Glycolipids from Gracilaria verrucosa

Phyrochemutry, Vol 29, No 1, pp 307 309, 1990 Prmted m Great Brltam

c

GLYCOLIPIDS

FROM

GRAClLARIA

0031 9422,90 $300+000 1989 Pergamon Press plc

VERRUCOSA

BYENG WHA SON Department

of Apphed

Chemistry,

Natlonal

Flsherles

Umverslty

of Pusan,

Pusan

608-023, Korea

(Received m reused form 24 May 1989) Key WordyTnndex--C;racilarfa terrucosa, marine reds alga, glycohpicf, galactolipid; suiphonogiycohpid

Abstract-From the marme red alga Graczlarta uerrucosa, galactopyranosyl] glycerol(acyl palmttate-oleate-arachtdonate vopyranosyl)glycerol(acyl myristateepalmitate-arachidonate

1,2-diacyl-3-O-[a-D-galactopyranosyl-(

1” 4 6’)-O-~-D5.1:4) and 1,2-dtacyl-3-O-(6’~sulpho-a-D-qumo4 15: 1) have been isolated

INTRODUCTION

Glycohpids have been shown to be widespread m organisms A number of glycohptds have been reported [l-7] and exhibit drverse biological functions As a part of a search for new biologically active substances from marine orgamsms, I have isolated a new glycohpid named GV-5 (1) from the marine red alga Gracthw uerrucosa and elucidated the stereostructure [8] This red alga is one of the marine organisms which contam prostaglandms A, and E, as secondary metabohtes [9], and as arachidonic acid, which is a biosynthetic precursor of prostaglandins, is abundantly contamed m this alga m the free and bound form, the biological significance of its metabolites 1s of interest

,kH20R’ 1

R’.R2=

Cbcd=419)

RESULTS AND DISCUSSION

Column chromatography of the ethyl acetate soluble portion gave, m order of polarity, apparently homogeneous 1,2 and 4 The IR spectrum of 2 shows the presence of hydroxyl groups and ester functions. Compartson of its rH and 13C NMR spectra (see Experimental and Table 1) with those reported for glycosylglycerides obtained from Medrcago satux~ [lo] and Sonchus urvensu [4] suggested that 2 was a digalactosyl diglyceride Alkaline hydrolysis of 2 provided a digalactosyl glyceride (3) and a mixture of fatty acid methyl esters In the secondary ion mass spectrum (SIMS) of 3, an ion peak at m/z 439 [M + Na]+ was observed. The anomertc proton and carbon signals of 2 were observed at 64.87 (lH, d, J= 3 5 Hz, 1”-H) and 64.24 (lH, d, J = 7 0 Hz, l’-H), and S, 105 3 and 100 7 (Table 1). Permethylatron of 3 (NaH, DMSO, MeI) followed by acid hydrolysis gave methyl-2,3,4,6-tetra-O-methyl galactopyranoside and methyl-2,3,4-tri-O-methyl galactopyranosrde identical with authentic materials Hence, the structure of the diglycosyl glycerol was 3-0-(LX-Dgalactopyranosyl)-(1” --* 6’)-O-fi-D-galactopyranosyl glycerol (3) [4, lo]. GC and GC-MS analyses of the abovementioned fatty acid methyl esters defined the composition as a mixture of palmnate, oleate and arachidonate m a ratio of 5: 1:4. Furthermore, i3CNMR analysis of the digalactosyl glyceride moiety for 2 m comparison with that of 3 showed that fatty acid residues were on C-l and

2

R’,R’

3

R’, R2 =

= (b c d = 5 14) H

C-2 of the digalactosyl glyceride moiety of 2 (Table 1) [3]. Based on the above evtdence, the chemical structure of the digalactosyl diglyceride was determined as 2, m which a mixture of fatty acid residues (b, c and d in a ratio of 5 : 1.4) is attached to C-l and C-2 of the glycerol moiety. The IR spectrum of 4 showed the presence of hydroxyl and ester functions. Comparison of the ‘H and i3C NMR spectra with those of M-6 [S] obtained from a marme sponge, and Ant-l [6] from the sea urchm suggested that 4 was a sulphonoglycohpid Alkahne hydrolyses of 4 as carried out for 2 furnished a glycerol glycoside (5) and a mixture of fatty acid methyl esters. In the SIMS of the 5 thus obtained, an ion peak at m/z 363 [M t Na]+ was observed The glycerol glycoside was shown to be 6’sulpho-O-a-qumovopyranosyl-(1’ -+ 3)-glycerol(5) on the

307

308

Short Report,

R’,KZ = a

-oc(cH~

b

--OC(Cti2h,,Me

11*Me

C ---OC(CHI

),Ct&-CIi(CH:

),Me

d --OC((‘ti,

12(CH,( H=CH

)4((‘11, )nMe

,CH,OR’

Table

4

RI,R~=

(a b c = 4 15 I)

5

R’, R’ =

H

1 13C NMR data for compounds 2a

3

4a

1 2 3

644 t* 124d” 688~

63 8 t 71 8d 722t

64 4 718d 613

1’ L, 3’

1053d 12 5 d” 74 8 d 70 2 db 74 6 d 68 0 t

1047d 72 3 6‘ 74 3 d 70 2 d 74 3 d

1002d 73 5d 7.506 750d 69 8 d 5441

1OO7d 716d 70 3 d” 713d 718d 630 t

100 0 cl 71 Id 69 8 d 70 9 ri 7228 62 5 t

5 6 1” 2” 3” 4”

5I, 6”

678 1

5

5

Carbon

4

and

Za, 3,4a

c

637 t

t

608 I

717d

99 72 73 73 69 53

2d 2d 9 Li 4d I d 1t

column LLKhroprep RP-8 (40 63 Inn)] (MeOHmHZO, 10 I) to afford 2 (100mg) dnd 4 (61 mg) 2, IRy(,‘,‘:“cm ’ 3375 (hr). 1725 1056. ‘H NMR (500 MHz. CD,OD, 01 5 36 ()n olefimcH).487(1H.rl,./-35H~. I”-H~.343(1H.rld,J-120.30Hz), 424(1H.d,J-7QE-Ir,I’-t~\.42!~11-1,dd,J=11-0,70Hrj.128 (hr. mcthylenes of tatty acid &un). 0 91. 0 90 (both r-like. 8J 173 1 termma methyls) ’ ‘c‘ NMR (22 5 MHL, d,-pqr&ne. (~),1729(\1),1304(‘~),110O(dj 129?,ld),1’7tcX(d),1287(d),1284 (di, 12X 3 (d). 128 I Cd), 177 X(d) 105 0 (d), 100 X (dl. 74 6 (d), 74 1 (d), 72 3 (d). 718 (d\ 71 3 (d I. 70 7 id\, 70 6 (d) 70 I (d\ 69 4 @I), 67 7 (t), 640 10. 63 I (IJ 62 2 ([I 3-1L tt), 34 0 jt), 33 4 (t). 31 Y (t,, 31 4 Ct),29 7 (I), 29 5 (I), 29 7 (I) 29 I it), 27 3 (I), 26 6 (1). 25 8 (tj, 250 (I), 22 7 (t), 22 5 (I) 140 (q) ’ ‘(‘NMR (125 MHz, CD,OD, 6,) for digaldctosyl glyceride molcty of 2 see Tdble 1

______ *Abbrcvlatmns denote the slgnal patterns ob.served m INEPT expcrlments “-‘The assignments for these signals wIthIn the same vertical column may be Interchanged

basis of acid hydrolysis, which hberated methyl-6’sulphoqumovose and glycerol, and by comparison of the 13C NMR data for that of M-6 [S] from a marme sponge Comparison of the 13CNMR data of 4 with that of 5 showed that fatty acid residues were attached to C-l and C-2 of the glycerol sulphonoglycoslde moiety of 4 Fmally, on the basis of the above evidence, the structure of 4 has been determined to be the sodium salt of 1,2-dlacyl3-0-(6’-sulpho-a-D-quinovopyranosyl)-glycerol(acyl, 4 15 1 mixture of myrlstate, palmltate and arachtdonate)

EXPERIMENTAL Isokztzm ofglyeukprds

Fmely cut alga (&&ted at Chungmu, Kyung-Nam Prefecture m May 1987) (2 kg) wab extracted with Me&O and MeOH at room temp The extracts were then partitIoned mto EtOAc-H,O to give the EtOAc extract (3 6 g) This extract (3 g) was subJected to slhca gel CC (70-230 mesh, Merck) developmg with CHCL-MeOH (10 1 -+ 3 1) to furmsh three frs contammg GV-4 (560 mg), GV-5 (I) (390 mg), and 2+4 (370 mg) The 2 + 4 fr was purified by successive CC on reverse phase silica gel (Waters. fi-Bondapak C,,) (MeOH) and d Lobar

Akzlrne hydrdystv of 2 A $oin of 2 (50 mg) m 10% dry NaOMe-MeOH (2 ml) w,is stlrred dt room temp (N, atm) for 2 hr The reactionmlvt was neutrahrcd with 2 M HCI-MeOH and partttloncd InLo tiKJAL~H,O E\apn of the H,O phase and succe9SI\e puillication on slltca gel (CHCI,~MeOH, 1 I). 11 Rondapak c’,, (MeOH Hz0 IO I) ,md Sephddex Lti-20 (M&H) gdve 3 I 18mgy f~nrrr-r tk FtOAc ph,l\e. r, mrxt of fatty acid Me esters (14 mg) ua\ obt~uned after ecdpn of solvent dnd silica gel (n-hexane FtOAc 15 I I purlhcatton Compound 3. mp 184--1X5 ( /Ix],‘%84 (H20, c 07) [4] SIMS (thmglycerol matrix) /?I/: 4iY [M + hdl+, ’ ‘C NMR (72 5 MHI, CD,OD~ D,O =1 1, ii,). see Table 1 CC analysla of fdntty acid Me esters (2% SE-30 on Chromosorh WAW DMCS 80 100 mesh, 3 mm x 2 m, column tcmp 210 , cdrrlcr N, How rate 30 ml!mm) R, (mm)- b 2 5X c 5 02 d 7 5X. b c d- 5 1 -1 Metltitnoly~~~ of 3 A soln oI 3 (X mg) m 9?h dry HC’l~-MeOH (1 5 ml) was retluxed (NZ atmj for 1 5 hr The redcllon rnixt was neutralzed wrth Ag,C’O, and filtered fiv,ipn of \olvenl dt red pres from the filtrate gdre ‘c rcbrdue. w-hrch wd\ Jrsbolred III pyrrdme (0 5 ml) and tre‘rtcd ~+rth 2 O-lrr\(trrln~thylsrlyl)tr~fluoroacetdtnide (BSTFA) (0 5 ml dt room temp for I hr The TM% derlvatlves thus obtdmed U‘LI Thown to be a m~xt ofTMSiglycerol and Me TMSI-gdlactosldc by GC dn,rlysls (2% SE:-30 on C’hromo\orb WAW DMCS, 8&100 mesh. 3 mm x 7 m) TM%-glycerol (column temp 120 . ,SzRou rdte 25 ml/mm R, (mm)==5 22) and Mc 7 MSi-g,dacto~ldc (column lcmp 170 . N, fiou r&e 30 ml mm R~~rnmL s 07 9 19. 10 39)

Short Reports Permethylatzon of 3 and successme methanolysls To a soln of 3 (15 mg) m dry DMSO (1 ml) was added dlmsyl carbamon (2 ml) [ll] After stlrrmg for 1 hr at room temp (N2 atm), MeI (5 drops) was added at 0” and stirring contmued at room temp for 12 hr The reaction mlxt was poured mto H,O and extd with EtOAc. Evapn of the washed and dried extract gave residues which were treated as described above for methanolysls of 3 The materials obtained were Identical with authentic Me-2,3,4,6-tetra-Omethylgalactose and Me-2,3,4-tn-0-methylgalactose by GC analysis (5% butane-1,4-dlol succmate on Umport B, 80-100 mesh, 3 mm x 2 m, column temp 170”, N, flow rate 35 ml/mm) R, (mm), Me-2,3,4,6-tetra-O-methylgalactose 3 29.4 10 and Me2,3,4-tn-O-methylgalactose 12 31 Alkalrne hydrolysis of4 Hydrolysis of 4 (50 mg) and successive treatment as described for 2 gave 5 (25 mg) and a mlxt of fatty acid Me esters (14 mg) Compound 5, [a]i’+55” (H,O, c 0 5) 167, SIMS (thloglycerol matnx) m/z 363 [M + Na]‘, ‘H NMR (90MHz, D,O, 6) 493 (lH, d, 5=3 1 Hz, 1’-H), ‘%NMR (125 MHz, D,O, 6,) 6, (TMS)=6, (tsp) - 1 6 ppm, see Table 1 Analytical condltlons for fatty acid Me esters by GC were the same as those described for 2 R, (mm)=a 1 43, b 2 55, d 7 53, a b d=4 15 1 Methanolysls of 5 Methanolysls of 5 (15 mg) and successive treatment as described for 3 gave TM%-glycerol GC analysis of TM%glycerol (the same as that described for 3) R, (mm)=4 46 Acknowledgements-I gratefully thank Professor I Kltagawa, Dr M Kobayashl, and Dr M Yoshlkawa (Osaka Umverslty) for

valuable discussions SIMS This research Foundation. 1988

309 I also thank Dr Y Ikemshl for measuring was financially supported by the Wolhae

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