the molecular formula of compound 1 was determined 
 as c22h27n5o10 ( 12 degrees 
 of unsaturation ) by hresims . analysis of its h and c nmr spectra ( tables 1 and 2 ) and hsqc experiments revealed the presence of 
 three methyl groups ( including a methyl ester ) , one methylene , eight 
 oxygenated methines , one sp oxygenated quaternary carbon 
 ( likely a hemiketal ) , six olefinic carbons , and two carbons for carboxylic 
 acids or derivatives . the h nmr data ( table 1 ) recorded 
 in methanol - d4 showed two singlet peaks 
 at h [ 7.98 ( 1h , s , h-8 ) and 8.23 ( 1h , s , h-2 ) ] , 
 which in conjunction with the five nitrogen atoms deduced by hresims 
 suggested the presence of an adenine unit ( fragment a ) in 1 ( figure 1 ) . this conclusion was further supported 
 by the hmbc correlations that were observed from h-8 to c-4/c-5 and 
 from h-2 to c-4/c-6 . a second subunit ( fragment b ) was assembled based 
 on analysis of the cosy spectrum , which connected an olefinic proton 
 that resonated at h 6.68 ( 1h , q , j = 7.2 hz , h-3 ) with a methyl doublet at h 1.89 ( 3h , d , j = 7.2 hz , h3 - 4 ) . the hmbc correlations observed from h-3 to c-4/c-1 
 and from h3 - 5 to c-2/c-3/c-1 
 in the hmbc experiments suggested a 2-methyl-2-butenoic ( tiglic ) group 
 was present in 1 . a methyl ester was deduced from the c nmr resonances at  169.8 ( c-11 ) and 51.3 
 ( 11-och3 ) and the hmbc correlation from 11-och3 ( h 3.62 ) to c-11. connections of 
 the tiglic and methoxycarboxyl groups were established through analysis 
 of the hmbc experiments . the hmbc correlations from h-8 to 
 c-7 ( the quaternary hemiketal carbon ) and c-1 indicated 
 the tiglic group was attached at the c-8 position ( figure 1 ) . this carbon ( c-8 ) could then be connected 
 to the carbonyl c-11 through a series of hmbc correlations : 
 h-9 to c-8 , c-7 , and c-11 ; h-10 
 to c-9 and c-11. fragment c was deduced as follows . the cosy data contained cross - peaks consistent with the connections 
 of h-1h-2 and h-3h-6. 
 this information , in combination with the hmbc correlations from h-1 
 to c-2 , c-3 , and c-4 and from h-2 
 to c-3 and c-4 suggested the presence of fragment 
 c ( figure 1 ) . these three fragments were then 
 assembled into a larger structure that fulfilled most of the structural 
 requirements . the observed hmbc cross - peaks of h-1 to c-4/c-8 
 suggested fragments a and c were connected at c-1 of the adenine 
 residue through an n  c glycosidic bond . the hmbc correlations 
 from both h-5 and h-6 to c-7 revealed a direct 
 c-6/c-7 linkage , and the hmbc correlation from h-1 
 to c-4 demonstrated the presence of a furan ring , while the 
 hmbc correlation from h-10 to c-6 established the 
 linkage of c-6 and c-10 via an ether bond to give 
 a pyran ring ( figure 1 ) . therefore , 
 one more ring was required to complete a planar structure . in principle , 
 three possible cyclic hemiketal structures could be generated : c2o  c7 , 
 c3o  c7 , and c9o  c7. 
 after carefully checking the literature and comparing our nmr spectroscopic 
 data to those reported for herbicidin f ( 8) , we concluded 1 had a c3o the only difference between the 
 two was the replacement of the 2-och3 in 8 with 2-oh in 1 . the physicochemical analyses and the key noesy correlations 
 from 
 h-8 to h-2/h-3 , from h-3 to h-6/11-och3 , and from h-1 to h-3/h-4 ( figure 2 ) of 1 supported the same relative 
 configuration compared to those of herbicidins b ( 5 ) and g. on the basis 
 of these data , the structure of 1 was determined to represent 
 a new compound and was named 2-o - demethylherbicidin 
 f. selected noesy correlations of 1 . its 
 molecular 
 formula was determined as c18h23n5o9 on the basis of the hresims data . comparison of the h and c nmr data of 2 and 1 ( tables 1 and 2 ) indicated that they were very closely related analogues , differing 
 only in the presence of an additional methoxy ( h 3.37 ; c 57.9 , 2-och3 ) , an oxygenated 
 quaternary carbon ( c 92.6 , c-8 ) , and a methylene 
 [ h 2.32 ( dd , j = 8.0 , 13.6 hz ) 
 and 2.19 ( br d , j = 13.6 hz)/c 38.4 
 ( c-9 ) ] in 2 , instead of the tiglyl group at c-8 
 and an oxygenated methine group at c-9 found in 1 . these conclusions were supported by the hmbc correlations from 
 h-9 to c-11/c-8/c-7 , of h-10 
 to c-9/c-6 , of 7-oh ( h 5.72 ) 
 to c-8/c-6 , and of two oh protons ( h 5.92 and 5.97 ) to c-9 , demonstrating the unique substitution 
 of an additional hydroxy group at c-8 and a methylene functionality 
 at the c-9 position , respectively ( figure 3 ) . the relative configuration of 2 was determined 
 to be the same as in 1 , after analyses of the h , c , and the noesy nmr spectroscopic data , and the 
 two hydroxy protons ( h 5.97 and 5.92 ) at c-8 
 were determined to have - and -orientations , respectively , 
 based on the noesy correlations between 8-oh ( h 5.97 ) and 7-oh (  axial ) and between 8-oh 
 ( h 5.92 ) and h-6 (  axial ) ( figure 3 ) . consequently , 2 was elucidated as 
 a new compound and named 9-deoxy-8,8-dihydroxyherbicidin 
 b. key hmbc and noesy correlations of 2 and 2a . the tricyclic sugar moiety with 
 the geminal dihydroxy functionality 
 in 2 was also reported in other aminoglycoside antibiotics 
 such as an adenylated spectinomycin analogue . as the presence of this c8-geminal diol ( a ketone hydrate ) 
 in 2 might affect its chemical stability , a set of nmr 
 experiments proton and carbon 
 nmr spectra were recorded at 1 day , 10 days , 20 days , and one month . interestingly , based on the analysis of the nmr spectra , 2 was found to gradually convert to 2a . for example , 
 after 10 days in dmso - d6 at room temperature 
 ( 20 c ) , the nmr spectrum of 2 was rerecorded , and 
 a second set of nmr signals was observed . this second set of signals 
 ( figure s1 ; supporting information ) was 
 consistent with 2a , which would be derived from dehydration 
 at c-8 in 2 . the result was further supported 
 by data obtained in hsqc and hmbc experiments , as well as by hresims , 
 which showed a minor ion peak at m / z 436.1452 [ m + h ] ( c18h22n5o8 ) . in the hmbc spectrum , the key correlations 
 from h-9 to c-8 ( ketone group ) , c-11 ( ester 
 carbonyl ) , and c-7 , from h-10 to c-9 and c-8 , 
 and from h-6 to c-8 , as well as of h-5 to 
 c-7 were observed ( figure 3 ) . finally , 
 the structure of compound 2a was elucidated and named 
 9-deoxy-8-oxoherbicidin b. compound 2 was further monitored using both h and c nmr every 10 days . analysis of the hresims and nmr data of 3 gave its 
 molecular formula as c18h23n5o9 . comprehensive interpretation of nmr spectra demonstrated 
 the structure of 3 was similar to herbicidin b ( 5 ) except for major chemical differences at c-6 , c-8 , 
 and c-10 , which seem to be shifted by the anisotropic effect 
 of the ester carbonyl group ( c-11 ) due to a change of configuration 
 on the outer pyran ring . the analyses of noesy spectra of 3 and 5 showed similar correlation peaks between h-8 
 and h-2/h-6. however , additional noesy correlation 
 peaks for 3 between h-8 and h-6/h-9 
 and between h-8 and h-8 revealed that h-8 was -oriented 
 and that an -hydroxy group was present at c-8 . in addition , 3 shares the same configurations with 5 in the 
 assignment of h-1 , h-6 , h-9 , 
 and h-10 , which were supported by analyses of the noesy 
 correlations ( figure 4 ) and a comparison of 
 their cd spectra ( figure 5 ) , in which the same 
 pattern of cotton effects was observed . subsequently , 3 was elucidated as an epimer of herbicidin b , named 8-epi - herbicidin b. key noesy correlations of 3 . in addition , three known 
 herbicidins , a ( 4 ) , b ( 5 ) , and f ( 8) , and 
 9-(-d - arabinofuranosyl)hypoxanthine were identified 
 by 1d and 2d nmr , esims , and/or comparisons of their h 
 nmr data with those in the literature . in particular 
 for ara - h , the noesy correlation between h-8/h-3 is shown 
 in figure s43 , suggesting an -orientation 
 of 3-oh , and it thus confirmed the existence of a d - arabinofuranosyl moiety in the structure . they are 
 composed of an adenine , an undecose moiety connected by a -glycosidic 
 bond , a -oriented h-6 , and hydroxy substitutions or 
 oxygenation at nine of the 11 undecose backbone carbons . their complex 
 structures have made their structure elucidation and data assignment 
 challenging , especially before the advent of 2d nmr techniques . our 
 work represents the first complete nmr assignment of the herbicidin 
 family of natural products and the first isolation of a naturally 
 occurring herbicidin with an 8-oh ( 3 ) . compounds 2 and 2a , with partial structural 
 similarity to spectinomycin and adenylated spectinomycin , are of special interest , as they are the first 
 herbicidin analogues without a hydroxy substituent at the c-9 
 position . the discovery of 2 led us to speculate the 
 herbicidins may be formed in a biogenic pathway originally from adenosine 
 and a deoxyhexose , possibly derived from glucose , as its nitrogen 
 and carbon sources . compounds 15 and 8 were evaluated for their inhibitory activities 
 against streptomyces 85e in the hfi assay , according 
 to an established protocol . however , 
 this series of nucleoside antibiotics 
 were inactive in the hfi assay . on the other hand , when evaluated 
 in a mammalian cell - based assay designed to monitor tnf--induced 
 nf-b activity , 15 and 8 were found to mediate inhibitory responses with ic50 values in a range of about 0.54.9 m ( table 3 ) . with the exception of 5 , when tested 
 using the same conditions as the nf-b assay , each of the remaining 
 compounds mediated a cytotoxic response with ic50 values 
 ranging from 2.2 to 43.7 m ( table 3 ) . 
 in the absence of a cytotoxic response , inhibition of tnf--induced thus , since 5 inhibits nf-b with some 
 specificity , it remains of interest as a cancer chemopreventative 
 agent . it is also 
 of interest to compare the inhibitory responses of the 
 remaining compounds with growth inhibitory activity . as shown with 
 the positive control compounds selected for this investigation ( tpck 
 and bay-11 ) , nf-b is inhibited in the low micromolar range , 
 but growth inhibition is observed with concentrations only 2 to 4 
 times higher . a similar response pattern was observed with 2 , 3 , 4 , and 8 , with nf-b 
 and growth inhibitory potency roughly equivalent to the positive control 
 compounds . compound 1 was an exception since the growth 
 inhibitory response was mediated with lower concentrations than the 
 nf-b inhibitory response , so we can suggest that nf-b 
 inhibition was due to cytotoxic effect of compound 1 . all the compounds failed to show any inhibitory effect against inducible 
 nitric oxide synthase ( inos ) or antioxidant activity with dpph ( data 
 not shown ) . in addition , growth inhibitory activity was not observed 
 with the hormone - responsive breast cancer cell line mcf-7 ( data not 
 shown ) . thus , the effect of these herbicidins on nf-b appears 
 somewhat unique and worthy of further investigation . in summary , a h nmr - guided fractionation of the culture 
 extract of terrestrial streptomyces sp . l-9 - 10 led 
 to the isolation and elucidation of three new herbicidin congeners , 
 one epimer of known herbicidin b , and three known analogues as well 
 as 9-(-d - arabinofuranosyl)hypoxanthine . moreover , 
 this work represents the first report of the complete nmr assignments 
 for the herbicidins and the first report on the isolation of an 8-oh 
 herbicidin from nature . biological testing showed that the herbicidins 
 exhibited modest inhibition against tnf--induced nf-b 
 activity . further work will be performed to design and develop novel 
 herbicidin analogues with potential improved efficacy and selectivity . nmr spectra 
 were recorded on bruker avance ( 400 mhz ) and inova unity ( 500 mhz ) 
 varian spectrometers . mass spectra and high - resolution ms spectra 
 were obtained with a biotof ii esi mass spectrometer . semipreparative 
 hplc was conducted on a beckman coulter gold-168 system equipped with 
 a photodiode array detector using an alltech reversed - phase econosil 
 c18 column ( 10 m , 10  250 mm ) with a flow 
 rate of 2.0 ml / min . column chromatography was carried out on merck 
 silica gel 60 ( 70230 mesh ) and preparative tlc plates ( 0.2 
  20  20 cm ) . the organism 
 was isolated from the surface of the lichen platismatia glauca growing on a douglas fir in the arboretum at the university of british 
 columbia malcolm knapp research forest , maple ridge , british columbia , 
 in february 2003 . the strain was isolated using isp#4 ( international 
 streptomyces project 4 , inorganic salts starch agar ) media plates 
 supplemented with 50 g / ml cycloheximide and 20 g / ml 
 nalidixic acid . the streptomyces sp . l-9 - 10 is maintained 
 in the laboratory collection at the university of british columbia 
 under the accession number dca 2501 . the strain shared > 99% identity 
 ( 835 base pairs of 836 ) with the partial 16s rrna gene of an unpublished 
 strain , streptomyces sp . the same method as previously described 
 was used to culture and harvest streptomyces sp . 
 l-9 - 10 . for detailed protocols , the fermentation 
 broth ( 100 
 l ) was centrifuged , and the supernatant was partitioned successively 
 with n - hexane , etoac , and n - buoh . the etoac - soluble partition exhibited inhibitory activity in the hyphae - formation 
 assay , giving an 18 mm clear zone of inhibition at a concentration 
 of 80 g / disk . l-9 - 10 was subjected to silica gel cc ( 500 
 g , 400600 mesh ) and eluted with a gradient of chcl3meoh ( 0:11:1 ) to afford 10 fractions , e1e10 . fraction e4 ( 5.2 g ) , with a 17 
 mm clear zone of inhibition in the hfi assay , was separated using 
 sephadex lh-20 ( 280 g , meoh ) to afford six subfractions , e4a  e4f . fraction e4d ( 1.2 g ) was further 
 purified by silica gel cc eluting with hexane fraction e4d2 was further separated with 
 silica gel cc eluting with chcl3meoh ( 40:1 ) to 
 obtain fractions e4d2a subfraction d2e ( 40 mg ) was separated by preparative 
 tlc ( 2.0 mm , 20  20 cm ) using chcl3etoh ( 5:1 , rf 0.6 ) to yield 8 ( 7.0 mg ) . then h nmr - guided fractionation was used for 
 fractions e5 and e6 , which exhibited characteristic 
 proton signals at  8.00 and 4.00 ppm in their h 
 nmr spectra . fraction e5 ( 3.6 g ) was separated by sephadex 
 lh-20 ( 280 g , meoh ) to obtain seven subfractions ( e5a  e5 g ) . fraction e5e ( 1.1 g ) was further subjected 
 to silica gel cc eluting with hexane  acetone ( 1:2 ) to yield 
 the main portion , then by a preparative tlc ( 2.0 mm , 20  20 
 cm ) purification using chcl3etoh ( 5:1 , rf 0.4 ) to give 4 ( 13.5 mg ) . fraction e6 ( 5.8 g ) was subjected to silica 
 gel cc eluting with etoac  etoh ( 20:15:1 ) to obtain 
 12 subfractions ( e6a  e6l ) . fraction e6e was separated by sephadex lh-20 ( 70 g , meoh ) to give four 
 fractions ( e1e4 ) . fraction e6i ( 0.8 g ) was separated with 
 sephadex lh-20 ( 70 g , meoh ) to obtain four subfractions ( e6i1e6i4 ) , and fraction e6i3 was repeatedly 
 separated by reversed - phase hplc eluting with meoh  h2o ( 55:45 ) to afford 2 ( 2.8 mg , tr 32.8 min ) , 3 ( 11.4 mg , tr 19.8 min ) , and 5 ( 27.8 mg , tr 24.2 min ) . fraction e9 ( 2.2 g ) was subjected 
 to sephadex lh-20 ( 280 g , meoh ) to yield four subfractions ( e9a  d ) . fraction e9c was applied to silica 
 gel and eluted with chcl3meoh ( 5:2 ) to afford crude 
 ara - h , which was purified by recrystallization from meoh . white , 
 amorphous solid ; [ ]d25 78.1 ( c 0.032 , 
 meoh ) ; uv ( meoh ) max ( log  ) 212 ( 4.27 ) , 259 
 ( 4.03 ) nm ; ir ( kbr ) 3340 , 3204 , 2951 , 1710 , 1634 , 1597 , 1574 , 1416 , 
 1249 , 1133 , 1053 , 1008 , 970 cm ; h 
 and c nmr ( cd3od , 400/100 mhz ) see tables 1 and 2 ; hresims m / z 522.1821 [ m + h ] ( calcd for c22h28n5o10 , 522.1836 ) . white solid ; 
 [ ]d25 48.0 ( c 0.050 , meoh ) ; 
 uv ( meoh ) max ( log  ) 210 ( 4.13 ) , 259 ( 4.00 ) 
 nm ; ir ( kbr ) 3332 , 3212 , 2849 , 1732 , 1642 , 1597 , 1574 , 1299 , 1197 , 
 1072 , 1019 , 948 cm ; h and c nmr ( dmso - d6 , 400/100 mhz ) see tables 1 and 2 ; hresims m / z 454.1560 [ m + h ] ( calcd for c18h24n5o9 , 454.1574 ) . not formally isolated ; h 
 and c nmr ( dmso - d6 , 400/100 
 mhz ) see tables 1 and 2 ; hresims m / z 436.1452 [ m + h ] ( calcd for c18h22n5o8 , 436.1468 ) . white , amorphous powder ; [ ]d25 9.4 ( c 0.040 , meoh ) ; uv 
 ( meoh ) max ( log  ) 209 ( 4.44 ) , 259 ( 4.05 ) 
 nm ; ir ( kbr ) 3396 , 3193 , 2951 , 2921 , 1729 , 1638 , 1597 , 1249 , 1057 , 
 955 cm ; cd ( 1.0 , meoh ) max (  ) 
 262 ( 9.0 ) , 216 ( 5.5 ) nm ; h and c nmr ( cd3od , 400/100 mhz ) see tables 1 and 2 ; hresims m / z 454.1555 [ m + h ] ( calcd for c18h24n5o9 , 454.1574 ) . white solid ; [ ]d25 58.3 ( c 0.075 , 
 meoh ) [ lit . [ ]d 61.7 ( c 1.0 , meoh ) ] ; h 
 and c nmr ( cd3od , 400/100 mhz ) see tables 1 and 2 . white , amorphous powder ; 
 [ ]d25 55.0 ( c 0.050 , meoh ) [ lit . [ ]d 63.0 ( c 1.0 , meoh ) ] ; 
 cd ( 1.0 , meoh ) max (  ) 260 ( 5.2 ) , 
 218 ( 0.5 ) nm ; h and c nmr ( cd3od , 400/100 mhz ) see tables 1 and 2 . white 
 solid ; [ ]d25 75.0 ( c 0.025 , meoh ) ; h and c nmr ( cd3od , 400/100 mhz ) see tables 1 and 2 . we employed human embryonic kidney 
 cells 293 panomic for monitoring changes occurring along the nf-b 
 pathway . stable constructed cells were 
 seeded into 96-well plates at 20  10 cells per well . cells were maintained in dulbecco s modified eagle s 
 medium ( dmem ) ( invitrogen co. ) , supplemented with 10% fbs , 100 units / ml 
 penicillin , 100 g / ml streptomycin , and 2 mm l - glutamine . 
 after 48 h incubation , the medium was replaced and the cells were 
 treated with various concentrations of test substances . tnf- 
 ( human , recombinant , e. coli , calbiochem ) was used 
 as an activator at a concentration of 2 ng / ml ( 0.14 nm ) . the plate 
 was incubated for 6 h. spent medium was discarded , and the cells were 
 washed once with pbs . cells were lysed using 50 l ( for 96-well 
 plate ) of reporter lysis buffer from promega , by incubating for 5 
 min on a shaker , and stored at 80 c . the gene 
 product , luciferase enzyme , reacts with luciferase substrate , emitting 
 light , which was detected using a luminometer ( lumistar galaxy bmg ) . data for nf-b inhibition are expressed as ic50 values 
 ( i.e. , concentration required to inhibit tnf - induced nf-b activity 
 by 50% ) . as a positive control , the inhibition of hyphae - formation in streptomyces 85e was performed on pure compounds as described 
 previously .
four new undecose nucleosides ( herbicidin 
 congeners ) , three known 
 herbicidins , and 9-(-d - arabinofuranosyl)hypoxanthine 
 ( ara - h ) were isolated from the organic extract of a fermentation culture 
 of streptomyces sp . 
 l-9 - 10 using proton nmr - guided 
 fractionation . 
 their structures were elucidated on the basis of comprehensive 
 1d and 2d nmr and mass spectrometry analyses . 
 these structures included 
 2-o - demethylherbicidin f ( 1 ) , 
 9-deoxy-8,8-dihydroxyherbicidin b ( 2 ) , 9-deoxy-8-oxoherbicidin b ( 2a ) , and 
 the 8-epimer of herbicidin b ( 3 ) . 
 this is the 
 first detailed assignment of proton and carbon chemical shifts for 
 herbicidins a , b , and f. the isolated compounds were evaluated for 
 cancer chemopreventive potential based on inhibition of tumor necrosis 
 factor alpha ( tnf-)-induced nuclear factor - kappa b ( nf-b ) 
 activity .