uic 10339 was obtained from a sample 
 collected from raven lake in northern wisconsin in august 2010 and 
 cultured in z media .
the lyophilized cells were extracted with ch2cl2 and meoh ( 1:1 ) and dried in vacuo .
this extract showed antiproliferative activity against the human 
 colon cancer cell line ht-29 and was subjected to a bioassay - guided 
 fractionation scheme using diaion vacuum liquid chromatography .
the 
 fraction eluting with 60% isopropyl alcohol ( ipa ) displayed antiproliferative 
 activity .
lc / ms and h nmr dereplication indicated the 
 presence of two potentially new peptides with molecular weights of 
 1184 and 1446 da .
subsequent reversed - phase hplc separations yielded 
 trichormamides a ( 1 ) and b ( 2 ) .
trichormamide 
 a ( 1 ) was obtained as a white , amorphous 
 powder .
the molecular formula of 1 was deduced as c58h93n11o15 based on hresims 
 analysis ( m / z 1184.6978 [ m + h ] ) .
the h nmr 
 spectra of 1 in cd3oh with suppression at 
 4.9 ppm exhibited sharper peaks , but the resonances close to 4.9 ppm 
 were also suppressed .
therefore , h nmr , cosy , and tocsy 
 experiments were conducted in both cd3od ( without signal 
 suppression , but nh signals absent ) and cd3oh ( suppressed 
 at 4.9 ppm , but nh signals present ) , respectively , to obtain both 
 nh signals and clear signals close to 4.9 ppm ( supporting information s4s12 ) .
the values of chemical 
 shifts were reported using 1d and 2d nmr spectra obtained in cd3oh .
the h nmr spectrum of 1 exhibited 
 a signal pattern characteristic of a lipopeptide , including exchangeable 
 amide signals ( h 6.78.9 ) , signals typical 
 of -protons ( h 4.15.0 ) , aliphatic 
 methylene signals ( h 1.11.4 ) , and several 
 doublet and triplet methyl signals ( h 0.71.0 ) . 

the two downfield doublets ( h 7.25 , j = 8.7 hz ; h 6.71 , j = 8.7 ) were 
 attributed to a para - substituted phenyl moiety .
analysis 
 of cosy and tocsy correlations of 1 revealed the presence 
 of 11 amino acid residues , including 10 standard -amino acid 
 residues : three serines ( ser1 , ser2 , ser3 ) , two prolines ( pro1 , pro2 ) , 
 two isoleucines ( ile1 , ile2 ) , tyrosine ( tyr ) , leucine ( leu ) , and glycine 
 ( gly ) .
sequential cosy correlations between nh ( h 7.44 ) , methine h-3 ( h 4.36 ) , and methylene h2 - 2 ( h 1.59 ; 2.06 ) , as well as cosy correlations 
 between methine h-3 ( h 4.36 ) and methylene h2 - 4 ( h 1.50 ; 1.58 ) , suggested the presence 
 of a -amino acid residue .
sequential cosy correlations from 
 h2 - 4 ( h 1.50 ; 1.58 ) to the region of 
 highly overlapped methylene signals ( h2 - 59 , h 1.281.33 ) , which corresponded to five carbons in 
 the hsqc spectrum ( c 32.9 , 30.3 , 30.2 , 27.2 , and 
 23.6 ) , and finally to the methyl triplet h-10 ( h 0.86 ) identified this -amino acid residue as -aminodecanoic 
 acid ( ada ) .
assignment of the amino acid sequence was carried 
 out by a combination 
 of hmbc , roesy , and esims / ms analysis ( figure 1 ) .
hmbc correlations from ile2-nh ( h 8.84 ) to ile1 
 c-1 ( c 174.1 ) , from ile1-nh ( h 6.95 ) to leu c-1 ( c 173.6 ) , from leu - nh ( h 7.71 ) to tyr c-1 ( c 175.1 ) , from tyr - nh 
 ( h 7.30 ) to ser3 c-1 ( c 173.0 ) , 
 and from ser3-nh ( h 7.57 ) to pro1 c-1 ( c 172.2 ) suggested a partial sequence of ile2-ile1-leu - tyr - ser3-pro1 . 
 hmbc correlation from pro1 h2 - 5 ( h 3.60 ; 
 3.88 ) to ser2 c-1 ( c 174.3 ) extended this sequence 
 to ile2-ile1-leu - tyr - ser3-pro1-ser2 . similarly , the hmbc correlations 
 from ser1-nh ( h 6.79 ) to ada c-1 ( c 172.2 ) , from ada - nh ( h 7.44 ) to gly c-1 ( c 170.4 ) , and from gly - nh ( h 8.75 ) to pro2 
 c-1 ( c 176.1 ) established the partial sequence of 
 ser1-ada - gly - pro2 . this partial sequence was confirmed by roesy correlations 
 between ser1-nh ( h 6.79 ) and ada h2 - 2 
 ( h 1.59 ; 2.06 ) and between ada - nh ( h 7.44 ) and gly h2 - 2 ( h 3.42 ; 3.97 ) . 
 a roesy correlation between ser2-nh ( h 7.88 ) and 
 ser1 h2 - 3 ( h 4.00 ; 4.27 ) linked the two 
 partial sequences together into ile2-ile1-leu - tyr - ser3-pro1-ser2-ser1-ada - gly - pro2 . 

the 18 degrees of unsaturation and the molecular formula suggested 
 that 1 was a cyclic peptide .
the chemical shifts of ile2 
 c-1 ( c 173.8 ) , c-2 ( c 52.1 ) and 
 pro2 c-2 ( c 64.5 ) , c-5 ( c 49.5 ) , 
 h2 - 5 ( h 4.07 ; 4.13 ) suggested the presence 
 of an amide moiety at ile2 c-1 and pro2 n. therefore , the cyclic undecapeptide ring was closed between ile2 
 and pro2 .
mass fragmentation analysis using a quadrupole - time - of - flight 
 ( qtof ) tandem mass spectrometer confirmed the sequence of amino acid 
 residues determined by nmr analysis . the parent ion [ m + h ] 1184.6 was fragmented using a 40 ev collision energy .
as expected , 
 protonation and breakage of the proline amide bond is a highly favored 
 process , and the macro ring of 1 opened between ile2 
 and pro2 , forming a linear acylium ion .
the acylium ion was continuously fragmented to generate fragment 
 ions as illustrated in figure 2 .
the esims / ms 
 fragments were in complete agreement with the structure determined 
 by nmr studies , and the planar structure of 1 was determined 
 as cyclo[ile2-ile1-leu - tyr - ser3-pro1-ser2-ser1-ada - gly - pro2 ] .
key 2d nmr 
 correlations of trichormamide a ( 1 ) and 
 trichormamide b ( 2 ) .
the absolute configurations of 
 the amino acids were determined 
 using acid hydrolysis followed by the advanced marfey s method 
 ( supporting information s14 ) .
the chromatographic comparison between marfey s derivatives 
 of the hydrolysate of 1 and appropriate amino acid standards 
 assigned the l configurations for ser1/2/3 and pro1/2 and 
 the d configuration for tyr .
the absolute configuration of 
 ada was determined as 3r by comparison of the elution 
 order of hydrolysate ada l- and d - fdla derivatives 
 with those reported in the literature .
the stereoisomers of leucine and isoleucine standards were analyzed 
 by marfey s method , and retention times were compared with 
 marfey s derivatives of hydrolysate of 1 . as
leucine 
 and isoleucine isomers have identical molecular weight and very similar 
 retention behavior in hplc , a rigorous co - injection scheme was utilized 
 to assign leu as d and ile1/2 as l ( 2s , 3s ) ( supporting information 
 s14 ) .
the final structure of 1 was determined 
 as cyclo[l - ile  l - ile  d - leu  d - tyr  l - ser  l - pro 
trichormamide b ( 2 ) was obtained 
 as a white , amorphous 
 powder .
the molecular formula was determined as c69h115n13o20 ( m / z 1446.8508 [ m + h ] ) by hresims analysis .
the h nmr spectrum of 2 in dmso - d6 ( table 2 ) 
 also exhibited signals characteristic of a lipopeptide .
signal doubling 
 was observed for some of the signals in both the h nmr 
 and deptq spectra , indicating the presence of two conformers of 2 in solution ( integration ratio 2:1 in dmso - d6 , according to integration of the phenyl protons from 
 tyrosine ) .
this phenomenon has been reported for cyclic peptides with 
 one or more n - methylated amino acid residues .
an elevated - temperature nmr experiment was attempted to generate 
 one average set of signals . however , the coalescence temperature of 2 was higher than the highest operating temperature permitted 
 by a cryogenically cooled probe ( t = 328 k ) , and 
 no signal coalescence was observed .
nevertheless , the elevated - temperature 
 nmr experiment showed improved peak shapes and better resolved coupling 
 patterns as compared to the room - temperature experiments .
thus , the 
 structure elucidation of 2 was performed using the signals 
 from the major conformer at elevated temperature ( t = 328 k ) . combined analysis of the cosy and
tocsy spectra identified 
 the structures of seven standard amino acids : isoleucine ( ile ) , glutamine 
 ( glu ) , serine ( ser ) , proline ( pro ) , tyrosine ( tyr ) , and two threonines 
 ( thr1 and thr2 ) ( figure 1 and table 2 ) .
an n - methylated isoleucine ( nmeile ) 
 was deduced by tocsy and the hmbc correlation observed from n - me ( h 3.00 ) to nmeile c-2 ( c 59.8 ) .
the presence of homoserine ( hse ) was evident by sequential 
 cosy correlations from nh ( h 8.01 ) to hse h2 - 4 ( h 3.42 ; 3.50 ) .
the downfield chemical 
 shifts of the hse c-4 methine ( c 57.3 ) and h2 - 4 ( h 3.42 ; 3.50 ) indicated the attachment 
 of a hydroxy group to c-4 . a broad hydroxy signal ( h 5.05 ) with a cosy correlation to a downfield methine ( h 3.52 ) , as well as sequential cosy correlations between nh 
 ( h 8.16)/h-2 ( h 4.39)/h-3 ( h 3.52)/h-4 ( h 1.61)/h3 - 5 ( h 0.93 ) , combined with hmbc correlations from 4-me ( h 0.81 ) and h3 - 5 ( h 0.93 ) to both 
 c-3 ( c 76.3 ) and c-4 ( c 30.5 ) , 
 indicated the presence of a 3-hydroxyleucine ( 3-ohleu1 ) .
an additional 
 3-hydroxyleucine ( 3-ohleu2 ) was identified using a similar approach 
 ( table 2 ) .
the presence of a -aminodecanoic 
 acid was suggested from cosy , tocsy , and hsqc correlations as described 
 for trichormamide a ( 1 ) .
carbon chemical 
 shifts were assigned 
 from the deptq spectrum recorded at 226 mhz .
carbon chemical 
 shifts were assigned 
 from the deptq spectrum recorded at 226 mhz .
the sequence of the 12 amino acid 
 residues was established by analysis 
 of the hmbc and roesy spectra ( figure 1 and 
 table 2 ) .
hmbc correlations from 3-ohleu1-nh 
 ( h 8.01 ) to ile c-1 ( c 170.8 ) , 
 from ile - nh ( h 8.16 ) to ada c-1 ( c 171.2 ) , from ada - nh ( h 7.52 ) and ada h-3 ( h 4.08 ) to thr2 c-1 ( c 169.6 ) , from thr2-nh 
 ( h 7.60 ) to tyr c-1 ( c 170.8 ) , 
 and from tyr - nh ( h 7.97 ) to pro c-1 ( c 171.5 ) indicated a partial sequence of 3-ohleu1-ile - ada - thr2-tyr - pro . 

roesy correlations between 3-ohleu1-nh ( h 8.01 ) 
 and ile h-2 ( h 4.19 ) , between ile - nh ( h 8.16 ) and ada h2 - 2 ( h 2.31 ; 
 2.49 ) , between ada - nh ( h 7.52 ) and thr2 h-2 ( h 4.05 ) , between thr2-nh ( h 7.60 ) and tyr 
 h-2 ( h 4.45 ) , and between tyr - nh ( h 7.97 ) and pro h-2 ( h 4.33 ) confirmed this partial 
 sequence .
similarly , a partial sequence of ser - nmeile - gln-3-ohleu2-hse 
 was established by the hmbc correlations from ser - nh ( h 7.83 ) to nmeile c-1 ( c 169.8 ) , from nmeile 
 n - me ( h 3.00 ) to gln c-1 ( c 172.3 ) , 
 from gln - nh ( h 7.93 ) to 3-ohleu2 c-1 ( c 171.3 ) , and from 3-ohleu2-nh ( h 7.76 ) to 
 hse c-1 ( c 171.3 ) . this partial sequence was also 
 confirmed by the roesy correlations between ser - nh ( h 7.83 ) and nmeile h-2 ( h 4.73 ) , between gln - nh 
 ( h 7.93 ) and 3-ohleu2 h-2 ( h 4.32 ) , 
 and between 3-ohleu2-nh ( h 7.76 ) to hse h-2 ( h 4.35 ) . the roesy correlations between thr1-nh ( h 7.51 ) and ser h-2 ( h 4.33 ) and between 
 thr1-nh ( h 7.51 ) and ser h2 - 3 ( h 3.60 ) further expanded this partial sequence to thr1-ser - nmeile - gln-3-ohleu2-hse . 
 an hmbc correlation from hse - nh ( h 7.95 ) to 3-ohleu1 
 c-1 ( c 172.3 ) , together with a roesy correlation 
 between hse - nh ( h 7.95 ) and 3-ohleu1 h-2 ( h 4.39 ) , connected those two partial sequences together into 
 thr1-ser - nmeile - gln-3-ohleu2-hse-3-ohleu1-ile - ada - thr2-tyr - pro .
the 
 molecular formula and degree of unsaturation indicated the cyclic 
 nature of 2 .
the chemical shifts of thr1 c-1 ( c 168.7 ) and c-2 ( c 55.6 ) suggested the presence 
 of an amide moiety at thr1 c-1 . similarly , 
 the chemical shifts of pro c-2 ( c 59.5 ) , c-5 ( c 47.0 ) , and h2 - 5 ( h 3.61 ; 3.67 ) 
 also indicated that pro n was part of an amide bond . therefore , the cyclic dodecapeptide ring was closed between 
 thr1 and pro .
protonation on 
 the amide nitrogen at pro generated amide bond cleavage to form a 
 linear acylium ion , which produced series of fragments ( figure 3 ) .
these observed fragments were consistent with 
 the amino acid sequence determined by nmr analysis , and the planar 
 structure of 2 was assigned as cyclo[thr1-ser - nmeile - gln-3-ohleu2-hse-3-ohleu1-ile - ada - thr2-tyr - pro ] .
acid hydrolysis of trichormamide b ( 2 ) followed by 
 marfey s derivatization with l- and d - fdla 
 and lc / ms analysis assigned the l configurations for gln , 
 pro , ile ( 2s , 3s ) , thr1/2 ( 2s , 3r ) , and the nonproteinogenic amino 
 acid hse , d configurations for tyr and ser , and 3r configuration for ada .
the four stereoisomers of nmeile 
 were synthesized according to the literature . on the basis of these standards , the absolute configuration of nmeile 
 was determined to be l ( 2s , 3s ) .
the 2s , 3r and 2s , 3s isomers of 3-hydroxyleucine were synthesized 
 following bonnard s procedure and 
 were derivatized with l- and d - fdla respectively . 
 because enantiomers exhibit identical retention behavior under nonchiral 
 hplc conditions , the ( 2s,3r)-d - fdla derivative shows the same retention time as ( 2r,3s)-l - fdla , and the ( 2s,3s)-d - fdla derivative shows 
 the same retention time as ( 2r,3r)-l - fdla ( detailed explanations can be found in supporting information s26 ) . by comparing the 
 above four retention times with retention times of 3-ohleu1 and 3-ohleu2 l - fdla derivatives , both 3-ohleu1 and 3-ohleu2 were assigned 
 as 2r , 3s .
the complete structure 
 of 2 was determined as cyclo[l - thr  d - ser  l - nmeile  l - gln(2r,3s)-3-ohleu  l - hse(2r,3s)-3-ohleu  l - ile(3r)-ada  l - thr  d - tyr  l - pro ] .
trichormamide a ( 1 ) is a cyclic undecapeptide 
 characterized 
 by the presence of -aminodecanoic acid and two d - amino 
 acid residues ( d - tyr and d - leu ) .
trichormamide a 
 ( 1 ) is a lipid - containing compound and displays amphiphilic 
 character with half of the amino acid sequence being hydrophobic ( ada - gly - pro2-ile2-ile1-leu ) 
 and the other half of the sequence hydrophilic ( tyr - ser3-pro1-ser2-ser1 ) . 

the amphiphilic nature of lipopeptides has been found to be strongly 
 correlated with their plasma membrane interactive potential .
trichormamide a ( 1 ) is structurally related to hormothamnin 
 a , lobocyclamide a , and laxaphycin a with conserved amino acid residues 
 of leu , ile1 , ile2 , and gly .
the amino acid residues homoserine , 
 4-hydroxyproline , ,-didehydro--aminobutyric acid , 
 and -aminooctanoic acid in the east portion of the three related 
 compounds were replaced by structurally related residues of ser , pro , 
 ser , and ada in trichormamide a ( 1 ) ( supporting information s27 ) .
trichormamide b ( 2 ) is a cyclic dodecapeptide characterized by the presence of a -amino 
 acid residue ( -aminodecanoic acid ) , two d - amino acid 
 residues ( d - tyr and d - ser ) , and four nonstandard 
 amino acid residues ( 3-ohleu1 , hse , 3-ohleu2 , nmeile ) .
trichormamide 
 b ( 2 ) is also an amphiphilic molecule containing a hydrophobic 
 ada residue and seven hydroxy groups .
trichormamide b ( 2 ) is structurally related to the cyclic dodecapeptides lobocyclamides 
 b and c , laxaphycins b , b2 , and b3 , and lyngbyacyclamides a and b. a comparison of 2 to these structures revealed that 
 the presence and location of 3-ohleu1 , gln , nmeile , thr1 , and thr2 
 are conserved in all these peptides , indicating these amino acid residues 
 may be crucial for the biological functions of these molecules .
trichormamides a ( 1 ) and b ( 2 ) were evaluated 
 for their antiproliferative activities against the human melanoma 
 cell line mda - mb-435 and the human colon cancer cell line ht-29 .
trichormamide 
 b ( 2 ) displayed significant cytotoxic activity against 
 mda - mb-435 and ht-29 cell lines with ic50 values of 0.8 
 and 1.5 m , respectively .
trichormamide a ( 1 ) was 
 less active than 2 and showed ic50 values 
 of 9.9 and 16.9 m against mda - mb-435 and ht-29 , respectively . 

compounds structurally related to 1 and 2 were reported to have 23-fold increased synergistic activities 
 in antimicrobial and cytotoxicity assays . however , the 
 limited supply of 1 stopped us from obtaining statistically 
 significant data in synergistic assays . in summary
, we have 
 obtained two new cyclic lipopeptides , trichormamides 
 a ( 1 ) and b ( 2 ) , each containing the -amino 
 acid residue -aminodecanoic acid , from the cultured freshwater 
 cyanobacterium trichormus sp .
their planar 
 structures were determined by analysis of the hresims , ms / ms , and 
 1d and 2d nmr experiments .
the stereoconfigurations of the amino acid 
 residues were assigned by the advanced marfey s method using 
 synthesized amino acid standards .
trichormamide b ( 2 ) was found to be more active against 
 mda - mb-435 and ht-29 human cancer cell lines than trichormamide a 
 ( 1 ) , with ic50 values of 0.8 and 1.5 m , 
 respectively .
optical rotations were 
 measured on a perkinelmer 241 polarimeter at 22 c in meoh .
1d 
 and 2d nmr spectra including h nmr , cosy , tocsy , hsqc , 
 hmbc , and roesy were obtained on a bruker avance drx 600 mhz spectrometer . 

a bruker avii 900 mhz nmr spectrometer was used to acquire the deptq 
 spectra .
h and c nmr chemical shifts were 
 referenced to the dmso - d6 ( h 2.50 and c 39.51 , respectively ) and cd3od / cd3oh ( h 3.31 and c 49.0 , respectively ) residual solvent signals .
the hmbc spectrum was recorded with an 
 average jch of 8 hz , and the 
 hsqc spectrum was recorded with an average jch of 145 hz .
high - resolution esi mass spectra and lc - ms 
 data were obtained using a shimadzu hplc - it - tof spectrometer .
the 
 hplc experiments were performed using an agilent 1100 series instrument 
 equipped with a diode array detector , and a waters instrument equipped 
 with a waters 600 pump and a waters 2487 detector .
uic 10339 was isolated from a sample collected at raven lake , wisconsin , 
 in 2010 ( n 4558.7 , w 8951.5 ) .
the 
 unialgal ( not axenic ) strain uic 10339 was obtained through micropipette 
 isolation techniques .
the strain was 
 cultured in 8  2 l of z medium in 2.8 l fernbach flasks with 
 sterile air aeration .
cultures were illuminated 
 with fluorescent lamps at 1.03 klx with an 18/6 h light / dark cycle . 

the biomass of uic 10339 was harvested after 8 weeks of growth by 
 centrifugation and freeze - dried .
morphological 
 observation was performed using a zeiss axiostar plus light microscope 
 equipped with a canon powershot a620 camera .
the following characters 
 were utilized for identification : structure of colony , morphology 
 of thallus , size , shape , and motility of trichomes , shape of vegetative 
 cells , presence or absence of mucilage or sheath , presence and arrangement 
 of heterocytes , presence and relative size of akinetes , and morphology 
 of the end cell ( supporting information s1 , s2 ) .
the morphological characterization and initial taxonomical designation 
 of uic 10339 were made according to the system by komrek et 
 al .
prior to dna extraction , 
 8 ml of a static culture of trichormus sp .
uic 10339 
 was combined with 1.5 ml of lysozyme buffer and 0.5 
 ml of 20 mg / ml lysozyme stock solution and incubated at 35 c 
 for 1 h. after incubation , the cell mass was centrifuged and transferred 
 to a 2 ml microcentrifuge tube . a wizard genomic dna purification 
 kit ( promega )
a partial sequence 
 of the 16s rdna gene was pcr - amplified using the cyanobacteria - specific 
 primers 106f and 1509r . for a total volume 
 of 25 l ,
the reaction mixture contained dna ( 1 l , approximately 
 300 ng ) , gotaq reaction buffer ( 5 l , 5 ) , dntp mix ( 0.5 
 l , 10 mm ) , upstream and downstream primer ( 1 l each , 
 10 m ) , gotaq dna polymerase ( 0.25 l , 2.5 u/l ) , 
 and h2o ( 16.25 l ) .
the reaction was performed in 
 a bio - rad c1000 thermal cycler with the following reaction program : 
 initial denaturation for 30 s at 98 c , 35 amplification cycles 
 of 10 s at 98 c , 30 s at 53 c , 30 s at 72 c , and 
 a final extension for 10 min at 72 c .
pcr products were purified 
 using a minelute pcr purification kit ( qiagen ) and sequenced using 
 the cyanobacteria - specific primers 106f , 359f , and 1509r .
the resulting 16s rdna gene sequence was deposited 
 in the ncbi genbank under the accession no .
lyophilized biomass ( 6.14 
 g ) from eight 2 l cultures of uic 10339 was extracted with 1:1 ch2cl2/meoh three times and concentrated in 
 vacuo to yield 650.9 mg of extract , which was fractionated 
 using diaion hp-20ss resin with an h2o / ipa step gradient 
 ( 1:0 , 4:1 , 3:2 , 2:3 , 3:7 , 1:4 , 1:9 , 0:1 v / v ) to yield eight fractions . 

the fraction eluting with h2o / ipa ( 2:3 v / v ) ( 58.8 mg ) displayed 
 significant antiproliferative activity against the human melanoma 
 cell line mda - mb-435 with 100% growth inhibition at a concentration 
 of 25 g / ml .
lc - ms combined with h nmr dereplication 
 of this fraction revealed the presence of new lipopeptides .
the fraction 
 was subjected to semipreparative reversed - phase hplc ( varian c8 semipreparative column , 10  250 mm , 3 ml / min ) with 
 a linear gradient from 80% to 100% meoh with h2o over 20 
 min , and the subfraction eluting from 9 to 10 min was collected and 
 dried in vacuo .
h nmr and ms flow injection 
 analysis revealed that this subfraction consisted of two compounds . 

the subfraction was subjected to semipreparative reversed - phase hplc 
 ( varian c18 semipreparative column , 10  250 mm , 3 
 ml / min ) using isocratic 50% aqueous ch3cn .
trichormamide 
 a ( 1 , 0.8 mg , 0.01% of dry weight ) was eluted at 8 min , 
 and trichormamide b ( 2 , 3.7 mg , 0.06% of dry weight ) 
 was eluted at 5 min .
white , amorphous 
 powder ; [ ]d22 + 20 ( c 0.06 , meoh ) ; uv ( meoh ) max ( log  ) 240 ( 3.07 ) , 278 ( 2.95 ) nm ; ir ( neat ) max 3312 ( br ) , 2984 , 2942 , 2831 , 1449 cm ; h and c nmr see table 1 ; hr - esi - tof - ms ( + ) m / z 1184.6978 
 [ m + h ] ( calcd for c58h94n11o15 , 1184.6931 ) .
white , amorphous 
 powder ; [ ]d22 25.9 ( c 0.32 , meoh ) ; uv ( meoh ) 
 max ( log  ) 240 ( 3.16 ) , 278 ( 2.97 ) nm ; ir 
 ( neat ) max 3317 ( br ) , 2943 , 2831 , 1449 , 1414
, 1114 
 cm ; h and c nmr see table 2 ; hr - esi - tof - ms 
 ( + ) m / z 1446.8508 [ m + h ] ( calcd for c69h116n13o20 , 1446.8460 ) .
the amino 
 group of l - ile was boc - protected using a previously published 
 protocol .
the methylation of nboc - l - ile was carried out using malkov s 
 method .
the boc - protected nmeile was 
 subjected to acid hydrolysis using the general peptide hydrolysis 
 scheme as described below , to yield l - n - methylisoleucine 
 ( 73.11 mg ) , which was analyzed by the advanced marfey s method . 

although citric acid from the workup was observed as a minor impurity 
 in the h nmr spectrum of l - n - methylisoleucine , it did not interfere with the advanced marfey s 
 analysis .
d - n - methylisoleucine , l - allo - n - methylisoleucine , and d - allo - n - methylisoleucine 
 were synthesized using the same protocol with d - ile , l - allo - ile , and d - allo - ile as starting compounds .
all were analyzed using the advanced 
 marfey s method as described for the peptide hydrolysate .
l - n - methylisoleucine : [ ]d22 + 17.6 ( c 0.39 , meoh ) ; h nmr data ( dmso - d6 , 400 mhz )  3.84 ( 1h , d , j = 
 3.0 hz ) , 2.59 ( 3h , s ) , 1.95 ( 1h , m ) , 1.50 ( 1h , m ) , 1.32 ( 1h , m ) , 0.91 
 ( 3h , d , j = 8.5 hz ) , 0.90 ( 3h , t , j = 7.1 hz ) ; deptq data ( dmso - d6 , 400 
 mhz )  169.5 , 64.6 , 35.3 , 32.5 , 26.4 , 14.5 , 12.0 . nmr spectra 
 of d - ile , l - allo - ile , and d - allo - ile can be found in supporting 
 information s24 .
we obtained 148.0 mg of major 
 precursor ( r)-1-((2s,5r)-5-isopropyl-3,6-dimethoxy-2,5-dihydropyrazin-2-yl)-2-methylpropan-1-ol 
 and 54.5 mg of minor precursor ( s)-1-((2s,5r)-5-isopropyl-3,6-dimethoxy-2,5-dihydropyrazin-2-yl)-2-methylpropan-1-ol . 

the hydroxyleucine precursors were hydrolyzed following the general 
 peptide acid hydrolysis process as described below .
the major precursor 
 gave a mixture of ( 2s,3r)-3-hydroxyleucine 
 and d - valine , and the minor precursor gave a mixture of ( 2s,3s)-3-hydroxyleucine and d - valine . 

these two mixtures were derivatized with l - fdla and d - fdla , respectively , and the resulting four derivatization mixtures 
 were analyzed using the general lc - ms method as described for peptide 
 hydrolysate analysis .
h nmr data of major precursor ( cdcl3 , 300 mhz ) :  4.08 ( 1h , dd , j = 3.2 
 hz , 2.9 hz ) , 3.98 ( 1h , dd , j = 3.6 hz , 3.4 hz ) , 3.71 
 ( 3h , s ) , 3.67 ( 3h , s ) , 3.61 ( 1h , br ) , 2.24 ( 1h , m ) , 1.99 , ( 1h , m ) , 
 1.69
( 1h , br ) , 1.03 ( 3h , d , j = 6.9 hz ) , 1.02 ( 3h , 
 d , j = 6.9 hz ) , 0.97 ( 3h , d , j = 
 6.9 hz ) , 0.70 ( 3h , d , j = 6.9 hz ) .
h 
 nmr data of minor precursor ( cdcl3 , 300 mhz ) :  4.17 
 ( 1h , dd , j = 4.3 hz , 4.0 hz ) , 3.98 ( 1h , dd , j = 3.7 hz , 3.6 hz ) , 3.71 ( 3h , s ) , 3.70 ( 3h , s ) , 3.65 ( 1h , 
 br ) , 2.27 ( 1h , m ) , 1.82 , ( 1h , m ) , 1.68 ( 1h , br ) , 1.04 ( 3h , d , j = 6.9 hz ) , 0.90 ( 3h , d , j = 6.9 hz ) , 
 0.89 ( 3h , d , j = 6.9 hz ) , 0.69 ( 3h , d , j = 6.9 hz ) . approximately 0.3 mg of 1 and 2 was hydrolyzed with 1 ml of 6
n hcl for 
 20 h at 110 c 
 in separate teflon - sealed ace pressure tubes .
the cooled hydrolysate 
 mixtures were evaporated to dryness , and traces of hcl were removed 
 from the reaction mixtures by repeated evaporation .
each acid hydrolysate ( approximately 
 0.3 mg ) was separated into two equal portions for derivatization with 
 either l - fdla or d - fdla .
each portion was dissolved 
 in 110 l of acetone and 50 l of h2o , followed 
 by mixing with 20 l of 1 n nahco3 .
finally , 20 l 
 of l - fdla or d - fdla ( 10 mg / ml in acetone ) was added , 
 and the mixtures were heated to 40 c for 1 h. the reaction mixtures 
 were then cooled to room temperature , and 20 l of 1 n hcl was 
 added to quench the reaction .
the cooled reaction mixtures were evaporated 
 to dryness and redissolved in 300 l of ch3cn .
lc - ms 
 analysis was carried out using a reversed - phase column ( phenomenex 
 kinetex c18 , 250  4.6 mm , 5 m , 1.0 ml / min ) 
 with a linear gradient from 25% to 65% aqueous ch3cn containing 
 0.1% formic acid over 50 min .
the selective ion chromatograms of l - fdla and d - fdla derivatives of each amino acid were 
 compared for the assignment of amino acid configurations .
detailed 
 reports of retention times of each amino acid can be found in supporting information s14 and s23 .
cytotoxicity assays against 
 the mda - mb-435 and ht-29 cancer cell lines were conducted according 
 to established protocols .
 two new cyclic lipopeptides , trichormamides 
 a ( 1 ) 
 and b ( 2 ) , were isolated from the cultured freshwater 
 cyanobacterium trichormus sp . 
 uic 10339 . 
 the strain 
 was obtained from a sample collected in raven lake in northern wisconsin . 
 
 the planar structures of trichormamides a ( 1 ) and b ( 2 ) were determined using a combination of spectroscopic analyses 
 including hresims and 1d and 2d nmr experiments . 
 the absolute configurations 
 of the amino acid residues were assigned by the advanced marfey s 
 method after acid hydrolysis . 
 trichormamide a ( 1 ) is 
 a cyclic undecapeptide containing two d - amino acid residues 
 ( d - tyr and d - leu ) and one -amino acid residue 
 ( -aminodecanoic acid ) . 
 trichormamide b ( 2 ) is 
 a cyclic dodecapeptide characterized by the presence of four nonstandard 
 -amino acid residues ( homoserine , n - methylisoleucine , 
 and two 3-hydroxyleucines ) and one -amino acid residue ( -aminodecanoic 
 acid ) . 
 trichormamide b ( 2 ) was cytotoxic against mda - mb-435 
 and ht-29 cancer cell lines with ic50 values of 0.8 and 
 1.5 m , respectively . 
Results and Discussion
Experimental Section