orange tufts of a tropical marine symploca sp . 
 were collected in approximately 20 m of water near kimbe bay , papua 
 new guinea , in july 2007 .
the preserved collection was repetitively 
 extracted ( 2:1 ch2cl2/meoh ) and fractionated 
 using normal - phase vacuum liquid chromatography ( vlc ) .
previously , 
 a middle polarity fraction ( 60% hexanes/40% etoac ) of this extract 
 yielded several biologically active metabolites , including kimbeamides 
 a  c , kimbelactone a , and tasihalide c. additionally , a relatively polar fraction eluting with 25% meoh / etoac 
 exhibited cytotoxic activity against h-460 human lung cancer cells 
 ( 81% toxicity at 3 g / ml ) . further chromatography of this fraction 
 using normal - phase solid - phase extraction ( spe ) and reversed - phase 
 hplc yielded 1.9 mg of tasiamide c ( 1 ) , 2.5 mg of tasiamide 
 d ( 2 ) , and 0.7 mg of tasiamide e ( 3 ) .
hr - esitofms of 1 yielded an [ m + na ] at m / z 839.4541 , giving a molecular formula 
 of c41h64n6o11 , with 13 
 degrees of unsaturation .
the ir spectrum featured absorptions indicative 
 of the presence of nh or oh protons 
 and the presence of amide or ester carbonyls ( 3371 and 1737 cm , respectively ) .
further evidence of ester or amide 
 carbonyls was present as eight downfield signals in the c nmr spectrum ( c 167.8 , 169.2 , 169.9 , 170.8 , 172.1 , 
 172.5 , 173.7 , and 175.7 ) . additionally , the presence of a monosubstituted 
 phenyl group was evident from four downfield carbon signals , two of 
 which were composed of two carbons each as indicated by their relative 
 peak height ( c 126.4 , 128.0  2 , 129.2  
 2 , and 136.4 ) .
further analysis of the h nmr spectrum 
 revealed the presence of three singlet methyls at shifts indicative 
 of an o - methyl ( h 3.67 ) and two n - methyl groups ( h 2.93 and 3.03 ) , along 
 with two broad downfield signals suggestive of nh 
 protons ( h 6.69 and 6.78 ) . analysis of the 
 2d nmr data ( cosy , tocsy , roesy , hsqc , and hmbc ) 
 enabled the assignment of eight cosy spin systems consisting of five 
 amino and two hydroxy acid residues [ proline methyl ester ( pro - me 
 ester ) , n - mephe , ala , ile , n - megln , 
 and two 2-hydroxy-3-methylbutyric acids ( hmba ) ] , accounting for all 
 13 degrees of unsaturation and thereby signifying an overall linear 
 arrangement ( figure 1 ) .
hmbc correlations from 
 the nh and n - me groups to the carbonyl 
 of the neighboring residues allowed for the assignment of connections 
 between n - mephe and ala ( c-16 to c-17 ) , n - megln and hmba-1 ( c-31 to c-32 ) , ala and ile ( nh-1 
 to c-20 ) , and ile and n - megln ( n - h-2 to c-26 ) , leading to three partial structures , pro - me ester , n - mephe  ala  ile  n - megln  hmba-1 , and hmba-2 .
key roesy correlations revealed 
 further connections between these fragments , one between h-5a / b (  
 3.34/3.15 ) and h-8 (  5.52 ) connecting pro - me ester to n - mephe and the other between h-34 (  2.15 ) and h-38 
 (  4.09 ) , making the final connection between the two hmba residues . 

thus , tasiamide c was deduced to have an overall linear structure 
 consisting of pro - me ester 
this 
 planar constitution was supported by tandem mass spectroscopy analysis 
 ( figure 2 ) .
select low - resolution ms fragmentation cleavages for tasiamides 
 c  e ( 13 ) .
the absolute configurations of several of the amino 
 acids ( pro , n - mephe , ala , and n - megln ) 
 were determined 
 by marfey s analysis .
authentic d and l standards 
 for pro , n - mephe , and ala were each derivatized with d-(1-fluoro-2,4-dinitrophenyl-5-d - alanine amide ) ( d - fdaa ) . unfortunately , authentic d - n - meglu was unavailable ; therefore , chromatographic standards were 
 prepared by derivatizing l - n - meglu with 
 both d- and l - fdaa ( upon acidic hydrolysis , n - megln becomes n - meglu ) .
compound 1 was hydrolyzed and derivatized with d - fdaa and 
 analyzed by lc - ms in comparison with the retention times of authentic 
 standards . from this analysis it was clear that three of the four 
 amino acids ( pro , ala , and n - megln ) were of the l configuration , while the n - mephe residue 
 was of the d configuration ( supporting 
 information ) .
the absolute configuration of the ile residue 
 was determined by 
 chiral - phase gc - ms comparison of n - boc , o - me - derivatized authentic standards against the similarly derivatized 
 ile residue released by acid hydrolysis of 1 .
the four 
 protected standards of l - ile , l - allo - ile , d - ile , and d - allo - ile were 
 prepared by first synthesizing the n - boc - protected 
 amino acids , followed by methyl esterification of the carboxylic acid 
 using diazomethane . from retention time comparison and co -
injection 
 experiments it was clear that the ile residue was of the d - allo configuration ( supporting 
 information ) .
the absolute configuration of the final 
 two hmba stereocenters 
 in 1 presented a challenge .
comparison of authentic standards 
 of s- and r - hmba with the methylated 
 hydrolysate revealed that the natural product contained both s- and r - hmba residues .
a similar situation 
 has been previously reported in closely related metabolites of this 
 compound family .
following mild base 
 treatment ( 1:1 0.5 n naoh(aq)/meoh ) , only the terminal hmba residue 
 was released ; this residue was subsequently methyl esterified using 
 diazomethane and , by retention time comparison 
 and co - injections with authentic standards , was identified as the s configuration .
the above experiments 
 established that tasiamide c ( 1 ) possessed a 2s , 8r , 18s , 21r , 22s , 27s , 33r , and 38s absolute configuration .
hr - esitofms of 2 yielded an [ m + na ] at m / z 825.4371 for a molecular formula of 
 c40h62n6o11 .
the ir and h and c nmr spectra were similar to those of 1 ; however , the molecular formula indicated a reduction of 
 14 amu ( e.g. , a ch2 unit ) relative to tasiamide c. inspection 
 of the h nmr spectrum revealed the presence of only two 
 singlet methyl groups ( e.g. , one n - methyl at h 3.02 and one o - methyl at h 3.58 ) and three nh protons ( h 6.77 , 6.89 , and 6.98 ) . from the 2d nmr data ,
it was clear that the 
 only modification between 1 and 2 was the 
 loss of the n - methyl on the phe residue , thus yielding 
 a planar constitution of pro - me ester  phe  ala 
the absolute configurations 
 of the residues in compound 2 were determined in an identical 
 fashion to that described above 
 for compound 1 .
analysis of the retention times of the 
 marfey s derivatized hydrolysate and authentic standards by 
 lc - ms revealed that three of the four amino acids ( pro , ala , and n - megln ) were of the l configuration and that the 
 phe residue was of the d configuration .
chiral - phase gc - ms 
 analysis of the ile residue , n - boc and o - methylated as with 1 , showed by retention time comparison 
 and co - injections that this residue was of the l configuration . 

chiral - phase gc - ms analysis of the methylated hydrolysate of 2 exhibited peaks matching both s- and r - hmba .
analysis of the methylated mild base hydrolysate 
 confirmed that , as with 1 , the terminal hmba was of s configuration , and so the penultimate hmba was of r configuration , establishing the absolute configuration 
 of tasiamide d ( 2 ) as 2s , 8r , 17s , 20s , 21s , 26s , 32r , and 37s .
hr - esitofms of 3 yielded an [ m + na ] at m / z 852.4842 in agreement with 
 a molecular 
 formula of c42h67n7o10 , requiring 13 degrees of unsaturation .
the ir and h 
 and c nmr spectra again featured similarities to 1 and 2 ; however , the molecular weight was 13 
 and 27 amu greater than 1 and 2 , respectively , 
 and thus could not be readily attributed to a single modification . 

closer inspection of the h nmr spectrum revealed the presence 
 of three singlet methyls ( h 2.98 , 2.99 , and 3.72 ) , 
 similar to 1 , and three amide protons ( h 6.78 , 6.82 , and 7.01 ) , as seen in 2 , suggesting that 
 one of the hydroxy acids was replaced by an amino acid .
further evidence 
 of this was seen by comparison of the c nmr spectra of 1 and 3 ; in tasiamide c there were two -carbons 
 with shifts indicative of hydroxy acids at c 74.6 
 and 76.4 , whereas there was only one such peak in tasiamide e at c 71.1 .
further analysis of 1d and 2d nmr data ( h , c , cosy , tocsy , roesy , hsqc , hmbc , and h2bc ) confirmed the presence of seven residues , six 
 amino acids , and 
 one hydroxy acid [ pro - me ester , n - mephe , gly , ile , n - megln , leu , and 2-hydroxy-4-methylpentanoic acid ( h4mpa ) ] . in a similar fashion to 1 , each 
 of the connections between residues in 3 were revealed 
 by key hmbc and roesy correlations .
hmbc correlations from both n - methyl groups to the carbonyls on the neighboring residues 
 allowed for the assignment of connections between n - mephe to gly ( c-16 to c-17 ) and n - megln to leu ( c-30 
 to c-31 ) .
correspondingly , hmbc correlations from amide protons to 
 adjacent carbonyls allowed for the assignment of connections between 
 ile and n - megln ( nh-2 to c-25 ) and 
 between leu and h4mpa ( nh-3 to c-37 ) , leading to 
 three partial structures , pro - me ester , n - mephe 
two 
 roesy correlations provided the final connections between these fragments , 
 one between h-5a / b (  3.36/3.31 ) and h-8 (  5.55 ) , connecting 
 pro - me ester and n - mephe , and the other between h-18a / b 
 (  4.05/3.81 ) and h-21 (  1.82 ) , making the final connection 
 between gly and ile .
thus , tasiamide e was deduced to have a planar 
 linear structure consisting of pro - me ester 
h4mpa , and this was supported by tandem mass spectrometry 
 analysis ( figure 2 ) .
the absolute configurations 
 of the residues in 3 were 
 determined as described above for compounds 1 and 2 .
analysis of the marfey s derivatized hydrolysate 
 with the retention times of authentic standards on lc - ms revealed 
 that three of the four amino acids ( pro , leu , and n - megln ) were of the l configuration , while the n - mephe residue was of the d configuration .
as for the chiral 
 gc - ms analysis of the ile residue , retention time comparison and co - injections 
 confirmed that it was also of the l configuration .
the absolute 
 configuration of the h4mpa was determined using chiral - phase gc - ms , 
 both comparing retention time and co - injections with authentic standards , 
 thus confirming its s configuration and establishing 
 the absolute configuration of tasiamide e ( 3 ) as 2s , 8r , 20s , 21s , 26s , 32s , and 38s .
tasiamides c  e ( 13 ) are 
 of close structural relation to several families of known metabolites , 
 the grassystatins , tasiamides , and symplocin a. in this regard , 13 are structurally most similar to tasiamide 
 ( 4 ) , with 3 varying only in the terminal 
 residue , while compounds 1 and 2 possess 
 other relatively simple modifications . in 2008 ,
li and co - workers 
 revised the original configuration of the n - megln 
 residue in 4 from l to d based on the 
 comparison of analytical data ( c nmr and specific rotation ) 
 of the natural product with four stereoisomers obtained from a total 
 synthesis ( containing both l and d stereoisomers 
 of n - megln and leu ; table 2 ) .
however , the original 
 assignments for all of the residues in 4 are consistent 
 with that of 3 , and a comparison of c shifts 
 revealed no significant differences between 3 and 4 , along with the four additional synthetic stereoisomers 
 ( a  d , table 2 ) ( supporting information ) .
furthermore , the opposite specific rotation 
 signs for tasiamides c and d suggest a structural variance between 
 these two metabolites ( 3 , [ ]d 22.2 ; 4 , [ ]d + 15.0 ) . therefore ,
a more detailed analysis of the c nmr data for tasiamide and the four synthetic analogues was conducted 
 using dp4 probability calculations and revealed that the carbon data 
 alone are insufficient to deduce the correct configuration in tasiamide 
 ( 4 ) .
the probability of 
 the original experimental data matching each analogue was calculated 
 as 1.3% for analogue a ( 4 ) , 56.0% for analogue b , 3.0% for analogue c , and 39.7% for analogue d. because analogues b and d have 
 the same sign and magnitude of specific optical rotation and are indistinguishable 
 by dp4 calculations , it is not possible to unequivocally assign the 
 configuration of 4 based solely on these data .
this also 
 suggests that the misassignment in tasiamide ( 4 ) may 
 involve residues other than just the n - megln , and 
 thus , a broader investigation into the configuration of 4 will be necessary to clarify its correct absolute configuration .
stereochemical 
 assignments for tasiamide 
 and tasiamide b intentionally omitted due to unresolved stereochemical 
 questions ( see discussion ) .
matches original configuration proposed 
 by williams et al . proposed revised structure of tasiamide 
 according to ma et al .
it is interesting to note that 
 there appears to be considerable 
 biosynthetic flexibility in this family of metabolites .
insights are 
 thus provided by comparing the amino or hydroxy acid residues in each 
 member of this family ( table 2 ) .
the first 
 three residues ( a , b , and c ) starting from the carboxy terminus of 
 these molecules are rather well conserved , with each analogue containing 
 a pro - me ester followed by the incorporation of d - phe , which 
 is n - methylated in all but one analogue , and then 
 the incorporation of an ala , aba ( 2-aminobutyric acid ) , or gly as 
 the third residue . residues
e , h , and j are also conserved residues 
 ( statine , hmba , and an n , n - dimethylated amino acid , 
 respectively ) , but they occur only in a subset of the natural products . 

residues f and i are slightly less conserved , with three different 
 polar amino acids ( n - megln , asn , and ser ) incorporated 
 into residue f and four different hydroxy acids ( hmpa , h4mpa , hpa , 
 and h3mpa ) in residue i. also observed 
 are variations in the absolute configurations of these residues . for 
 example
, residues d and j in eight of nine metabolites incorporate 
 an l residue , while a single metabolite has a d - allo residue at this position . at residue
f all of the amino 
 acids were originally deduced as l ; however , in tasiamide 
 and tasiamide b these residues were reassigned as d based 
 on total synthesis . although non - ribosomal peptide synthetase ( nrps)-derived 
 peptides can show variations in the incorporated amino acid , it is 
 difficult to imagine a single biosynthetic pathway that would be capable 
 of producing all of these metabolites , especially those of varying 
 absolute configurations , which usually requires an epimerase .
both the grassystatins and symplocin a 
 were reported to possess 
 exceptional inhibitory activity toward cathepsin e ( grassystatin a : 
 ic50 = 886 pm , symplocin a : ic50 = 300 pm ) ; however , this activity is likely due to the presence of a statine 
 residue , which is absent in the tasiamides .
additionally , tasiamide 
 and tasiamide b both showed moderate cytotoxicity against kb cells , 
 with ic50 values of 0.48 and 0.8 m , respectively . due to limited isolated quantities of tasiamide e ( 3 ) ,
only tasiamides c ( 1 ) and d ( 2 ) were 
 evaluated for their cytotoxicity against the hct-116 colon cancer 
 cell line and were found to be inactive ( tasiamide c , ic50 > 25 m ; tasiamide d , ic50  25 m ) .
the new metabolites tasiamides c  e ( 13 ) were isolated from a kimbe bay , papua new guinea , collection 
 of the tropical marine cyanobacterium symploca sp . 

these metabolites add to the growing family of structurally homologous 
 natural products [ tasiamide ( 4 ) , grassystatins ( 5 , 6 ) , and symplocin a ] that have all been isolated 
 from similar tuft - forming cyanobacteria .
this particular symploca sp . is a prolific producer of secondary metabolites , as several 
 structurally diverse natural products were isolated from this collection 
 including kimbeamides a  c , kimbelactone , tasihalide c , and 
 the three tasiamides reported herein .
optical rotations were 
 measured on a jasco p-2000 polarimeter , uv spectra on a beckman coulter 
 du-800 spectrophotometer , and ir spectra on a nicolet ir-100 ft - ir 
 spectrophotometer using kbr plates .
nmr spectra were recorded with 
 chloroform as an internal standard ( c 77.0 , h 7.26 for chcl3 ) on a varian unity 500 mhz spectrometer 
 ( 500 and 125 mhz for h and c nmr , respectively ) , 
 a varian unity 300 mhz spectrometer ( 300 and 75 mhz for h and c nmr , respectively ) , a varian vnmrs ( varian nmr 
 system ) 500 mhz spectrometer equipped with a cold probe ( 500 and 125 
 mhz for h and c nmr ) , and a bruker 600 mhz 
 spectrometer equipped with a 1.7 mm microcyroprobe ( 600 and 150 mhz 
 for h and c nmr ) .
lr- and hr - esims data were 
 obtained on thermofinnigan lcq advantage max and thermo scientific 
 ltq orbitrap - xl mass spectrometers , respectively .
tandem mass spectroscopy 
 experiments were run with a biversa nanomate ( advion biosystems ) electrospray 
 source for a finnigan ltq - fticr - ms instrument ( thermo - electron corporation ) 
 running tune plus software version 1.0 .
hplc was carried out using 
 a waters 515 pump system with a waters 996 pda detector .
acid hydrolysis 
 was performed using a biotage ( initiator ) microwave reactor equipped 
 with high - pressure vessels .
the tasiamides 
 c  e - producing 
 cyanobacterium ( collection code : png 07/14/07 - 6 ) was collected by 
 hand on reefs 20 m deep from approximately eight nearby sites located 
 in kimbe bay off the north coast of new britain , papua new guinea 
 ( s 526.292 , e 15040.813 ) .
field notes 
 identified the puffballs as a consortium of schizothrix sp . with a minor amount of lyngbya sp .
the collected samples were stored in 1:1 etoh / seawater in the field , 
 and the supernatant was decanted and discarded before shipment . as 
 was previously reported , a portion of the cyanobacterial biomass was 
 used for 16s rrna sequencing , which revealed it claded 
 with  tropical marine symploca 
jq388599 ) . the cyanobacterial biomass 
 ( 101.7 g dry wt ) was extracted with 2:1 ch2cl2/meoh to afford 1.8 g of dried extract .
a portion of the extract 
 was fractionated by silica gel vlc using a stepwise gradient solvent 
 system of increasing polarity starting from 100% hexanes to 100% meoh 
 ( nine fractions , a 
the fraction eluting with 25% meoh/75% 
 etoac ( fraction h ) was separated further using rp hplc [ 4  
 synergi hydro , isocratic 65% mecn/35% h2o ] to yield pure 
 tasiamide c ( 1 , 1.9 mg ) , tasiamide d ( 2 , 
 2.5 mg ) , and tasiamide e ( 3 , 0.7 mg ) . white , amorphous solid ; 
 [ ]d 37 ( c 1.2 , 
 chcl3 ) ; uv ( meoh ) max ( log  ) 211.0 
 ( 3.84 ) ; ir ( neat ) max 3371 , 2965 , 2933 , 2877 , 1737 , 
 1644 , 1521 , 1453 , 1263 , 1201 , 1178 , 1098 , 1031 cm ; h nmr ( 600 mhz , cdcl3 ) and c nmr ( 125 mhz , cdcl3 ) , see table 1 ; hresims m / z 839.4541 [ m + na ] ( calcd for c41h64n6o11na , 839.4525 ) .
white , 
 amorphous solid ; 
 [ ]d 85 ( c 1.6 , 
 chcl3 ) ; uv ( meoh ) max ( log  ) 220.0 
 ( 3.86 ) nm ; ir ( neat ) max 3318 , 
 2965 , 2931 , 1739 , 1648 , 1526 , 1453 , 1203 , 1033 cm ; h nmr ( 500 mhz , cdcl3 )  7.17 ( 2h , 
 m ) , 7.13 ( 1h , m ) , 7.09 ( 2h , m ) , 6.98 ( 1h , d , j = 
 6.8 ) , 6.89 ( 1h , d , j = 7.7 ) , 6.77 ( 1h , d , j = 6.7 ) , 5.98 ( 1h , bs ) , 5.62 ( 1h , bs ) , 5.08 ( 1h , dd , j = 7.6 , 6.7 ) , 4.85 ( 1h , d , j = 7.5 ) , 4.76 
 ( 1h , q , j = 6.6 ) , 4.43 ( 1h , dq , j = 8.0 , 6.8 ) , 4.13 ( 1h , m ) , 4.12 ( 1h , m ) , 4.11 ( 1h , m ) , 3.58 ( 3h , 
 s ) , 3.47 ( 1h , dd , j = 8.7 , 5.1 ) , 3.02 ( 3h , s ) , 2.94 
 ( 2h , m ) , 2.68 ( 1h , m ) , 2.48 ( 1h , m ) , 2.32 ( 1h , m ) , 2.25 ( 1h , m ) , 2.14 
 ( 1h , m ) , 2.07 ( 1h , m ) , 1.97 ( 1h , m ) , 1.81 ( 1h , m ) , 1.78 ( 2h , m ) , 1.35 
 ( 2h , m ) , 1.26 ( 3h , d , j = 7.2 ) , 1.07 ( 2h , m ) , 0.99 
 ( 3h , d , j = 6.1 ) , 0.97 ( 3h , d , j = 6.2 ) , 0.93 ( 3h , d , j = 7.0 ) , 0.82 ( 3h , t , j = 6.8 ) , 0.82 ( 6h , d , j = 6.3 ) ; c nmr ( 75 mhz , cdcl3 )  175.8 , 174.6 , 172.2 , 171.8 , 
 170.7 , 170.4 , 136.2 , 129.1 , 128.5 , 127.0 , 76.7 , 75.1 , 59.4 , 59.1 , 
 57.1 , 52.5 , 52.2 , 48.8 , 46.9 , 39.0 , 35.9 , 33.0 , 32.1 , 31.1 , 29.9 , 
 29.0 , 24.7 , 24.6 , 24.0 , 18.7 , 18.4 , 18.1 , 16.1 , 15.7 , and 11.5 ; hresims m / z 825.4371 [ m + na ] ( calcd 
 for c40h62n6o11na , 825.4369 ) .
white , amorphous solid ; 
 [ ]d 22 ( c 0.5 , 
 chcl3 ) ; uv ( meoh ) max ( log  ) 207.0 
 ( 4.10 ) nm ; ir ( neat ) max 3329 , 
 2926 , 2958 , 1743 , 1645 , 1521 , 1454 , 1281 , 1177 cm ; h nmr ( 600 mhz , cdcl3 )  7.26 ( 2h , 
 m ) , 7.22 ( 2h , m ) , 7.20 ( 1h , m ) , 7.01 ( 1h , d , j = 
 9.3 ) , 6.82 ( 1h , d , j = 9.4 ) , 6.78 ( 1h , d , j = 4.4 ) , 5.61 ( 1h , bs ) , 5.55 ( 1h , t , j = 7.4 ) , 5.25 ( 1h , bs ) , 5.06 ( 1h , t , j = 7.4 ) , 4.95 
 ( 1h , q , j = 7.9 ) , 4.39 ( 1h , dd , j = 8.1 , 5.2 ) , 4.29 ( 1h , dd , j = 8.8 , 6.3 ) , 4.09 
 ( 1h , dd , j = 9.1 , 4.5 ) , 4.05 ( 1h , dd , j = 17.7 , 3.3 ) , 3.81 ( 1h , dd , j = 17.7 , 3.3 ) , 3.72 
 ( 3h , s ) , 3.36 ( 1h , m ) , 3.31 ( 1h , m ) , 3.27 ( 1h , dd , j = 13.6 , 8.2 ) , 2.99 ( 3h , s ) , 2.98 ( 3h , s ) , 2.82 ( 1h , dd , j = 13.6 , 6.9 ) , 2.31 ( 1h , m ) , 2.24 ( 1h , m ) , 2.18 ( 1h , m ) , 
 2.12 ( 1h , m ) , 1.99 ( 1h , m ) , 1.93 ( 1h , m ) , 1.87 ( 2h , m ) , 1.86 ( 1h , 
 m ) , 1.82 ( 1h , m ) , 1.80 ( 1h , m ) , 1.60 ( 2h , m ) , 1.54 ( 2h , m ) , 1.43 ( 1h , 
 m ) , 1.11 ( 1h , m ) , 0.96 ( 6h , d , j = 6.6 ) , 0.95 ( 3h , 
 d , j = 6.4 ) , 0.92 ( 3h , d , j = 6.4 ) , 
 0.88 ( 3h , d , j = 7.2 ) , 0.87 ( 3h , t , j = 7.7 ) ; c nmr ( 125 mhz , cdcl3 )  175.2 , 
 174.4 , 174.0 , 172.5 , 171.5 , 169.7 , 167.8 , 136.8 , 129.4 , 128.4 , 126.8 , 
 71.1 , 58.9 , 57.6 , 56.9 , 56.2 , 52.3 , 46.8 , 43.1 , 41.2 , 37.2 , 35.1 , 
 31.9 , 31.0 , 29.7 , 28.8 , 25.0 , 24.8 , 24.7 , 24.6 , 23.4 , 23.0 , 22.7 , 
 22.3 , 21.4 , 15.6 , 11.3 ; hresims m / z 852.4842 [ m + na ] ( calcd for c42h67n7o10na , 852.4842 ) .
tasiamides c  e 
 ( 13 , 0.2 mg ) were treated separately 
 with 400 l of 6 n hcl in a microwave reactor at 160 c 
 for 5 min .
an aliquot of the reaction product was dissolved in 500 
 l of a 1 mg / ml solution of d - fdaa ( 1-fluoro-2,4-dinitrophenyl-5-d - alanine amide ) in acetone followed by the addition of 20 l 
 of 1 n nahco3 .
the solution was maintained at 40 c 
 for 1 h , at which time the reaction was quenched by the addition of 
 40 l of 1 n hcl .
the reaction mixture was then dried down under 
 n2(g ) and resuspended in 200 l of 50% h2o/50% mecn , and 10 l of the solution was analyzed by lc - esims .
the marfey s derivatives of the hydrolysate and standards 
 reacted with d - fdaa ( ala , phe , n - mephe , 
 pro , and leu ) were analyzed by rp hplc using a phenomenex luna 5 m 
 c18 column ( 4.6  250 mm ) .
the hplc conditions began 
 with 10% mecn/90% h2o acidified with 0.1% formic acid ( fa ) 
 ( aqueous ) followed by a gradient profile to 50% mecn/50% h2o acidified with 0.1% fa(aq ) over 85 min at a flow of 0.4 ml / min , 
 monitoring from 200 to 600 nm .
because authentic d - nmeglu was not available , l - nmeglu 
 standard was derivatized with both the l - fdaa and d - fdaa .
the nmeglu residue was analyzed by rp hplc 
 using a kinetex 5  c18 100a column ( 4.6  100  mm ) . 

the hplc condition began with 5% meoh/95% h2o acidified 
 with 0.1% fa(aq ) followed by a gradient profile to 45% meoh/55% h2o acidified with 0.1% fa(aq ) over 125 min at a flow of 0.4 
 ml / min , monitoring from 200 to 600 nm .
the retention times of the 
 derivatives of the authentic amino acids when analyzing for 1 were d - ala ( 64.8 min ) , l - ala ( 71.1 min ) , d - pro ( 66.6 min ) , l - pro ( 69.4 min ) , d - nmephe ( 78.4 min ) , l - nmephe ( 79.7 
 min ) , l - nmeglu reacted with l - fdaa 
 ( 93.3 min ) , and l - nmeglu reacted with d - fdaa ( 91.9 min ) ; the derivatives of the hydrolysate product 
 of 1 gave peaks with retention times of 71.5 , 69.8 , 78.3 , 
 and 91.8 min , according to l - ala , l - pro , d - nmephe , and l - nmeglu , 
 respectively .
the retention times of the derivatives of the authentic 
 amino acids when analyzing for 2 were d - ala 
 ( 59.3 min ) , l - ala ( 63.6 min ) , d - pro ( 60.4 min ) , l - pro ( 62.6 min ) , d - phe ( 78.7 min ) , l - phe 
 ( 83.8 min ) , l - nmeglu reacted with l - fdaa ( 93.3 min ) , and l - nmeglu reacted 
 with d - fdaa ( 91.9 min ) ; the derivatives of the hydrolysate 
 product of 2 gave peaks with retention times of 64.0 , 
 62.6 , 83.8 , and 91.7 min , according to l - ala , l - pro , d - phe , and l - glu .
the retention times of the derivatives 
 of the authentic amino acids when analyzing for 3 were d - leu ( 68.4 min ) , l - leu ( 85.8 min ) , d - pro 
 ( 60.4 min ) , l - pro ( 62.6 min ) , d - nmephe ( 78.6 min ) , l - nmephe ( 79.2 min ) , l - nmeglu reacted with l - fdaa ( 107.6 
 min ) , and l - nmeglu reacted with d - fdaa ( 103.9 min ) ; the derivatives of the hydrolysate product of 3 gave peaks with retention times of 85.7 , 62.2 , 78.2 , and 
 104.5 min , according to l - leu , l - pro , d - nmephe , and l - nmeglu .
an aliquot 
 ( 0.1 mg ) of the above hydrolysate product was dissolved in 
 100 l of h2o and then treated with 0.26 mg ( 0.43 
 mol ) of nahco3 followed by 0.94 mg ( 0.43 mol ) 
 of di - tert - butyl dicarbonate .
after 16 h at room 
 temperature ( rt ) , the reaction mixture was quenched by 500 l 
 of 5% khso4 and back extracted with 3  1 ml of etoac , 
 and the combined organic layers were dried over mgso4 . 

the derivatized hydrolysate product and standards 
 were analyzed by chiral - phase gc - ms using a chirasil - val ( agilent 
 technologies j&w scientific , 30 m  0.25 mm ) under the following 
 conditions : the initial oven temperature was 40 c , kept for 
 2 min , followed by a ramp from 40 to 75 c at a rate of 10 c / min , 
 kept for 5 min , followed by another ramp to 110 c , at a rate 
 of 0.5 c / min , followed by a final ramp to 200 c , at a 
 rate of 25 c / min , kept for 2 min . the retention times for the 
 four authentic standards when analyzing 1 were d - allo - ile ( 49.6 min ) , l - allo - ile ( 49.9 ) , d - ile ( 51.5 min ) , and l - ile ( 51.7 
 min ) ; the derivatized hydrolysis product of 1 yielded 
 a peak at 49.6 min , according to d - allo - ile . 

the retention times for the four authentic standards when analyzing 2 and 3 were d - allo - ile ( 47.7 min ) , l - allo - ile ( 48.2 ) , d - ile ( 50.1 min ) , and l - ile ( 50.5 min ) ; the derivatized 
 hydrolysis products of 2 and 3 each yielded 
 a peak at 50.6 min , according to l - ile .
tasiamides 
 c ( 1 ) and d ( 2 ) ( 0.15 mg ) were treated separately 
 with 150 l of 1:1 0.5 n naoh(aq)/meoh ( 1:1 ) solution at room 
 temperature for 72 h. the reaction mixture 
 was neutralized by the addition of 40 l of 1 n hcl(aq ) and 
 back extracted with 3  1 ml of etoac , and the combined organic 
 layers were dried over mgso4 .
the product was then treated 
 with freshly prepared diazomethane in diethyl ether for 5 min at rt . 

the derivatized acid hydrolysate was analyzed by chiral - phase gc - ms 
 using a cyclosil b column ( agilent technologies j&w scientific , 
 30 m  0.25 mm ) under the following conditions : the initial oven 
 temp was 35 c , kept for 15 min , followed by a ramp to 60 c , 
 at a rate of 1.5 c , followed by a ramp to 170 c , at a 
 rate of 5 c / min , kept for 5 min .
the retention times for the 
 authentic standards were r - hmba ( 34.9 min ) and s - hmba ( 35.8 min ) ; the derivatized hydrolysis product for 1 and 2 each exhibited a single peak at 35.8 
 and 35.9 min , respectively , according to s - hmba .
the derivatized hydrolysate product used in the ile analysis from 3 was also used in the h4mpa analysis .
the derivatized hydrolysis product and authentic 
 standards were analyzed by chiral - phase gc - ms using a cyclosil b column 
 under the following conditions : the initial oven temp was 40 c , 
 kept for 15 min , followed by a ramp to 90 c , at a rate of 1.5 
 c / min , followed by a ramp to 200 c , at a rate of 10 c / min , 
 kept for 5 min .
the retention times for the authentic standards were r - h4mpa ( 43.2 min ) and s - h4mpa ( 43.8 min ) ; 
 the derivatized hydrolysis product yielded a peak at 43.7 min , consistent 
 with s - h4mpa .
 a collection of the tropical marine 
 cyanobacterium symploca sp . , collected near kimbe 
 bay , papua new guinea , previously yielded 
 several new metabolites including kimbeamides a  c , kimbelactone 
 a , and tasihalide c. investigations into a more polar cytotoxic fraction 
 yielded three new lipopeptides , tasiamides c  e ( 13 ) . 
 the planar structures were deduced by 2d 
 nmr spectroscopy and tandem mass spectrometry , and their absolute 
 configurations were determined by a combination of marfey s 
 and chiral - phase gc - ms analysis . 
 these new metabolites are similar 
 to several previously isolated compounds , including tasiamide ( 4 ) , grassystatins ( 5 , 6 ) , and symplocin 
 a , all of which were isolated from similar filamentous marine cyanobacteria . 
Results and Discussion
Conclusion
Experimental Section