during our continuing search for structurally unique secondary metabolites from marine dinoflagellates , we have isolated a series of cytotoxic macrolides , amphidinolides , as well as long chain polyhydroxyl metabolites from dinoflagellates amphidinium sp . .
( strain number y-52 ) , which was isolated from the inside cells of the okinawan marine acoel flatworm pseudaphanostoma luteocoloris , and isolated polyhydroxyl metabolites , luteophanols a ~ c [ 23 ] .
further investigation of extracts of the cultured dinoflagellate ( y-52 ) led to the isolation of a new polyhydroxyl metabolite , luteophanol d ( 1 ) , possessing two tetrahydropyran rings and twenty three hydroxy groups on c63-linear aliphatic chain . in this paper
the dinoflagellate was unialgally cultured at 25 c for two weeks in seawater medium enriched with 1% es supplement .
the extract was partitioned between hexane and 1 m nacl aq , and the aqueous phase was successively extracted with chcl3 and then n - buoh .
the n - buoh soluble materials were subjected to gel filtration on sephadex lh-20 ( meoh and then meoh / h2o , 1:1 ) followed by purification with reversed - phase hplc ( meoh / h2o ) to afford luteophanol d ( 1 , 0.00023 % , wet weight )
. esims of luteophanol d { 1 , [ ]d + 4.7 ( c 0.25 , meoh ) } showed the pseudomolecular ion peak at m / z 1329 ( m+na ) , and its molecular formula , c66h114o25 , was established by hresims [ m / z 1329.7522 ( m+na ) , -2.5 mmu ] .
the uv and ir spectra indicated the presence of conjugated diene chromophore ( max 232 nm ) and hydroxy group ( max 3430 cm ) , respectively .
the h and c nmr data revealed that 1 contained two sp quaternary carbons , twelve sp methines , two sp methylenes , twenty - seven sp methines , of which twenty - six were oxymethines , twenty - one sp methylenes including one oxymethylene , and two methyl groups .
since eight out of ten elements of unsaturation implied by the molecular formula were accounted for , 1 was inferred to possess two rings .
this suggested that luteophanol d ( 1 ) was a congener of luteophanol b or c. the structure of luteophanol d ( 1 ) was elucidated by extensive 2d nmr experiments including h - h cosy , hohaha , roesy , hsqc , and hmbc .
detailed analyses of h - h cosy , hohaha , and hsqc spectra revealed the proton - proton connectivities from h2 - 1 to h2 - 27 , from h3 - 65 to h2 - 39 , and from h-41 to h2 - 63 .
the hmbc spectrum of 1 showed cross - peaks for h-29/c-27 , h2 - 66/c-39 , h2 - 66/c-41 , and h2 - 39/c-40 , indicating connecitivities of c-27 to c-28 , c-39 to c-40 , and c-40 to c-41 .
two tetrahydropyran rings ( c-32 to c-36 and c-43 to c-47 ) were assigned by hmbc cross - peaks for h-32/c-36 and h-47/c-43 .
five di- and one tri - substituted double bonds were indicated to have all e geometries by roesy data ( h-30/h3 - 65 , h-54/h-56 , h-55/h-57 , h-56/h-58 , and h-57/h-59 ) and h - h coupling constants ( j12,13 = 16 hz , j16,17 = 15 hz , and j51,52 = 15 hz ) .
relative stereochemistry of the two tetrahydropyran rings ( c-32 to c-36 and c-43 to c-47 ) was elucidated on the basis of roesy data and h - h coupling constants ( j32/33 = ~0 hz , j33/34 = ~0 hz , j34/35a = 10 hz , j34/35b = 2 hz , j35a/36 = 10 hz , j35b/36 = 2 hz , j43/44a = 10 hz , j43/44b = 2 hz , j44a/45 = 9 hz , j44b/45 = 2 hz , j45/46 = ~0 hz , and j46/47 = ~0 hz ) of 1 ( figure .
thus , luteophanol d ( 1 ) was assigned as a luteophanol b congener lacking one methylene between c-52 and c-54 of luteophanol b. luteophanol d ( 1 ) possesses two tetrahydropyran rings and twenty - three hydroxyl groups on a c63 - 1inear carbon chain with one exo - methylene and two methyl branches .
luteophanol d ( 1 ) contains a hydrophilic diene portion at c-54 ~ c-59 , whereas known polyhydroxyl metabolites , amphidinols [ 48 ] and lingshuiols [ 910 ] , previously isolated from dinoflagellates amphidinium sp .
since the polyketide chain of 1 was shorter than those of luteophanols b and c by only one carbon , this truncation of the carbon chain might be occurred by unusual dinoflagellate polyketide biosynthesis .
luteophanol d ( 1 ) exhibited antibacterial activity against micrococcus luteus ( mic , 33 g / ml ) .
ir and uv spectra were recorded on jasco ft / ir-5300 and jasco ubest-35 spectrophotometers , respectively .
the 3.35 and 49.8 ppm resonances of residual cd3od were used as internal references for h and c nmr spectra , respectively .
( strain number y-52 ) was unialgally cultured at 25 c for two weeks in seawater medium enriched with 1% es supplement .
the harvested cells of the cultured dinoflagellate ( 385 g wet weight , from 725 l of culture ) were extracted with meoh ( 1 l x 3 ) . the meoh extract ( 15.19 g )
was partitioned between hexane ( 500 ml x 3 ) and 1 m nacl aq . , and the aqueous phase was successively extracted with chcl3 ( 500 ml x 3 ) and then n - buoh ( 500 ml x 3 ) . the n - buoh soluble fraction ( 4.47 g ) was subjected to a sephadex lh-20 column [ meoh and then meoh / h2o , ( 1:1 ) ] followed by reversed - phase hplc ( develosil ods-5 , 10 x 250 mm ; eluent : 55% meoh / h2o then 45% meoh / h2o ; flow rate : 2.5 ml / min ; uv detection at 226 nm ) to afford luteophanol d ( 1 , 0.9 mg , 0.00023 % wet weight ) .
colorless amorphous solid ; [ ]d + 4.7 ( c 0.25 , meoh ) ; uv ( meoh ) max 232 nm (  22800 ) ; ir max 3430 , 2920 , 1640 , and 1050 cm ; esims m / z 1329 ( m + na ) ; hresims m / z 1329.7522 ( m + na)+ . calcd . for c66h114o25na , 1329.7547 ; h nmr ( cd3od / c5d5n 2:1 )  3.62 ( 2h , h2 - 1 ) ; 3.75 ( m , 1h , h-2 ) ; 1.50 ( 1h , h-3a ) ; 1.65 ( 1h , h-3b ) ; 1.47 ( 1h , h-4a ) ; 1.75 ( 1h , h-4b ) ; 1.50 ( 2h , h2 - 5 ) ; 3.63 ( 1h , h-6 ) ; 1.50 ( 2h , h2 - 7 ) ; 1.47 ( 1h , h-8a ) ; 1.75 ( 1h , h-8b ) ; 1.47 ( 1h , h-9a ) ; 1.58 ( 1h , h-9b ) ; 3.68 ( 1h , h-10 ) ; 2.25 ( 2h , h2 - 11 ) ; 5.80 ( 1h , h-12 ) ; 5.63 ( 1h , h-13 ) ; 4.17 ( 1h , h-14 ) ; 2.35 ( 2h , h2 - 15 ) ; 5.67 ( 1h , h-16 ) ; 5.76 ( 1h , h-17 ) ; 2.30 ( 1h , h-18a ) ; 2.70 ( 1h , h-18b ) ; 3.75 ( 1h , h-19 ) ; 3.77 ( 1h , h-20 ) ; 2.67 ( 1h , h-21 ) ; 3.77 ( 1h , h-22 ) ; 3.95 ( 1h , h-23 ) ; 1.70 ( 1h , h-24a ) ; 2.13 ( 1h , h-24b ) ; 4.02 ( 1h , h-25 ) ; 1.70 ( 2h , h2 - 26 ) ; 2.10 ( 1h , h-27a ) ; 2.32 ( 1h , h-27b ) ; 5.70 ( 1h , h-29 ) ; 4.80 ( 1h , h-30 ) ; 3.90 ( 1h , h-31 ) ; 4.33 ( 1h , h-32 ) ; 4.40 ( 1h , h-33 ) ; 4.20 ( 1h , h-34 ) ; 2.02 ( 1h , h-35a ) ; 2.11 ( 1h , h-35b ) ; 3.68 ( 1h , h-36 ) ; 3.77 ( 1h , h-37 ) ; 1.75 ( 1h , h-38a ) ; 2.19 ( 1h , h-38b ) ; 2.32 ( 1h , h-39a ) ; 2.69 ( 1h , h-39b ) ; 4.45 ( 1h , h-41 ) ; 3.58 ( 1h , h-42 ) ; 4.30 ( 1h , h-43 ) ; 1.70 ( 1h , h-44a ) ; 2.40 ( 1h , h-44b ) ; 4.26 ( 1h , h-45 ) ; 4.39 ( 1h , h-46 ) ; 4.08 ( 1h , h-47 ) ; 4.28 ( 1h , h-48 ) ; 4.70 ( 1h , h-49 ) ; 5.90 ( 1h , h-50 ) ; 5.86 ( 1h , h-51 ) ; 2.18 ( 2h , h2 - 52 ) ; 1.65 ( 1h , h-53a ) ; 1.70 ( 1h , h-53b ) ; 4.18 ( 1h , h-54 ) , 5.73 ( 1h , h-55 ) ; 6.30 ( 1h , h-56 ) , 6.30 ( 1h , h-57 ) , 5.78 ( 1h , h-58 ) , 4.18 ( 1h , h-59 ) , 1.60 ( 1h , h-60a ) ; 1.65 ( 1h , h-60b ) ; 2.15 ( 2h , h2 - 61 ) ; 5.83 ( 1h , h-62 ) ; 4.93 ( 1h , h-63a ) ; 5.01 ( 1h , h-63b ) ; 1.18 ( d3h , h3 - 64 ) ; 1.75 ( 3h , h3 - 65 ) ; 5.03 ( 1h , h-66a ) ; 5.18 ( 1h , h-66b ) ; c nmr ( cd3od / c5d5n 2:1 )  c-1 , 68.3 ; c-2 , 74.0 ; c-3 , 35.5 ; c-4 , 23.8 ; c-5 , 39.4 ;
c-6 , 72.8 ; c-7 , 39.4 ; c-8 , 23.8 ; c-9 , 38.8 ; c-10 , 72.8 ; c-11 , 42.3 ; c-12 , 129.4 ; c-13 , 137.7 ; c-14 , 74.0 ; c-15 , 43.0 ; c-16 , 131.2 ; c-17 , 131.7 ; c-18 , 39.2 ; c-19 , 74.0 ; c-20 , 80.4 ; c-21 , 35.9 ; c-22 , 81.1;c-23 , 73.2 ; c-24 , 42.3 ; c-25 , 72.5 ; c-26 , 37.7 ; c-27 , 37.5;c-28 , 139.4 ; c-29 , 127.7 ; c-30 , 68.6 ; c-31 , 73.3 ; c-32 , 80.2 ; c-33 , 69.8 ; c-34 , 68.2 ; c-35 , 31.6 ; c-36 , 76.6 ; c-37 , 75.3 ; c-38 , 33.7 ; c-39 , 28.9 ; c-40 , 152.9 ; c-41 , 77.4 ; c-42 , 76.2 ; c-43 , 71.5 ; c-44 , 32.7 ; c-45 , 68.1 ; c-46 , 69.8 ; c-47 , 81.4 ; c-48 , 73.0 ; c-49 , 75.0 ; c-50 , 130.5 ; c-51 , 135.4 ; c-52 , 30.4 ; c-53 , 38.8 ; c-54 , 73.0 ; c-55 , 132.3 ; c-56 , 131.5 ; c-57 , 131.5 ; c-58 , 138.5 ; c59 , 73.0 ; c-60 , 38.5 ; c-61 , 31.4 ; c-62 , 140.4 ; c-63 , 115.9 ; c-64 , 7.8 ; c-65 , 18.2;c-66 , 113.5 .
ir and uv spectra were recorded on jasco ft / ir-5300 and jasco ubest-35 spectrophotometers , respectively .
the 3.35 and 49.8 ppm resonances of residual cd3od were used as internal references for h and c nmr spectra , respectively .
the dinoflagellate amphidinium sp . ( strain number y-52 ) was unialgally cultured at 25 c for two weeks in seawater medium enriched with 1% es supplement .
the harvested cells of the cultured dinoflagellate ( 385 g wet weight , from 725 l of culture ) were extracted with meoh ( 1 l x 3 ) . the meoh extract ( 15.19 g )
was partitioned between hexane ( 500 ml x 3 ) and 1 m nacl aq . , and the aqueous phase was successively extracted with chcl3 ( 500 ml x 3 ) and then n - buoh ( 500 ml x 3 ) . the n - buoh soluble fraction ( 4.47 g ) was subjected to a sephadex lh-20 column [ meoh and then meoh / h2o , ( 1:1 ) ] followed by reversed - phase hplc ( develosil ods-5 , 10 x 250 mm ; eluent : 55% meoh / h2o then 45% meoh / h2o ; flow rate : 2.5 ml / min ; uv detection at 226 nm ) to afford luteophanol d ( 1 , 0.9 mg , 0.00023 % wet weight ) .
colorless amorphous solid ; [ ]d + 4.7 ( c 0.25 , meoh ) ; uv ( meoh ) max 232 nm (  22800 ) ; ir max 3430 , 2920 , 1640 , and 1050 cm ; esims m / z 1329 ( m + na ) ; hresims m / z 1329.7522 ( m + na)+ . calcd . for c66h114o25na , 1329.7547 ; h nmr ( cd3od / c5d5n 2:1 )  3.62 ( 2h , h2 - 1 ) ; 3.75 ( m , 1h , h-2 ) ; 1.50 ( 1h , h-3a ) ; 1.65 ( 1h , h-3b ) ; 1.47 ( 1h , h-4a ) ; 1.75 ( 1h , h-4b ) ; 1.50 ( 2h , h2 - 5 ) ; 3.63 ( 1h , h-6 ) ; 1.50 ( 2h , h2 - 7 ) ; 1.47 ( 1h , h-8a ) ; 1.75 ( 1h , h-8b ) ; 1.47 ( 1h , h-9a ) ; 1.58 ( 1h , h-9b ) ; 3.68 ( 1h , h-10 ) ; 2.25 ( 2h , h2 - 11 ) ; 5.80 ( 1h , h-12 ) ; 5.63 ( 1h , h-13 ) ; 4.17 ( 1h , h-14 ) ; 2.35 ( 2h , h2 - 15 ) ; 5.67 ( 1h , h-16 ) ; 5.76 ( 1h , h-17 ) ; 2.30 ( 1h , h-18a ) ; 2.70 ( 1h , h-18b ) ; 3.75 ( 1h , h-19 ) ; 3.77 ( 1h , h-20 ) ; 2.67 ( 1h , h-21 ) ; 3.77 ( 1h , h-22 ) ; 3.95 ( 1h , h-23 ) ; 1.70 ( 1h , h-24a ) ; 2.13 ( 1h , h-24b ) ; 4.02 ( 1h , h-25 ) ; 1.70 ( 2h , h2 - 26 ) ; 2.10 ( 1h , h-27a ) ; 2.32 ( 1h , h-27b ) ; 5.70 ( 1h , h-29 ) ; 4.80 ( 1h , h-30 ) ; 3.90 ( 1h , h-31 ) ; 4.33 ( 1h , h-32 ) ; 4.40 ( 1h , h-33 ) ; 4.20 ( 1h , h-34 ) ; 2.02 ( 1h , h-35a ) ; 2.11 ( 1h , h-35b ) ; 3.68 ( 1h , h-36 ) ; 3.77 ( 1h , h-37 ) ; 1.75 ( 1h , h-38a ) ; 2.19 ( 1h , h-38b ) ; 2.32 ( 1h , h-39a ) ; 2.69 ( 1h , h-39b ) ; 4.45 ( 1h , h-41 ) ; 3.58 ( 1h , h-42 ) ; 4.30 ( 1h , h-43 ) ; 1.70 ( 1h , h-44a ) ; 2.40 ( 1h , h-44b ) ; 4.26 ( 1h , h-45 ) ; 4.39 ( 1h , h-46 ) ; 4.08 ( 1h , h-47 ) ; 4.28 ( 1h , h-48 ) ; 4.70 ( 1h , h-49 ) ; 5.90 ( 1h , h-50 ) ; 5.86 ( 1h , h-51 ) ; 2.18 ( 2h , h2 - 52 ) ; 1.65 ( 1h , h-53a ) ; 1.70 ( 1h , h-53b ) ; 4.18 ( 1h , h-54 ) , 5.73 ( 1h , h-55 ) ; 6.30 ( 1h , h-56 ) , 6.30 ( 1h , h-57 ) , 5.78 ( 1h , h-58 ) , 4.18 ( 1h , h-59 ) , 1.60 ( 1h , h-60a ) ; 1.65 ( 1h , h-60b ) ; 2.15 ( 2h , h2 - 61 ) ; 5.83 ( 1h , h-62 ) ; 4.93 ( 1h , h-63a ) ; 5.01 ( 1h , h-63b ) ; 1.18 ( d3h , h3 - 64 ) ; 1.75 ( 3h , h3 - 65 ) ; 5.03 ( 1h , h-66a ) ; 5.18 ( 1h , h-66b ) ; c nmr ( cd3od / c5d5n 2:1 )  c-1 , 68.3 ; c-2 , 74.0 ; c-3 , 35.5 ; c-4 , 23.8 ; c-5 , 39.4 ; c-6 , 72.8 ; c-7 , 39.4 ; c-8 , 23.8 ; c-9 , 38.8 ; c-10 , 72.8 ;
c-11 , 42.3 ; c-12 , 129.4 ; c-13 , 137.7 ; c-14 , 74.0 ; c-15 , 43.0 ; c-16 , 131.2 ; c-17 , 131.7 ; c-18 , 39.2 ; c-19 , 74.0 ; c-20 , 80.4 ; c-21 , 35.9 ; c-22 , 81.1;c-23 , 73.2 ; c-24 , 42.3 ; c-25 , 72.5 ; c-26 , 37.7 ; c-27 , 37.5;c-28 , 139.4 ; c-29 , 127.7 ; c-30 , 68.6 ; c-31 , 73.3 ; c-32 , 80.2 ; c-33 , 69.8 ; c-34 , 68.2 ; c-35 , 31.6 ; c-36 , 76.6 ; c-37 , 75.3 ; c-38 , 33.7 ; c-39 , 28.9 ; c-40 , 152.9 ; c-41 , 77.4 ; c-42 , 76.2 ; c-43 , 71.5 ; c-44 , 32.7 ; c-45 , 68.1 ; c-46 , 69.8 ; c-47 , 81.4 ; c-48 , 73.0 ; c-49 , 75.0 ; c-50 , 130.5 ; c-51 , 135.4 ; c-52 , 30.4 ; c-53 , 38.8 ; c-54 , 73.0 ; c-55 , 132.3 ; c-56 , 131.5 ; c-57 , 131.5 ; c-58 , 138.5 ; c59 , 73.0 ; c-60 , 38.5 ; c-61 , 31.4 ; c-62 , 140.4 ; c-63 , 115.9 ; c-64 , 7.8 ; c-65 , 18.2;c-66 , 113.5 .
 luteophanol d ( 1 ) , a new polyhydroxyl linear carbon - chain metabolite , has been isolated from the cultured marine dinoflagellate amphidinium sp . , which was isolated from okinawan marine acoel flatworm pseudaphanostoma luteocoloris . 
 the structure of 1 was elucidated by detailed analyses of 2d nmr spectra . 
 luteophanol d ( 1 ) possesses two tetrahydropyran rings and twenty - three hydroxyl groups on c63-linear aliphatic chain with one exo - methylene and two methyl branches . 
Introduction
Results and Discussion
Experimental
General
Cultivation and Isolation