the ascidians have become one of the most important sources of marine natural products in the last decades , and a significant number of compounds with unusual structures and bioactivities have been isolated from various ascidians .
the peptide metabolite didemnin b is an example of an important drug lead isolated from trididemnum solidum , a caribbean tunicate of the family didemnidae.(1 ) didemnin b was the first marine natural product to enter phase i and ii clinical trials as an anticancer agent.(1 ) the genus didemnum is an extremely productive source of bioactive cyclic peptides characterized by the presence of modified amino acids .
enzymatic heterocyclization of serine , threonine , and cysteine side chains with vicinal carbonyl groups results in the formation of five - ring heterocyclic oxazole and thiazole moieties .
thiazole- , oxazole- , and thiazoline - containing peptides have been isolated from the common indo - pacific ascidian didemnum molle from many different geographic locations , and they share the peculiar reverse prenylated ethers of serine and threonine amino acids .
they include the heptapeptides mollamide,(5 ) cyclodidemnamides , and myotamides a and b(8 ) and the hexapeptides comoramides a and b(8 ) and didmolamides a and b.(9 ) as the chemical composition of this genus changes from one collection to another , mller et al .
proposed that these metabolites are actually biosynthesized by symbiotic prochlorophytes , and they isolated prokaryotic algal symbionts often associated with the didemnid family.(10 ) furthermore , schmidt et al . reported the full sequence and functional expression of the related peptides , patellamides , from the obligate symbiont prochloron didemni.(11 ) recently , donia et al .
generate a diverse library of patellamides , and they used this information to engineer the production of a new cyclic peptide in escherichia coli.(12 )
an investigation of the tunicate d. molle collected from manado bay , indonesia , yielded two new cyclic hexapeptides , mollamides b ( 1 ) and c ( 2 ) , along with the known keenamide a ( 3 ) , which was previously reported from the notaspidean mollusk pleurobranchus forskalii collected from manado , indonesia .
this mollusk was shown to feed on the ascidian d. molle on the basis of the analysis of the gut content.(13 ) all three peptides share the peculiar reverse prenylated ethers of serine and threonine amino acids .
the freeze - dried invertebrate was extracted with water to remove the foaming aqueous - soluble polysaccharides .
the dried residue and water extracts were exhaustively extracted separately with dichloromethane and then ethanol .
the dichloromethane and the ethanol extracts were combined on the basis of h nmr analysis and were subjected to silica gel vacuum - liquid chromatography followed by reversed - phase and amino normal - phase hplc to yield the two new cyclic hexapeptides mollamides b ( 1 ) and c ( 2 ) and the known peptide keenamide a ( 3 ) .
mollamide b ( 1 ) was obtained as a light yellow powder , and hresims gave an [ m + h ] peak at 697.3713 for a molecular formula of c36h53n6o6s ( calc 697.3747 ,  3.4 mmu , mmu = millimass unit ) , requiring 14 degrees of unsaturation .
the peptidic nature of the compound was suggested by the ir , and h and c nmr spectra ; four amide protons were observed at  7.00 , 7.61 , 7.86 , and 9.14 ppm , and six c nmr signals resonated between  168 and 175 ppm , corresponding to amide carbonyls and indicating the presence of six amino acids .
in addition , the ir spectrum showed bands at 3256 , 3356 , and 1683 cm , confirming the presence of an amide functionality .
the compound was negative to ninhydrin , suggesting a blocked n - terminus or a cyclic peptide .
the structure of mollamide b ( 1 ) was determined using both one- and two - dimensional nmr spectral measurements including cosy , hmqc , hmbc , and roesy experiments .
six partial structures were constructed that were assigned as one phenylalanine , two valine units , one threonine carrying a dimethylallyl ether group , one proline unit , and one thiazoline unit .
the ether linkage between the isoprene moiety and the threonine amino acid was assigned on the basis of the hmbc correlation between the -proton of threonine resonating at  3.57 ppm and the quaternary carbon of the isoprene group resonating at  78.0 ppm .
the assignment of the thiazoline residue was accomplished using hmqc , hmbc , cosy , and hresims spectrometric techniques . the methylene protons (  4.03 , 3.46 ) showed hmbc correlations to the carbonyl signal at  174.5 , which was assigned as part of the valine 2 residue , and to the carbonyl signal at  170.8 .
the same protons also showed cosy correlations to a methine proton at  5.13 , which provides a strong hmbc correlation to the valine 2 carbonyl (  174.5 ) .
the cis / trans configuration of pro was determined to be cis on the basis of the  ( differential value of c chemical shifts of c and c in pro)(14 ) value of 8 ppm . the amino acid sequence and the connection of amino acid residues of 1 were accomplished by long - range hc correlation experiments ( hmbc ) and roesy .
the nh proton (  9.10 ) and -proton (  4.80 ) signals of threonine provide hmbc correlations to the carbonyl (  171.1 ) signal of proline .
the nh proton (  7.80 ) and -proton (  4.37 ) signals of valine 2 showed hmbc correlations to the threonine carbonyl resonance (  168.9 ) .
the nh resonating at  7.00 and belonging to the valine 1 residue correlated with the thiazoline carbonyl carbon (  170.8 ) in the hmbc spectrum connecting both residues .
the nh proton signal (  7.60 ) of the phenylalanine residue showed an hmbc correlation to the valine 1 carbonyl signal (  170.5 ) .
the connection between the proline and the phenylalanine amino acid residues was established by roesy correlations between the -proton of phenylalanine (  4.50 ) and the -proton of proline (  3.44 ) and from the aromatic protons of phenylalanine to the -proton of proline .
the absolute configuration of mollamide b ( 1 ) was determined by acid hydrolysis and derivatization with marfey s reagent(15 ) followed by comparative hplc analysis with derivatized standard d- and l - amino acids .
the analysis established the l - configuration for each amino acid residue ( l - valine , l - phenylalanine , l - proline , and l - threonine ) .
a minor peak was also observed for d - valine , which resulted from partial racemization of the l - valine from the l - valinylthiazoline moiety .
it has been reported that the thiazoline - based amino acid units in cyclic peptides tend to epimerize easily during hydrolysis .
the configuration at the thiazoline amino acid moiety using traditional degradation and derivatization techniques remains a challenge . during acid hydrolysis ,
the thiazoline moiety hydrolyzes to cysteine , which then incompletely derivatizes by fdaa ( n - alkylation , s - arylation , formation of bis - derivative , and oxidation to cystine ) , leading to unpredictable results.(16 ) the synthesis of these compounds has historically been the only stereochemical solution to these moieties , but it is time - consuming and costly . due to the inherent limitations of experimental data to assign the absolute configuration of functional groups such as these , molecular modeling coupled with
nmr - derived restraint information has exhibited significant potential for assigning relative and absolute configuration of many natural products and pharmaceutically relevant compounds .
a similar approach has been used to assign the unknown configuration at the -carbon of the thiazoline moiety of mollamide b. molecular modeling was utilized to provide a reasonable prediction of configuration at the -carbon of the thiazoline moiety based on conformational changes to the molecule associated with d- or l - configuration at this center . using the roesy data as distance restraints ( figure 1 ) , molecular mechanics calculations involving stepwise minimization followed by restrained simulated annealing
were performed to generate an ensemble of conformations for the two possible configurations at the -carbon ( d - tzn ( s ) : molb - s ; l - tzn ( r ) : molb - r ) .
additionally , since molecular mechanics energies are empirical in nature , more accurate semiempirical quantum mechanical calculations were performed to calculate the heats of formation ( hf ) for the lowest energy conformations .
molb - r appeared to be more stable than molb - s by 8.5 kcal / mol .
also , molb - r was found to be consistent with nmr - derived distance restraints , while molb - s showed two significant violations ( figure 2 ) .
thus , the configuration at the -carbon of the thiazoline moiety was assigned to be r , corresponding to the l - amino acid configuration .
energy - minimized conformations of molb - r ( 1 ) and molb - s ( 2 ) .
the hresims showed an [ m + h ] 619.3259 ( calc 619.3277 ,  1.8 mmu ) peak , indicating a molecular formula of c30h46n6o6s , which accounts for 15 degrees of unsaturation and one more additional unsaturation unit than the known peptide keenamide a ( 3 ) .
three h nmr resonances are lacking (  5.0 ( d ) , 3.60 , and 3.70 ( m ) ) corresponding to the thiazoline unit , and instead an additional singlet peak at  7.9 ppm appears .
two additional aromatic carbon resonances (  122.4 , 149.2 ppm ) were observed , indicating the presence of an aromatic ring system .
analysis of hmbc data revealed that the aromatic singlet proton resonating at  7.9 ppm correlates to the leucine carbonyl carbon (  170.7 ppm ) and to a quaternary aromatic carbon (  149.2 ppm ) .
the same proton showed a strong correlation to the carbonyl carbon forming the amide bond with the isoleucine amino acid , supporting the presence of the thiazole - modified amino acid .
careful analysis of cosy , hmqc , and hmbc nmr data indicated that the rest of the molecule is identical to keenamide a.  of the proline in mollamide c was calculated to be 2.8 ppm , showing that the proline amide bond is trans , which is the same as in keenamide a ( 3 ) (  3.1 ppm ) .
the absolute configuration of the thiazole - containing peptide mollamide c ( 2 ) was determined by the procedure described by mcdonald and ireland.(21 ) this procedure uses ozone to destroy the aromaticity of the thiazole in order to facilitate hydrolysis and prevent racemization . the analysis established the l - configuration of each amino acid residue ( l - isoleucine , l - leucine , l - serine , and l - proline ) .
mollamide b ( 1 ) and keenamide were examined for antimicrobial activity against mrsa , mycobacterium intracellulaire , candida albicans , c. glabrata , c. krusei , and cryptococcus neoformans and exhibited no activity .
mollamides b and c as well as keenamide have shown no anti - inflammatory activity in rat neonatal microglia , and neither of them inhibited cyclooxygenase enzyme ( cox-2 ) activity in a cell - based assay .
mollamide b ( 1 ) exhibited moderate antimalarial activity against plasmodium falciparum ( d6 clone and w2 clone ) , with ic50 values of 2.0 and 2.1 g / ml , respectively .
mollamide b also exhibited marginal activity against leishmania donovani , with ic50 and ic90 values of 18 and 35 g / ml , respectively , and against hiv-1 in human pbm cells with an ec50 value of 48.7 m in vitro .
mollamide b ( 1 ) has been tested at 100 m against four cancer cell lines : the non - small cell lung cancer cell line h460 , the breast cancer cell line mcf7 , and the cns cancer cell line sf-268 .
the percentage growth was h460 ( 29 ) , mcf7 ( 44 ) , and sf-268 ( 42 ) , which allowed the compound to be evaluated by the national cancer institute ( nci ) in the 60-cell - line panel .
mollamide c was tested in an in vitro disk diffusion assay that defines the differential cell killing among nine cell types examined .
these are two leukemias ( murine l1210 and human ccrf - cem ) , five solid tumors ( murine colon 38 , human colon hct-116 , human lung h125 , human breast mcf-7 , and human prostate lncap ) , and a murine and human normal cell ( hematopoietic progenitor cell , cfu - gm ) .
the assay is designed to determine large differences in the relative sensitivity of solid tumors , normal cells , and leukemias .
the greater the zone differential in the solid tumor colonies and only minor toxicity for both the leukemia and normal cells , the higher the selectivity of the compound to solid tumors .
mollamide c showed a unit zone differential value of 100 against l1210 , human colon hct-116 , and human lung h125 and a value of 250 against murine colon 38 and was not considered to be solid tumor selective .
ir and uv spectra were obtained using an aati mattson genesis series ftir and a perkin - elmer lambda 3b uv / vis spectrometer .
the h and c nmr spectra were recorded in cdcl3 using bruker spectrometers operating at 400 or 600 mhz for h and 100 or 150 mhz for c nmr .
chemical shifts are reported in parts per million ( ppm ) , and coupling constants ( j ) in hz .
the esi - ftms was acquired on a bruker - magnex bioapex 30es ion cyclotron fourier transform mass spectrometer by direct injection into an electrospray interface .
silica gel ( 200400 mesh ) and alumina ( 63200 m ) were obtained from natland international corporation and scientific adsorbents incorporation , respectively .
tlc analyses were carried out using precoated silica gel g254 or aluminum oxide alox-100 uv254 500 m .
the tunicate was collected at 1020 m depth from manado bay , indonesia , on september 28 , 2003 , where it was extraordinarily abundant .
the animal was easily identified as didemnum molle ( herdman , 1886 ) , the most commonly encountered sea squirt in much of the indo - pacific .
six kilograms of d. molle was collected in manado bay , indonesia , in 2003 . the 450 g dry wt of tunicate was extracted with h2o .
both the etoh and ch2cl2 extracts were combined on the basis of tlc , yielding 19 g of dried extract .
the extract was subjected to si gel vlc using a step gradient elution system of hexanes / etoac / meoh in 25% increments to yield 10 fractions .
fractions 6 and 7 eluted with etoac / meoh ( 9:1 ) and etoac / meoh ( 7.5:2.5 ) , respectively , were enriched with peptides based on h nmr analysis .
both fractions were purified separately with reversed - phase hplc ( phenomenex prodigy 10 m , ods 250  50 mm ;  230 nm ) eluting with a gradient system from 20% ch3cn in h2o to 100% ch3cn .
mollamide b ( 1 ) ( 40 mg , 8.8  10 % dry wt ) was eluted with 2% h2o in ch3cn .
a mixture of keenamide a and a minor peptide was eluted with 14% h2o in ch3cn .
the mixture was further purified using a nh2 hplc column ( phenomenex luna 5 m , 250  21.2 mm ) and an isocratic elution system of 20% ch2cl2/hexanes , yielding mollamide c ( 2 ) ( 2.0 mg , 4.4  10 % dry wt ) and keenamide a ( 3 ) ( 40 mg , 8.8  10 % dry wt ) .
yellow solid ; [ ]d 119.9 ( c 0.25 , chcl3 ) ; uv ( meoh ) max ( log  ) 202 ( 4.53 ) nm ; ir ( kbr film ) max 3356 , 3256 , 2966 , 2931 , 1683 , 1650 , 1530 , 1447 cm ; h nmr and c nmr , see table 1 ; lresims m / z 697 [ m + h ] ; hresims m / z [ m + h ] 697.3713 ( calcd for c36h53n6o6s , 697.3747 ,  3.4 mmu ) .
all spectra were measured in cdcl3 and referenced to the residual solvent signal at h 7.26 and c 77.4 .
white solid ; [ ]d 0 ( c 0.1 , chcl3 ) ; uv ( meoh ) max ( log  ) 200 ( 5.15 ) ; ir ( kbr film ) max 3318 , 2959 , 2926 , 2873 , 1729 , 1685 , 1628 , 1541 , 1456 , 1271 , 1140 , 1070 cm ; h
nmr and c nmr , see table 2 ; hresims m / z [ m + h ] 619.3259 ( calcd for c30h46n6o6s 619.3277 ,  1.8 mmu ) .
all spectra were measured in cdcl3 and referenced to the residual solvent signal at h 7.26 and c 77.4 .
hydrolysis of 1 ( 348 g ; 0.5 mol ) was achieved in 1 ml of 6 n hcl in a sealed vial at 100 c for 20 h. traces of hcl were removed under nitrogen .
the resulting hydrolysate was resuspended in 100 l of h2o and derivatized with 20 l of 10% n-(2,4-dinitro-5-fluorophenyl)-l - alaninamide ( fdaa ) in acetone and 10 l of 1 n nahco3 .
the mixture was heated at 40 c for 1 h.(15 ) the solutions were cooled to room temperature , neutralized with 5 l of 2 n hcl , and evaporated to dryness.(13 ) hplc analyses of the fdaa - derivatized hydrolysate of 1 and 2 and standard fdaa - derivatized amino acids were carried out using a waters novapak c18 ( 3.9  150 mm column ) with a linear gradient of triethylammonium phosphate ( 50 mm , ph 3.0)/mecn ( 90:10 to 60:40 ) in 60 min at 1 ml / min ( uv detection at  340 nm ) . because l - threonine and l - allo - threonine could not be resolved clearly using this method , they were separated using a luna c5 column ( 4.6  150 mm , 5 m ) with a gradient of 40 mm ammonium acetate , 70% ch3cn , and 30% meoh from 98:2 to 66:34 in 85 min at 0.8 ml / min , detected by microtof , and then analyzed for the extracted ion at m / z 372 for the marfey s derivative of threonine .
a slow stream of o3 was bubbled into a 7 ml ch2cl2 solution of mollamide c ( 0.3 mg ; 0.5 mol ) in a threaded bomb at 25 c for approximately 8 min .
after removal of the solvent under a stream of n2 , the residue was subjected to hydrolysis and derivatization as described above with mollamide b. the d / l - configuration for serine , proline , and leucine were assigned using the c18 column , and l - isoleucine and l - allo - isoleucine were assigned using the c5 column as described above.then the ion at m / z 384 was extracted for the four isoleucine isomers .
the analysis of mollamide b ( 1 ) using the c18 column established the presence of l - pro ( 34.47 ) [ d - pro ( 40.52 ) ] , l - val ( 43.47 ) , [ d - val ( 54.10 ) ] , and l - phe ( 52.41 ) [ d - phe ( 59.86 ) ] .
amino acid standards using lctof revealed retention times as follows : l - thr ( 36.42 min ) [ l - allo - thr ( 34.94 ) , d - thr ( 45.46 ) , d - allo - thr ( 41.37 ) ] .
the analysis of mollamide c ( 2 ) using the c18 column established the presence of l - ser ( 21.56 min ) [ d - ser ( 26.13 ) ] , l - pro ( 34.07 ) [ d - pro ( 40.25 ) ] , and l - leu ( 51.82 ) [ d - leu ( 60.85 ) ] .
amino acid standards using lctof revealed retention times as follows : l - ile ( 52.63 ) [ l - allo - ile ( 52.93 ) , d - ile ( 70.42 ) , d - allo - ile ( 71.37 ) ] . due to the close retention time of l - ile and l - allo - ile ,
mollamide c was co - injected with l - ile and l - allo - ile standards , which clearly revealed that ile has the l - configuration .
thus , the analysis of mollamides b ( 1 ) and c ( 2 ) established the presence of all l - amino acids . in order to investigate the chirality at the -carbon of the thiazoline moiety , two structures of molecule 1 were drawn using the builder module in insight ii ( accelrys inc . , san diego , ca ) .
all regular amino acids were drawn as s , while the -carbon of the thiazoline moiety was drawn with both s and r configurations to provide two initial configurations of molecule 1 : molb - r and molb - s ( denoting the chirality at the -carbon of the thiazoline moiety to be l - tzn and d - tzn , respectively ) .
the structures were first optimized using a series of concurrent minimizations employing the cvff forcefield to a gradient of 0.001 .
the optimized structures were then subjected to simulated annealing using the discover ( v 2.98 ) module of insight ii . all the simulations were performed using a distance - dependent dielectric . a forcing potential of 30 kcal / mol / rad was applied to the omega dihedrals for all amino acids , except for the cis - proline , to maintain the trans geometry .
also , nmr - derived distance restraints based on roesy correlations ( figure 1 ) were introduced during the md simulations with a force constant of 20 kcal / mol / rad .
the molecules were minimized beginning with 300 steps of steepest descents and 1000 steps of conjugate gradients to relieve any strain in the starting structures .
the structures were then  heated  gradually from 100 to 1000 k in steps of 100 k with 1500 fs of simulation at each step . at 1000 k ,
the simulation was further continued for 20  000 fs , during which 20 conformations were sampled at an interval of 1000 fs .
the conformations were then gradually  cooled  to 300 k in steps of 100 k with 2500 fs simulation at each step .
the structures were finally minimized using 500 steps of steepest descents , 1500 steps of conjugate gradients , and 500 steps of va09a . at this stage , the nmr - derived distance restraints were removed , and all 20 conformations were again subjected to the same minimization protocol as given above .
the structures were analyzed , and the lowest energy conformation of molb - r obtained without any restraints appeared to maintain important interproton distances in accordance with the nmr data .
the am1 hamiltonian was used to calculate the heat of formation ( hf ) using the mopac suite of programs ( v 6.0 ) .
the semiempirical calculations were carried out without any restraints , and all degrees of freedom in the molecule were optimized .
minimization was run with the ef algorithm to a gnorm of 0.01 . since it is well known that most semiempirical methods underestimate the barrier to rotation in peptides , the key word mmok was used to allow for a molecular mechanics correction .
stable points on the potential energy surface ( pes ) of the molecule were identified by running a force calculation and were confirmed by the absence of any imaginary ( negative ) frequencies .
the hf values were calculated to be 175.2 kcal / mol for molb - r and 166.7 kcal / mol for molb - s .
thus , molb - r appeared to be more stable by 8.5 kcal / mol .
also , significant violations were observed for two important nmr - derived distance restraints in case of molb - s ( figure 2 ) .
the distances between the -proton of valine-1 and valine-2 were 5.95 and 6.86  , while those between the -proton of valine-2 and the side chain methyl of threonine were 5.16 and 5.45  for molb - r and molb - s , respectively .
the former restraint violation appears to be critical since the two atoms involved in the restraint flank the thiazoline ring , and thus their positions ( coordinates ) are expected to be affected by the chirality at the -carbon of the ring . in other words ,
the optimized conformation of molb - r appeared to match the experimental distances derived from the nmr data , while molb - s showed two violations and was also energetically less stable than the former .
on the basis of these observations , the chirality at the -carbon of the thiazoline ring was assigned to be r , corresponding to the l - amino acid configuration for the thiazoline moiety .
 two new cyclic hexapeptides , mollamides b ( 1 ) and c ( 2 ) , were isolated from the indonesian tunicate didemnum molle along with the known peptide keenamide a ( 3 ) . 
 the structures were established using 1d and 2d nmr experiments . 
 the relative configuration of mollamide b at the thiazoline moiety was determined using molecular modeling coupled with nmr - derived restraints . 
 their absolute configuration was determined using marfeys method . 
 the new peptides have been evaluated for their antimicrobial , antimalarial , anticancer , anti - hiv-1 , anti - mtb , and anti - inflammatory activities . 
 keenamide a and mollamide b show cytotoxicity against several cancer cell lines . 
None
Results and Discussion
Experimental Section