the secondary metabolite profile 
 of s. caribica has been investigated previously by 
 hplc . in our screening for antifungal 
 and cancer cell - inhibitory marine 
 secondary metabolites , we examined several samples ( n = 10 ) of s. caribica collected from the bahamas 
 in 2007 and 2008 . most showed in vitro antifungal activity against 
 fluconazole - resistant candida albicans , can . gatti . a single sample of s. caribica ( 08 - 045q ) 
 collected from sweetings cay , with a reversed - phase lcms profile different 
 from other samples , lacked antifungal activity , but showed significant 
 cytotoxic activity toward cultured human colon tumor cells ( hct-116 ) . while mainly 3 and smaller amounts of 2a were evident in most samples of s. caribica , these two compounds were not detected in 08 - 045q 
 ( lcms analysis ) . instead , the latter sample contained hymenidin ( 2b ) as the major pai along with late - eluting components that 
 lacked br and with m / z values in 
 the range expected for stylissamides . sequential solvent partitioning 
 of a meoh extract of the sample separated the constituents by polarity , 
 to give an n - buoh - soluble fraction that was further 
 purified by preparative hplc to provide a cytotoxic fraction ( ic50 = 5.5 gml ) . rechromatography 
 of the latter gave pure samples of two new heptapeptides , stylissamides 
 g ( 1 g ) and h ( 1h ) . the molecular formula 
 of 1 g , c45h61n7o7 , was established from hresims data . the 
 high n content of 1 g and preliminary analysis of h nmr data ( table 1 ) suggested a heptapeptide ; 
 this was further supported by the presence of cross - peaks in the hsqc 
 spectrum consistent with seven -ch signals (  51.661.6 
 ppm ) and seven amide carbonyl signals (  170.6171.7 
 ppm ) . analysis of cosy and hmbc spectra of 1 g provided 
 assignments of spin systems due to one each of leu and ile , two phe , 
 and three pro residues ( table 1 ) . all three 
 pro residues could be assigned cis conformations 
 based on empirical rules that correlate 
 with c nmr chemical shifts (  c < 23.3 ppm ) and differences between the  and  carbons 
 [ (cc ) > 
 8.0 ppm ] . see structures 
 of 1 g and 1h for the amino acid residue 
 key . c nmr shifts from indirect 2d nmr ( hsqc , hmbc , and bscthmbc ) . sequence 
 assignments of 1 g were obtained by analysis 
 of hmbc and noesy data . a band - selective constant - time hmbc experiment ( c 161181 
 ppm , optimized for j = 8 hz ) showed correlations 
 from  4.37 ( pheh ) to  
 169.9 ( proc = o ) ,  4.34 ( proh ) to  171.7 ( ilec = o ) ,  7.88 ( ilenh ) and  4.26 ( ileh ) to  170.6 ( leuc = o ) ,  8.83 ( leuh ) to  169.9 ( proc = o ) ,  3.27 ( proh ) to  168.8 
 ( phec = o ) , and  8.94 ( phenh ) to  171.4 ( proc = o ) . the foregoing data were sufficient to define the sequence pro - phe - pro - phe - pro - leu - ile . cross - peaks in the noesy spectrum of 1 g were observed 
 between the following pairs of amino acid residue nh and -ch 
 nmr signals : phenh / proh , proh/pheh , proh/ileh , ilenh / leuh , leunh / proh , and proh/pheh ( figure 1 ) . selected noesy 
 and hmbc correlations for stylissamide g ( 1 g ) . stylissamide g ( 1 g ) is a variant on the heptapeptide 
 motif detected in cyclic peptides , from not only stylissa ( family dictyonellidae ) but other sponges in the family axinellidae . cytotoxic phakellistatins constitute a family of over a dozen peptides 
 characterized from different samples of phakellia spp . from the indian ocean , western 
 pacific , and the south china sea mostly with the typical heptapeptide motif of 
 stylissamides . in terms of sequence and amino acid composition , peptide 1 g most closely resembles phakellastatin-2 ( 4 ) and stylisins-1 ( 5a ) 
 and -2 ( 5b ) from s. caribica collected 
 in jamaica . the h nmr 
 spectrum of cyclopeptide 1h exhibited conformational 
 heterogeneity in several solvents ( cdcl3 , dmso - d6 , and cd3od ) that significantly 
 complicated interpretation and assignment . similar solvent - dependent 
 complexity has been observed in the nmr spectra of 4 . a relatively well - resolved h nmr 
 spectrum of 1h was obtained in cd2cl2 , and analysis of the spin systems by proton , cosy , hmqc , and hmbc 
 nmr ( table 2 ) showed the presence of trp , ser , 
 ile , val , phe , and two pro residues . pro was assigned 
 the cis conformation , while pro was assigned 
 as trans according to empirical rules . attempts to 
 sequence 1h by hmbc and noesy failed due to relatively 
 weak carbonyl signals dispersed between multiple conformers . h2o , 60 c , 90 min ) gave 
 a linear peptide from single amide bond hydrolysis that was identified 
 as pro - trp - pro - ile - ser - phe - val by lc / ms / ms 
 ( figure 2 ) . the composition and amino acid 
 sequence of 1h are identical to those of euryjanicin 
 a ( 6 ) , which was isolated 
 by rodriguez and co - workers from the caribbean sponge prosuberites 
 laughlini ( daz , alvarez & van soest , 1987 ) . however , 
 peptides 1h and 6 are configurational isomers : 
 pro is in the trans configuration in 1h , while both proline residues in 6 adopt the cis configuration . no interconversion of 1h into 6 was observed upon heating the former in dmso 
 ( 70 c , 30 min ) , suggesting that 1h is the thermodynamically 
 favored isomer . lc / ms / ms analysis of the partial hydrolysis product of 
 stylissamide 
 h ( 1h ) . c nmr chemical shift assignments from 
 indirect 2d nmr ( hsqc , hmbc , 
 and bscthmbc . the amino acid residues in all stylissa peptides 
 reported to date are of the l - configuration . pure samples 
 of 1 g and 1h were separately hydrolyzed 
 ( 6 m hcl , 95 c , 15 h ) , and , after removal of volatiles , the 
 residues were subjected to marfey s analysis . the configurations of all amino acid residues in both peptides 
 ( except trp in 1h , which decomposed during hydrolysis ) 
 were also found to be l. natural product cyclic heptapeptides and octapeptides are presumed 
 to adopt rigid , conformationally constrained structures ordered by 
 minimized torsional strain , especially those imposed by cis and trans pro residues . aside from computational 
 modeling studies that determined the minimized energy structures , no experimental data have been reported on the 
 conformational dynamics of the stylissamides . we briefly examined 
 the solution conformational dynamics of 1 g and 1h by temperature - dependent cd spectra ( figure 4 ) and amide signal ( nh ) h nmr chemical shifts ( table 3 ) . cd and uv spectra 
 ( ch3cn , 23 c ) : ( a ) stylissamide 
 g ( 1 g ) and ( b ) stylissamide h ( 1h ) . temperature - dependent cd spectra ( ch3cn ) : ( a ) stylissamide 
 g ( 1 g ) and ( b ) stylissamide h ( 1h ) . at room temperature 
 ( 23 c ) , the cd spectrum of 1 g ( ch3cn , 
 figure 3 ) was characterized 
 by two cotton effects ( ces ) (  196 (  + 23.2 ) , 223 
 (  7.2 ) nm ) , while that of 1h showed 
 more complex band structure due to the presence of trp . at elevated 
 temperatures ( 50 and 70 c , figure 4 ) , 
 little difference was observed in the cd spectra of 1 g and 1h , although at lower temperature ( 0 c ) moderate 
 increases in the magnitudes of both ces of 1 g were observed . in their study of temperature - dependent h nmr amide 
 nh signals and exchange rates in proteins , baxter and williamson concluded 
 that temperature coefficients ( /t ) greater than 4.5 ppb / k were correlated with hydrogen - bonded 
 amide protons , while nh protons with values less than 4.5 
 ppb / k were not hydrogen - bonded . measurement 
 of temperature - dependent h nmr amide nh chemical shifts 
 of 1 g ( t = 25 , 50 , 70 c ) gives 
 /t coefficients ( table 3 ) that are greater than 4.5 ppb / k for phe and leu , but lower than 4.5 ppb / k for phe and ile , showing that nh of phe and leu are strongly 
 hydrogen bonded . these temperature coefficients support a generally 
 tightly constrained amide backbone for 1 g with weaker 
 hydrogen bonding at the phe and ile residues . taking both 
 cd and /t measurements into 
 consideration , the data support conformations of 1 g and 1h with relatively rigid amide backbones , constrained by intramolecular 
 hydrogen bonds , but higher mobility of the ile and phe residues . pettit and others have reported potent cytotoxicity of cyclic heptapeptides 
 related to 1 g and 1h , for example , phakellistatin-2 , related phakellistatins , and hymenistatins g , h , and j. kobayashi and co - workers showed that stylissamide inhibited egf - induced migration of hela cells in the concentration 
 range 0.1 to 10 m . in the present 
 work , the fraction of s. caribica extract containing 
 stylissamides 1 g and 1h was cytotoxic to 
 hct-116 cells . peptide 1h exhibited modest in vitro cytotoxicity 
 against hct-116 ( ec50 5.7 m ) while 1 g was essentially inactive ( ec50 > 200 m ) . in conclusion , two new heptapeptides , stylissamides g ( 1 g ) and h ( 1h ) , were isolated from a cytotoxic fraction 
 of the sponge s. caribica from the bahamas and characterized 
 by integrated spectroscopic methods . studies of the solution dynamics 
 of 1 g and 1h by temperature - dependent cd 
 and amide nh h nmr temperature coefficients suggested 
 peptide conformations ordered by a relatively rigid , hydrogen - bonded 
 backbone structure with limited mobility . uv  vis spectra 
 were recorded on a jasco uv v630 dual beam instrument in 1 cm quartz 
 cells . cd spectra were recorded on a jasco 810 spectropolarimeter 
 in quartz cells ( 1 or 2 mm path length ) . h nmr and 2d 
 nmr spectra of submilligram samples were measured on a bruker 600 
 nmr spectrometer under control of an avance iii console and equipped 
 with a 1.7 mm { c / n}h microcryoprobe , 
 operating at 599.5556 mhz . additional h and c nmr spectra were measured on a jeol eca 500 spectrometer operating 
 at 500.1599 and 125.6940 mhz , respectively , with samples dissolved 
 in cd3od or dmso - d6 ( 99.8 atom 
 % d , cambridge isotopes ) . low - resolution ms spectra were recorded 
 on a thermofisher msq plus surveyor single quadrupole mass spectrometer , 
 operating in positive ion esi mode , coupled to a thermo fisher accela 
 ultra - high - performance liquid chromatograph ( uhplc ) . hrms measurements 
 were made using an agilent 6230 tofms under positive ion esi tofms 
 conditions and provided by the ucsd small molecule ms facility and 
 the torrey pines scripps research institute , la jolla , california . semipreparative hplc was carried out using an agilent 1100 hplc under 
 specified columns and gradient conditions . a sample 
 of stylissa caribica ( accession 08 - 08 - 045 ) was collected 
 at sweetings cay , bahamas ( 49.646 s , 7754.054 
 w ) using scuba from a depth of 27 m in june 2008 and kept 
 frozen ( 20 c ) until required . the frozen sponge ( wet 
 wt 107.1 g ) was cut into 3 cm cubes and extracted with 1:1 
 ch2cl2meoh with stirring ( rt , overnight , 
 2  500 ml ) . following removal of the solvent under reduced pressure , 
 the residue was partitioned between hexanes ( 3  250 ml ) and 
 9:1 meoh the aqueous meoh layer was 
 concentrated and further partitioned between n - buoh 
 ( 3  250 ml ) and h2o ( 250 ml ) . removal of the volatile 
 solvent from the upper layer gave a brown semisolid ( 1.68 g ) , a portion 
 of which was adsorbed onto a solid - phase extraction cartridge ( c18 ) and eluted with meoh . the meoh eluate was concentrated 
 under reduced pressure to give a solid ( 870 mg ) , which was separated 
 by semipreparative reversed - phase hplc ( c18 , gradient from 
 10:90 to 60:40 ch3cn  h2o + 0.1% tfa ) 
 to give 10 fractions . a portion ( 8.6 mg ) of the eighth fraction ( 36.8 
 mg ) was further purified by semipreparative hplc ( c18 , 
 gradient 34:8:58 ch3cn  iproh the tenth fraction ( 72.3 mg ) was found to be 
 cytotoxic against hct-116 cells ( ic50 = 5.5 g.ml ) . a portion of the tenth fraction ( 22 mg ) was further 
 purified by reversed - phase hplc ( c18 phenomenex 250  
 10 mm 5 m , 100  column , 3.0 mlmin under the following gradient profile ( a = h2o0.1% 
 cf3cooh , b = ch3cn ; 40% b , t = 0 min ; 40% b , t = 5 min ; 60% b , t = 20 min ) and gave stylissamide h ( tr = 12.6 min , 0.50 mg , 1h , 0.015% wet wt ) , stylissamide 
 g ( 1 g , tr = 15.1 min , 1.6 
 mg , 0.049% wet wt ) , and known compounds stylisin-1 ( 5 , tr = 9.78 min ) , stylissamide a ( 1a , tr = 18.5 min ) , and stylissamide 
 c ( 1c , tr = 18.5 min ) as 
 colorless solids . colorless solid ; 
 uv ( ch3cn , 23 c ) max ( log  ) 
 258 ( 2.88 ) , 264 ( 2.87 ) nm ; cd ( 0.322 mm , ch3cn , 23 c ) 
 max (  ) 196 ( + 23.2 ) , 223 ( 7.2 ) 
 nm , see also figures 3 and 4 ; h and c nmr , see table 1 ; hresims m / z 812.4704 
 [ m + h ] , 834.4522 [ m + na ] ( calcd for c45h62n7o7 812.4705 ; 
 calcd for c45h61n7nao7 , 834.4525 ) . colorless solid ; 
 uv ( ch3cn , 23 c ) max ( log  ) 
 272 ( 3.39 ) , 289 ( 3.25 ) nm ; cd ( 0.189 mm , ch3cn , 23 c ) 
 max (  ) 203 ( 3.2 ) , 221 ( 7.8 ) 
 nm , see also figures 3 and 4 ; h and c nmr , see table 2 ; hresims m / z 827.4417 
 [ m + h ] ( calcd for c44h59n8o8 827.4456 ) . a band - selective 
 constant - time hmbc spectrum of a sample of 1 g ( 1.0 
 mg ) in cdcl3 was recorded under 
 the following conditions : the band - selective pulse was a g3 gaussian 
 cascade of 500 s duration centered at  172 ppm . the c spectral width was set to 20 ppm , and h - detected 
 fids ( 64 t1 increments ) were collected 
 at 600 mhz for a total experiment time of 84 min . the delay for selection 
 of long - range coupling was set to j = 8 hz , and the 
 2-fold low - pass j filter was optimized to suppress 
 one - bond couplings between j = 120 and 180 hz .
two new peptides , stylissamides g 
 and h , were isolated from extracts 
 of a sample of stylissa caribica collected in deep 
 waters of the caribbean sea . 
 a single sample of s. caribica among a collection of 10 samples that were examined by lc - ms appeared 
 to be a different chemotype from the others in that it lacked the 
 familiar pyrrole-2-aminoimidazole alkaloids , stevensine and oroidin , 
 and contained peptides of the stylissamide class . 
 the structures of 
 the title compounds were solved by integrated analysis of the ms and 
 nmr spectra and chemical degradation . 
 the solution conformation of 
 stylissamide g was briefly examined by electronic circular dichroism 
 and temperature - dependent 1h nmr chemical shifts of amide 
 nh signals , which supported a conformationally rigid macrocycle .