with the equatorial c-6 methyl (  0.81 ppm ) , which exhibited 
 same intensity roesy correlations with both h-7ax and h-7eq and with h-5ax and h-5eq (  
 1.67 ppm ) .
on the other hand , h-6ax was seen to correlate 
 with h-4ax (  1.45 ppm ) , h-7eq , and h-5eq   the fused cyclohexenyl ring also seems to assume a 
 pseudochair configuration with h-3 , c-2 methyl , and h-8 in equatorial 
 positions , which was deduced from their roesy correlations .
correlations 
 were also observed between h-4ax and h-12 since c-4 and 
 c-12 are in pseudoaxial positions .
likewise , the pseudoaxial h-9 (  
 5.09 ppm ) also correlated with h-6ax .
the relative 
 configuration of 3 differs from that of 2 at stereocenters c-8 and c-11 . the first indication was the upfield 
 shifts of the proton signals h-3 (  1.57 ppm ) and h-8 (  
 1.79 ppm ) as mentioned above .
h-8 was also more deshielded than h-3 
 compared to cis - fused decalins , where h-3 is more 
 deshielded .
the chair conformation 
 of the cyclohexyl ring was also deduced from the following roesy correlations : 
 h-3 correlated with h-5ax (  1.71 ppm ) and h-7ax (  0.82 ppm ) ; h-8 correlated with h-4ax (  1.00 ppm ) and h-6ax (  1.49 ppm ) .
the equatorial 
 c-6 methyl also correlated equally to h-5ax (  1.71 
 ppm ) and h-5eq (  1.00 ppm ) and to h-7eq (  1.77 ppm ) .
the fused cyclohexenyl ring in 3 also seems to adopt a pseudochair configuration where h-3 and the 
 c-2 methyl are anti to each other in pseudodiaxial 
 positions .
h-11 and c-10 methyl appeared to be in pseudoequatorial 
 and pseudoaxial positions , respectively , due to the observed correlation 
 between the c-10 methyl and c-2 methyl .
the 
 absolute configuration of c-5 was determined by oxidative 
 bond cleavage of the tetramic ring followed by acid hydrolysis ( figure 3 ) .
the crude extract 
 containing both compounds 2 and 3 was reacted 
 with sodium hypochlorite and sodium hydroxide at room temperature 
 for 8 h. the oxidation products were 
 extracted using ethyl acetate and evaporated in vacuo .
the residue was hydrolyzed using 6 m hcl for 15 h at 110 c 
 to yield serine .
marfey derivatization of the hydrolytic product was performed using fdla ( 1-fluoro-2,4-dinitrophenyl-5-l - alanine amide ) .
the resulting hydrolytic product serine - fdla 
 ( hps - fdla ) was analyzed by analytical hplc together with reference 
 standards l- and d - serine , which were derivatized 
 in the same way as the hydrolytic product serine .
the hps - fdla eluted 
 at 25.00 min , while d - serine - fdla injected alone and l - serine - fdla injected alone eluted at 24.83 and 24.63 min , 
 respectively .
the hps - flda that was spiked with l - serine - fdla 
 showed two peaks , at 24.54 and 24.99 min , while the hps - fdla that 
 was spiked with d - serine - fdla showed one peak , at 24.96 min . 

the hps - fdla was also analyzed by lc - ms and showed the expected ion 
 peak at m / z 339 [ m + h ] .
( a ) oxidative bond cleavage ; ( b ) acid 
 hydrolysis ; ( c ) derivatization with marfey s reagent .
each compound was reacted with ( r)- and ( s)-mtpa chloride to form ( s)-mtpa and ( r)-mtpa esters , respectively .
r calculated ( figure 4 ) . both 2 and 3 were found to have 
 the 17r configuration .
r values obtained in pyridine - d5 of the mtpa esters of 1 and 2 . without any
derivatization 
 step , the exciton cd method was used 
 to elucidate the absolute configuration 
 of 2 .
this was possible because two existing chromophores 
 can possibly interact and exhibit exciton split cd spectra , which 
 can then be observed as a positive or negative cotton effect .
the 
 positive cotton effect that is predicted from the configuration of 
 the two chromophores of 2 ( figure 5a ) was observed in the experimental cd spectra of 2 ( figure 5b ) .
the tetramic acid ring attached at c-2 and the 
 conjugated diene attached at c-11 are two chromophores that coupled 
 to cause the positive cotton effect that was observed in the experimental 
 cd spectrum of 2 .
the absolute configuration of 2 was therefore assigned as 2r , 3s , 6s , 8s , 17r , and 5r .
this method has been applied to 
 establish the absolute configuration of natural products with pre - existing 
 chromophores , including abscisic acid , quassin , dendryphiellin f , 
 and arnottin ii .
( a ) expected cotton effect 
 of 2 according to the exciton 
 cd method of determining absolute configuration .
the optical rotation [ ]d = + 130 and the cd spectrum 
 of 3 ( figure 6 ) were almost identical 
 to that of phomasetin ( [ ]d = + 93.9 ) and ent - equisetin ( table 2 ) and opposite 
 of that of altersetin / coniosetin ( figure 7 ) .
the c-5r configuration of the tetramic 
 ring as determined above is also consistent with that of phomasetin . 

the absolute configuration of 3 was therefore assigned 
 as 2r , 3s , 6s , 
 8r , 17s , and 5r .
experimental cd spectrum 
 of coniosetin ( reprinted with permission 
 from ref ( 8) ) .
compound 1 , which was isolated 
 as an off - white oil , was a minor compound 
 in the recombinant expression .
its hplc retention time ( figure 8a and b ) , high - resolution esims , uv , h , c , cosy , hmbc , and hsqc nmr spectra , and cd data 
 matched those of 1 from the wild - type fungus nrrl 50135 . 

determination of the absolute configuration of 1 was 
 achieved by comparison of its roesy and cd spectra with 3 .
the roesy spectrum of 1 was very similar to that of 3 , which indicates that 1 and 3 have 
 the same relative configuration .
the similarity of the cd spectra 
 of these two compounds further revealed that 1 and 3 have the same absolute configuration .
the 
 co - occurrence of 1 , 2 , and 3 seems to suggest that 2 and 3 are cis- and trans - adducts originating from 
 an intramolecular diels  alder reaction in the biosynthetic 
 process .
chemically , this reaction usually prefers 
 to go through an endo transition state , which leads 
 to the cis - decalin isomer , while the less favored exo transition state leads to the trans - decalin . in secondary metabolism , the favored stereochemical route 
 is usually controlled by enzymes , rather than by solution chemistry . 

for example , the fungal polyketide solanapyrone is cyclized by a fairly 
 novel enzyme after release of the intermediate from the polyketide 
 synthase ( pks ) . in a more relevant example
the 
 fungal decalin lovastatin is produced by a pks that is similar to 
 the pyrrolocin pks . in lovastatin biosynthesis , it is the pks itself 
 that controls decalin stereochemistry , leading to strict production 
 of the trans - decalin .
the relevant biosynthetic intermediate 
 is proposed to contain an ene that is activated by conjugation to 
 an enzyme - bound thioester . by contrast
, the synthetic analogue of 
 the proposed intermediate leads to a mixture of cis- and trans - decalin compounds , reflecting a mixture 
 of endo- and exo - diels 
equisetin and its related compounds 
 are thought to be synthesized by a similar mechanism , wherein the 
 pks itself would dictate whether the diels  alder reaction follows 
 the endo or exo pathway . in the 
 present study , 1 was found as the major product by nrrl 
 50135 , and only trace amounts of 3 and none of 2 were detected ( figure 8a ) , revealing 
 that only trans - decalins were produced in the wild - type 
 fungus . on the other hand ,
the heterologous expression yielded 2 and 3 in a 2:1 ratio , which is what 
 one would expect for a nonenzymatic reaction ( figure 8b ) .
although there are many reasons for this , the result implies 
 that a separate diels  alderase domain might be involved in 
 the biosynthesis of equisetin - like compounds , which in the present 
 study might not be present in the heterologous expression . as postulated 
 by other authors
, it seems that lovb 
 ( the lovastatin nonaketide synthase ) and eqxs ( equisetin pks - nrps 
 hybrid ) , at the very least , contain a binding pocket to direct the 
 stereochemical outcome of the reaction . on the other hand , the direct 
 product of the pyrrolocin pks still contains a carbonyl in the correct 
 position to activate an ene for the diels  alder reaction . by 
 contrast , in lovastatin biosynthesis , more advanced pks intermediates 
 no longer contain an activated ene .
this result implies that perhaps 
 pyrrolocins are enzymatically cyclized in the wild - type host , but 
 not in the recombinant host . of additional interest , despite 
 the structural similarities between 
 pyrrolocins and compounds such as equisetin ,
while this fact is very convenient in terms 
 of assigning the absolute configuration of the compound series as 
 we have done here , it is curious
. why is the d - amino acid 
 always found with one decalin absolute configuration and the l - amino acid with the other ?
first , 
 the presence of the d- or l - amino acid may be controlled 
 enzymatically ; the configuration would then dictate the stereochemical 
 course of the diels  alder reaction .
second , since the  
 position is relatively labile in the tetramic acid motif , the amino 
 acid may begin as l - configured and equilibrate to the thermodynamically 
 favored configuration based upon the stereochemistry of the decalin 
 ring .
these ideas provide a testable hypothesis that will enable understanding 
 of the timing and biochemical basis of the diels  alder reaction 
 in pyrrolocins and the many structural analogues .
( a ) hplc 
 chromatogram with pda detection of the original extract 
 of nrrl 50135 .
( b ) hplc chromatogram with pda detector of the extract 
 from the expression platform in mutated f. heterosporum .
compounds 13 were found to be active 
 against m. tuberculosis , with 1 and 3 being more potent than 2 ( table 3 ) .
the data seem to indicate 
 that a trans configuration and n - methylation enhance the potency of antimicrobial activity .
uv spectra were measured 
 on a hewlett - packard 8452a diode array spectrophotometer ( agilent , 
 santa clara , ca , usa ) .
ir spectra were recorded using a jasco ( easton , 
 md , usa ) ft / ir-400 spectrometer .
specific rotations were recorded 
 on a perkinelmer ( downers grove , il , usa ) 343 digital polarimeter 
 in meoh at 22 c .
cd spectra were recorded on an aviv model 420 
 cd spectrometer ( aviv biomedical , inc .
nmr spectra were recorded on a varian ( palo alto , ca , 
 usa ) inova at 500 mhz for h and 125 mhz for c using vendor - supplied pulse sequences .
accurate mass measurements 
 were performed by hresims on a micromass q - tof micro ( waters , milford , 
 ma , usa ) using positive ion mode and an ftms ( ltq - ft , thermofisher , 
 waltham , ma , usa ) .
hplc was performed on an agilent 1200 series equipped 
 with a pda detector ( agilent technologies ) .
a luna 5 m c18 column , 250  10 mm ( phenomenex , torrance , ca , usa ) , 
 was used for the isolation of the three compounds .
supelco diaion 
 hp20ss was purchased from sigma - aldrich ( st . louis , mo , usa ) .
tlc 
 was conducted using kieselgel 60 f254 ( merck , whitehouse 
 station , nj , usa ) .
was collected in watunou , 
 milne bay province , 
 papua new guinea , by a team from wyeth ( now pfizer ) .
a piece of the 
 stem was rigorously surface - sterilized , and the sterile stem was placed 
 on malt extract agar containing antibacterials and 1 g / ml cyclosporin 
 until fungal hyphae were observed to protrude from the stem .
the strain 
 was initially isolated with the name endo-0549 , and after axenic cultivation , 
 it was deposited in the nrrl culture collection as nrrl 50135 .
phylogenetic 
 analysis using molecular markers identified nrrl 50135 as a potentially 
 novel genus in the order diaporthales .
a 250 ml erlenmeyer flask 
 containing 50 ml of seed medium , difco potato dextrose broth , was 
 inoculated with agar - grown culture .
the seed was incubated at 22 c 
 with shaking at 200 rpm until sufficient cell density was achieved 
 to inoculate the production fermentation .
the production fermentation 
 medium consisted of 15 g of nongauze milk - filter paper ( kenag animal 
 care group , ashland , oh , usa ) cut into 8 mm wide by 60 mm long strips 
 and 102 g of milled long grain white rice wetted with 125 ml of 0.1% 
 yeast extract solution per 2.8 l fernbach flask .
the fermentation 
 was inoculated with 20 ml of the seed and incubated stationary at 
 22 c for 15 days .
the methanol extract 
 was then purified by reversed - phase hplc using a c18 column 
 ( phenomenex luna , 12 p , 50  250 mm ) and a gradient solvent 
 of 85100% acetonitrile in water ( 40 ml / min , 0 min run ) , with 
 both solvents containing 0.01% in volume of trifluoroacetic acid ( tfa ) . 

the peak at 18 min , monitored at 293 nm , was concentrated to give 
 partially purified pyrrolocin .
the compound was further chromatographed 
 by hplc using a different column ( ymc ods - a , 10 p , 30  
 250 mm ) and a gradient solvent of 80100% acetonitrile in water 
 ( 20 ml / min , 23 min run ) , with both solvents containing 0.01% in volume 
 of tfa .
the peak at 17 min was collected and evaporated under reduced 
 pressure to afford 1 as a colorless oil ( 7.2 mg ) .
mutant strains 
 of f. heterosporum carrying pyrrolocin 
 biosynthetic genes were maintained and grown as described .
three - week 
 cultures of f. 
 heterosporum grown on 500 g of corn grit agar at room temperature 
 in 2.5 l fernbach flasks were extracted twice with acetone for 12 
 h. extracts were pooled and dried invacuo using a rotary evaporator ( ika works , inc . ,
a portion of the crude extract ( 6.7% ) 
 was dissolved in meoh , mixed with diaion hp20ss , and dried .
the resin 
 was loaded into a column ( 8.5 cm  2.0 cm i.d . ) and fractionated 
 using 40 ml each of the following solvents : 100% water , 75% h2o/25% 2-propanol , 50% h2o/50% 2-propanol , 25% h2o/75% 2-propanol , and 100% meoh to yield five fractions , designated 
 fw , f1 , f2 , f3 , and f4 .
f3 was 
 purified by semipreparative hplc using a reversed - phase c18 column at a 3.5 ml / min flow rate as follows : 60% acn/20% 0.1%
aqueous 
 tfa from 0 to 5 min , linear gradient from 60% to 100% acn from 5 to 
 20 min .
compound 1 ( 1.5 mg ) eluted at 18.7 min , 2 ( 5.0 mg ) at 16.0 min , and 3 ( 11.5 mg ) at 13.0 
 min . 2 and 3 ( 0.5 
 mg each )
were separately dissolved in pyridine - d6 ( 150 l ) and transferred into clean nmr tubes . under 
 a n2 gas stream , (
r)-(+)--methoxy--(trifluoromethyl)phenylacetyl 
 chloride ( 5 l ) was added into each sample , and the nmr tubes 
 were then carefully shaken to mix the samples and the mtpa chloride 
 evenly .
the nmr tubes were allowed to stand at room temperature and 
 monitored every 2 h by h nmr .
the reaction was found to 
 be complete after 6 h. h nmr data of the ( s)-mtpa ester derivative of 2 ( 2a ) ( 500 
 mhz , pyridine - d6 , data were assigned on 
 the basis of its h  h cosy correlations ) : 
  0.81 ( 3h , d , j = 6.3 hz , h3 - 6-me ) , 
 1.21 ( 3h , s , h3 - 2-me ) , 1.31 ( 3h , d , j = 
 6.3 hz , h3 - 18 ) , 2.44 ( 2h , m , h2 - 16 ) , 5.12 ( 1h , 
 s , h-9 ) . 1.64 ( 3h , s , h3 - 10-me ) , 5.58 ( 1h , p , j = 7.3 hz , h-15 ) , 5.68 ( 1h , dd , j = 14.7 , 10.7 hz , 
 h-12 ) , 6.11 ( 1h , dd , j = 14.7 , 10.0 hz , h-13 ) , 6.20 
 ( 1h , dd , j = 10.0 , 14.0 hz , h-14 ) .
h 
 nmr data of the ( s)-mtpa ester derivative of 3 ( 3a ) ( 500 mhz , pyridine - d6 , data were assigned on the basis of its h  h cosy correlations ) :  0.81 ( 2h , d , j = 6.6 hz , h3 - 6-me ) , 1.17 ( 3h , d , j = 
 6.3 hz , h3 - 18 ) , 1.48 ( 3h , s , h3 - 2-me ) , 2.22 
 ( 2h , m , h2 - 16 ) , 3.31 ( 1h , m , h-11 ) , 5.40 ( 1h , m , h-15 ) , 
 5.58 ( 1h , m , h-12 ) , 5.91 ( 1h , m , h-14 ) , 6.13 ( 1h , m , h-13 ) . in the manner described for 2a and 3a , 2 and 3 ( 0.5 mg each ) were separately dissolved 
 in pyridine - d6 and reacted with ( s)-()--methoxy--(trifluoromethyl)phenylacetyl 
 chloride ( 5 l ) in nmr tubes at room temperature for 6 h to 
 afford the ( r)-mtpa esters of 2 ( 2b ) and 3 ( 3b ) .
h nmr 
 data of the ( r)-mtpa ester derivative of 2b ( 500 mhz , pyridine - d6 ) :  0.82 
 ( 1h , d , j = 6.4 hz , h3 - 6-me ) , 1.21 ( 3h , 
 d , j = 6.4 hz , h3 - 18 ) , 1.25 ( 3h , s , h3 - 2-me ) , 1.64 ( 3h , s , h3 - 10-me ) , 2.44 ( 2h , m , h2 - 16 ) , 5.09 ( 1h , s , h-9 ) , 5.60 ( 1h , p , j = 
 7.1 hz , h-15 ) , 5.69 ( 1h , m , h-12 ) , 6.04 ( 1h , dd , j = 10.7 , 14.7 hz , h-13 ) , 6.22 ( 1h , dd , j = 10.6 , 
 14.9 hz , h-14 ) .
h nmr data of the ( r)-mtpa 
 ester derivative of 3b ( 500 mhz , pyridine - d6 , data were assigned on the basis of its h  h cosy correlations ) :  0.80 ( 3h , d , j = 6.3 hz , h3 - 6-me ) , 1.10 ( 3h , d , j = 6.4 hz , h3 - 18 ) , 1.52 ( 3h , s , h3 - 2-me ) , 1.80 
 ( 3h , s , h3 - 10-me ) , 2.22 ( 2h , m , h2 - 16 ) , 3.39 
 ( 1h , m , h-11 ) , 5.45 ( 1h , m , h-15 ) , 5.58 ( 1h , m , h-12 ) , 5.96 ( 1h , m , 
 h-14 ) , 5.99 ( 1h , m , h-13 ) .
the crude methanol extract was obtained and dried , and 
 a portion ( 0.2 g ) was dissolved in methanol ( 6 ml ) .
then 1 m naoh 
 ( 0.75 ml ) was slowly added , followed by a naocl solution ( available 
 chlorine 8.513.5% , 3.5 ml ) .
the mixture was stirred at rt 
 for 8 h. then , 1 m aqueous na2so2 ( 3 ml ) was 
 added , and the mixture was neutralized by addition of 1 m hcl .
after 
 removal of the solvent , the residue was diluted with h2o , and then the resulting mixture was extracted with ethyl acetate . 
 the organic layer was dried in vacuo .
the residue 
 was dissolved in 6 n hcl ( 0.5 ml ) and heated in a sealed ampule vial 
 at 110 c for 15 h. the solvent was removed in vacuo .
the absolute stereochemistry of the resulting serine was determined 
 by the advanced marfey method .
the 
 acid hydrolysate was dissolved in 1 ml h2o . to a 
 100 l aliquot
were added 1% fdaa ( 1-fluoro-2,4-dinitrophenyl-5-l - alanine amide ) in acetone ( 200 l ) and 40 l of 
 1 n nahco3.the mixture was heated at 40 c for 1 h 
 in a water bath .
the vial was then cooled , and 2 m hcl ( 20 l ) 
 was added . this solution was analyzed by analytical hplc using the 
 following method : 0 min 5% acn/95% aqueous tfa ( 0.1% ) , 02 
 min 525% acn , 23 min 25% acn , 348 min 2570% 
 acn , 4850 min 70% acn .
m. tuberculosis h37ra growth inhibition was quantified 
 using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 
 bromide ( mtt ) assay modified from previously published methods .
plant extracts and control drugs were dissolved 
 in dimethyl sulfoxide ( dmso ) to produce test stock solutions , and 
 dmso served as a negative control .
m. tuberculosis cultures were dispensed in 200 l of adc ( remel , lenexa , ks , 
 usa)-enriched 7h9 medium into 96-well culture clusters at 100  000 
 cells per well . then 1 l of dmso ( control ) or dmso containing 
 extract or drug dilution was added in quadruplicate wells . after incubation 
 for 4 days at 37 c , 11 l of sterile mtt ( 5 mg / ml in pbs ) 
 was added and further incubated for 12 h. the insoluble formazan salt 
 formed was solubilized by the addition of 50 l of a solubilization 
 solution ( 5% sds w / v , 50% dmf v / v , 45% h2o v / v ) .
a570 was measured using a multiskan fc plate 
 reader ( fisher scientific , waltham , ma , usa ) .
all data were corrected 
 against media - only blank wells by subtracting media - only a570 .
the percent inhibition was calculated as the fraction 
 of the sum of the test wells over the sum of control wells subtracted 
 from 1 and multiplied by 100 .
the ic50 was determined from 
 the percent inhibition as the concentration of test material that 
 inhibited at least 50% of growth of the test organism .
the cytotoxicity assay utilized 
 cem - tart cells obtained from the nih aids reagent reference program 
 ( nih aids reagent program , division of aids , niaid , nih ) .
cells were grown in complete rpmi 1640 in a 
 jacketed incubator with 5% co2 at saturated humidity and 
 at 37 c .
compounds to be tested were added in quadruplicate wells , 
 while controls were as follows : solvent only ( dmso ) in 16 wells , of 
 which 8 received media only (  media only  blank ) and 
 8 received 20  000 cells / well cem - tart cells ( positive growth 
 control ) .
a further 8 wells received a decreasing amount of doxorubicin 
 in duplicate wells ( 4 concentrations ) and cem - tart cells serving as 
 positive cytotoxicity control .
after 4 days of incubation 11 l / well 
 of 5 mg / ml mtt in pbs was added , followed by 2 h of further incubation . 

cells were pelleted by centrifugation , and the medium in each cell 
 was aspirated off using a vacuum aspirator .
dmso ( 150 l / well ) 
 was added to dissolve the insoluble formazan salt .
to completely dissolve 
 the formazan precipitate , plates were usually agitated for 5 min using 
 a fisher scientific ms1 s7 plate shaker .
a570 was determined using a scientific multiskan fc ( fisher scientific ) 
 plate reader .
the resulting data were processed with a specially made 
 excel spreadsheet to calculate the percent inhibition as follows : 
 the average of the media - only blank was subtracted from all wells . 
 the average of the test wells for each compound was subtracted from 
 the average of the positive control wells and divided by the average 
 of the positive growth control , resulting in a fraction of control . 
 to determine the percent fraction of inhibition ,
the ic50 was determined from the percent inhibition as the concentration 
 of test material that inhibited at least 50% of tart cell growth .
brownish oil ; [ ]d + 56.1 ( c 0.03 , meoh ) ; uv ( meoh ) 
 max nm ( log  244 ( 3.48 ) , 295 ( 3.05 ) ; cd max nm (  ) 239 ( + 5.82 ) , 294 ( + 2.40 ) ; ir ( nacl 
 disk ) max 3317 , 2946 , 2832 , 2161 , 2012 , 1977 , 1679 ; h nmr ( dmso - d6 , 400 mhz ) and c nmr ( dmso - d6 , 100 mhz ) see 
 table 1 ; hresims m / z 458.2906 [ m + h ] ( calculated for c27h40no5 458.2906 ) .
off - white solid ; [ ]d 150 
 ( c 0.025 , meoh ) ; 
 uv ( meoh ) max nm ( log  ) 238 ( 4.4156 ) , 286 
 ( 4.0263 ) ; cd max nm (  236 ( 16.9 ) , 
 283 ( 3.0 ) ; ir ( nacl disk ) max 3325 , 2945 , 2834 , 
 2362 , 2340 , 1683 , 1653 , 1456 ; h nmr ( cd3od , 
 500 mhz ) and c nmr ( cd3od , 125 mhz ) see table 1 ; hresims m / z 444.2751 
 [ m + h ] ( calculated for c26h38no5 444.2750 ;  + 0.2 ppm ) .
off - white solid ; [ ]d + 130 
 ( c 0.025 , meoh ) ; uv ( meoh ) 
 max nm ( log  ) 247 ( 4.4776 ) , 286 ( 4.1049 ) ; 
 cd max nm (  ) 232 ( 6.46 ) , 288 ( 7.56 ) ; 
 ir ( nacl disk ) max 3300 , 2945 , 2916 , 2868 , 2843 , 
 2155 , 1678 , 1575 , 1453 ; h nmr ( dmso - d6 , 500 mhz ) and c nmr ( dmso - d6 , 125 mhz ) see table 1 ; hresims m / z 444.2762 [ m + h ] ( calculated 
 for c26h38no5 444.2750 ;  + 2.7 
 ppm ) .
 three new decalin - type tetramic acid 
 analogues , pyrrolocins a ( 1 ) , b ( 2 ) , and 
 c ( 3 ) , were defined 
 as products of a metabolic pathway from a fern endophyte , nrrl 50135 , 
 from papua new guinea . 
 nrrl 50135 initially produced 1 but ceased its production before chemical or biological evaluation 
 could be completed . upon transfer of the biosynthetic pathway to a 
 model host , 13 were produced . 
 all 
 three compounds are structurally related to equisetin - type compounds , 
 with 1 and 3 having a trans - decalin ring system , while 2 has a cis - fused decalin . 
 all were active against mycobacterium tuberculosis , with the trans - decalin analogues 1 and 3 exhibiting lower mics than the cis - decalin analogue 2 . 
 here we report the isolation , structure 
 elucidation , and antimycobacterial activities of 13 from the recombinant expression as well as the isolation 
 of 1 from the wild - type fungus nrrl 50135 . 
Results and Discussion
Experimental Section