De Riccardis, F.

Debitus, Cécile

Iorizzi, M.

Minale, L.

Riccio, R.

Horizon / Pleins textes

t 
Letters, Vol. 33, No. 8, pp. 1097-1100, 1992 0040-4039/92 $3.00 + .O0 Q3 
Pergatnon Press plc Prinled in Crent Britain 
The First Occurrence of Polyhydroxylated Steroids with Phosphate 
Conjugation from the Starfish Tremaster novaecaledoniae 
I Cécile Debitus 
Abslracl: Three steroid coiisliiuciiis hevc been isolaicd from ilic siarfish Trenrusier rioi,ul,f.uledo/~iul~ (Jangoux 1982) collecied aia dcpih 
of530 in off New C:ilcdonia. Compounds 1 - 3. designalcd as ireinasierol A - C. iue chmacicrizcd by lhe presence of 3ß-O-sulphaied. 
(ia-O-phosphaicd tuid 16~-O-aceiyl;iied groupings u11 a s1croid;il skclclon. In cornpound 1 thc inonophosphate residue is further linked 
IO l'-glucose (I'-glucosc tciracclatc in 2 and I'-glucose-G'-aceiaic in  3). 
-.. 
It is known that there are three groups of steroidal oligoglycosides in starfishes: sulphated steroidal penta- 
and hexa-glycosides (('asterosaponins"), steroidal cyclic glycosides (so far only found in two species of the genus 
Echaster) ,  and glycosides of polyhydroxysteroids consisting of a polyhydroxysteroid with one or two sugar units, 
which are found in both sulphated and non sulphated form. Often these saponins are accompanied by small amounts 
of polyhydroxylated steroids, also occurring in both sulphated and non-sulphated form2.). In our continuing 
investigation of the New Caledonian marine species we have examined the polar extracts of the starfish Trenzaster 
iio~ueculedorriao (Jangoux 1982), which is a "living fossil"species4, collected at depth of 530 m off New Caledonia, 
and wish to report the first isolation of phosphated polyhydroxysteroids from a natural source. Compounds 1 - 3 
are also sulphated. 
Compounds were isolated and purified by chroimtography on Sephadex LH-60 (methanol-water 2: 1 as 
eluant) of the methanol soluble portion of the acetone extracts, followed by droplet counter current chromatogra- 
phy [DCCC, butan-1-01 : acetone : water (3: 15); descending mode] and HPLC (p-Bondapak C,,, 30 cm x 7.8 mm 
i.d.; methanol-water 1:1 as eluant). 
Tremasterol A (l), [alD= t40" (c 0.5, MeON) exhibited, in its negative-ion FAB mass spectrum, molecular 
ion species at m/z 803 (80%) and 78 I (40%), corresponding to [MN,l-and [MJ, respectively, and intense fragment 
ion peaks a t  m/z 641 (100%) and 619 (90%) arising by loss of the glucosyl residue (=  162 m. u.). Fragment ion 
peaks at m/z 539 (20%) and 521 (50%) were interpreted as loss of SO, from m/z 619 and NaHSO, from m/z 641. 
The presence of a steroidal skeleton was deduced from 'H NMR methyl proton signals (Table 1) and one sugar unit 
(identified t is  glucose; anhydrous acid methanolysis) would be also considered for the signals in the 3.28 - 3.68 
region and the anomeric one at 6 4.89. 
The presence of an acetoxy group was shown from IR (vmnr 1735 cm-I), 'H NMR (ZII 2.02 s, 3H) and W N M R  
(6, 172.4 and 21.2 ppm) data. In addition to the sugar moiety and the acetoxy group, the IT NMR spectrum 
revealed carbon signals in agreement with a trioxygenated cholestane structure (Table 1). 2D-COSY experiment 
allowed the sequential assignement of the resonances for the ~ I U C O S Y I  residue and also the establishment of the 
connectivities C-1 to C-8 and C-15 to C-21 within the steroidal framework. The multiplet at 6 4.24 had the 
complexity normally seen for a'3ß-oxygenated group and its downfield chemical shift, virtually identical to that 
1097 
ORSTOM Fonds Documentaire 
N * ~ :  3569A :.,A U A -  
Cote 2 8 
* -  
1098 t 
seen i n  the spectrum of the Sa-cholestan-3ß-yl sulphate, suggested a sulphate group located there. The multiplet 
at 6 4.06 appearing as a dddd (J = 9.5,9.5,7.5 and 4.5 Hz), was assigned to an axial proton (HAß) in a cholestane 
skeleton. The axial proton associated with the 6a-hydroxyl group is usually seen as a triple doublets (J = 9.5 and 
4.5 Yz), and the shape of the 6ß-proton signal in 1 was suggestive for the presence of a phosphate (typical Ju.c.o. 
E= cw 8.4 HZ)~,  to which the ß-glucopyranosyl residue is glycosidally linked. This was confirmed by theproton noise 
decoupled ''C NMR spectrum, in which l', 2', 5 and 6 carbons appear as doublets with J,,o.c = 5.5 Hz and J E.o.c. 
= 7.4 Hz. The presence of a phosphate group linking C-6 of the steroid and C-1' of glucose was definitively 
confirmed by "P NMR spectrum, which showed a triplet (J = 7.5 Hz) signal at 3.54 ppm downfield from external 
standard (H3P0, 85% in D,O), converted into doublets on irradiation at SI, 4.06 (H-6) and at 6,4.89 (H-I'), The 
"C chemical shifts of the &~cosyl residue were also compatible with those of reference ß-O-glucopyranose-1- 
phosphate7, Under very mild acid treatment (reverse phase HPLC using 0.1% CH,CO,H in 60% aq. methanol) 
glucose was removed giving laR, negative-ion FAB mass spectrum, m/z 643 [MNJ and m/z 621 [M 1'. The 
presence of the sulphate group at C-3 was confirmed by solvolysis of 1 in a dioxane-pyridine mixture, 8fording 
the desulphated derivative 1 b9, negative-ion FAB mass spectrum, m/z 541 [MJ, 6,, 3.50 m (4.24 in 1); 6,, 72.0 
ppm (79.9 ppin in I). The remaining acetoxy function was located at the 16 position from the chemical shift of the 
H-16 signal (6 5.27 m), which showed coupling to signals at 6 2.42 (H-13, 1.32 (H-17) and 1.15 (H-15) ppm in 
the 2D-COSY spectrum. The ß-configuration was deduced from the downfield shift of the 18-methyl protons to 
Na+'O- P -0 
? 
Na+-O-{-OH 
O 
1 R = R ' = H  l a  R = SU3-Na+ 
2 R = R ' = A c  l b  R = H  
3 R = H ,  R '  = A c  
80.93, requiring the presence of a 1,3-.~yit interaction, and confirmed by theI'C NMR signal at 6 31.3 ppm assigned 
to C-20, significantly upfield relative to the model 5a-cholestan-3ß-o11@ (6, 36.0 ppm), as expected upon 
introdution of a substituent at l6ß-position. 
Therefore, based on the above results, tremasterol A possesses structure 1, that is 5a-cholesta-3ß,6a, 16ß- 
triol-3-sulphate,6-(plucose-l')-phosphate, 1 (,-acetate. 
Tremasterol B (2) is the 2',3',4',6'-tetraacetate derivative of 1. The negative-ion FAB mass spectrum showed 
molecular anion species at m/z 971 (100%) [M,,]-and 949 (20%) [M,1- accompanied by a fragment ion peak at 
m/z 641 (25701, corresponding to the loss of a glucosyl tetraacetate residue (330 m.u.1 from m/z 971. In addition 
to the signals for the aglycone already observed in the spectrum of 1, the 'H NMR spectrum of 211 contained four 
more methyl singlets in  the 1.99-2.09 region (four acetate groups) and the sugar signals were seen downfield shifted 
to 6 3.97 (m, H-57, 6 4.22 (dd, J = 12.5 and 2.5 Hz) - 4.36 (dd, J = 12.5,S.O Hz, -CH,-OAc) and as overlapping 
signals in the 4.9-5.3 ppm region (H-l', H-2', H-3' and H-4'). The "C NMR chemical shifts of the glucosyl 
tetraacetate residue in 2 werecompatible with those in references7. Reverse phase HPLC using O. 1% CH,CO,H in 
60% uy. methanol resulted in the removal of glucose tetraacetate affording la  and, upon solvolysis in a dioxane- 
pyridine mixture, 2 afforded the desulphated steroid lb.  
1099 d - 
Table 1. NMR daia (CD,OD) of tremasterol A (1 ). 
~~ ~~ 
Carbon 13C NMRI (ppm) Proton 'H NMRh (ppm) 
1 .  
2 
3 
4 
5 
6 
7 '  
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
18 
19 . 
20 
21 
22 
23 
24 
25 
26 
27 
GH,CO- 
CH,CO- . 
1' 
2' 
3' 
4' 
5' ' 
6' . 
37.0 
29-5 
79.9 
3 1.0 
52.0* 
76.0* 
. 40.9 
35.1 
55.5 
37.6 
22.0 
' 41.1 
44.1 
55.1 
35.8 
76.6 
61.6 
13.2 
13.9 
31.3 
1 X.6 
38.4 
25.3 
* 40.6 
29.0 
23.0 
22.8 
21.2 
172.4 
99.3* 
76.0* 
77.8 
71.8 
78.4 
63.0 
l a ,  lß  1.12 m, 132  m 
2a; 2ß 2.07 in, 1.61 m 
3a 4.24 m 
4a,  4ß 2,60 m, 1.50 m 
1.30 m 
6ß 4-06 dddd (9.5,9,5,7.5,4.5) 
7a,  7ß 1.08 m, 2.35 m 
1.65 m 
15a, ISß 2.42 in, 1.15 m 
16a 5.27 m 
1.32 m 
0.93 s, 3H 
0.92 s, 3H 
1.88 m 
1.00 d (71, 3H 
. 0.91 d (71, 3H 
0.91 d (7), 3H 
cli,co- 2.02 s, 3H 
4.x9 t (7.5) 
3.28 dd (957.5) 
.3.45 t (9.5) 
3.27 t (9.5) 
3.44 in 
3.68dd (11.2,6.2)-3.91 dd (11.2,2.5) 
At 62.9 MHz; values relative to 13CD,0D (?+! = 49 ppm). 
At 500.13 MHZ by'H-'H correlation spectroscopy; chemical shifts referred to CHD,OD (6, = 3.34), J (in Hz 
are given in parenthesis. 
*Signals appear as doublets in the proton noise decoupled spectrum. 
Tremasterol C (3) [negative-ion FAB mass spectrum: m/z 845 (30%) [MN,]-, 823 (100%) [M,]-and 619(loss 
of the glucosyl-O-acetate residue from m/z X23)j is a monoacetate derivative of the steroid 1. On very mild acid 
treatment 3 afforded la whereas on solvolysis in a dioxane-pyridine mixture it gave 1 b. The acetate group on 3 was 
located at C-6 of the glucopyranose moiety from the 'H NMR spectrum", which showed the C-6 H, signals 
downfield shifted to 6,4.23 (dd, J = 12.5 and 5 Hz) and 4.49 (dd, J = 12.5,2.5 Hz). 
6 1100 J 
AcknowZedgements.Tis contribution is part of the project SMIB [Substances Marines d'Intéret Biologique), 
ORSTOM-CNRS, Nouméa, New Caledonia. Chemical work was supported by the "Ministero dell'Universit8 e 
della Ricerca Scientifica e Tecnologica", MURST, ROME. We thank Professor M. Jangoux (Universite Libre de 
Bruxelles) for the identification of the starfish. FAB-MS spectra were provided by "Servizio di Spettrometria di 
Massa del C.N.R. e dell'Universitiì di Napoli", the assistance of the staff is gratefully acknowledged. 
REFERENCES AND NOTES 
1 
2 
3 
4 
5 
6 
7 
' X  
9 
1 o 
11 
,I2 
* 
Present address: Universitiì degli Studi del Molise, Facoltà di Agraria - 86100 - Campobasso, Italy. 
Minale, L.; Riccio R.; Pizza C.; Zoll0 F. Nuticru/ Product arid Biological Activities, NAITO Foundation 
Symposium; H. Imura, T. Goto, T. Murachi and T. Ndkajima Eds.; University of Tokyo Press, Elsevier 
Science, 1986, p. 39, 
Stonik,V. A.; Elyakov, G. B. Biorgmric Muriric~ Cliertiistry; Scheuer P. J. Ed.; Springer Verlag, New 
York,1988,2, p. 43. 
M. Jangoux, personal inforination. 
Bridgeman, J. E.; Cherry, P. C.: Clegg, A. S.; Evans J. M.; Jones, Sir Ewart R. H.; Kasal, A.; Kumar, V.; 
Meakins, G. D.; Morisawa, Y.; Richards, E. E. Woodgate, P.D../, Ch~nz. Soc. (C) 1970, p. 250. 
Pretsch, E.; Clerc, T.; Seibl, J.; Simon, W. Siiectrul Dutuj3i.StriictiIr.L. Deterniiriutiori oforganic Conipourids, 
Springer Verlag, 1989, p. H360. 
Breitmaier, E.; Voelter, W.; CUl60ii-13 NMR Spcfrnscopy, Third Edition, VCH Publisher, 1987, p. 388. 
Spectral data of l a :  'H NMR (CD,OD); E, ,  = 0.90 (GH, d, J = 7 Hz, CH,-26 and CH,-27), 0.91 (3H, s, CH,- 
7.5 and 4.5 Hz, H-6). 4-25 (IH, m, H-3) and 5.25 (IH, m, H-16) ppm. 
Spectral data of Ib: 1H NMR (CD,OD); 6, = 0.89 (GH, d, J = 7 Hz, CH,-26 and CH,-27), 0.91 (GH, s, CH,- 
1 X  and CH,-191, 1.00 (3H, d,  J = 7  Hz, CH,-21), 2.03 (3H, s, CH,CO-), 3.50 (lH, m, H-3), 3.98 (lH, dddd, 
J=Y.5,9.5,7.5and4.5Hz, H-6)and5.25(1H,m,H-16)ppin. "CNMR(CD,OD); 6,=C-1: 38.6,C-2: 31.4, 
19),0.93(3H,~,CH,-18), 1.00(3H,d,J=7H~,CH,-21),2.02(3H,~,CH,CO-),4.06(1H,dddd,J=9.5,9.5, 
C-3: 72.0, (2-4: 33.3, C-5: 52.1*, C-6: 76.7*, C-7: 41.0, C-8: 35.1, C-9: 55.6, C-IO: 37.0, (2-11: 22.1, (2-12: 
41.4, C-13: 44.0, C-14: 55.3, C-15: 35.8, C-16: 76.7, C-17: 61.7, C-18: 13.2, C-19: 13.9, C-20: 31.8, C-21: 
18.6, C-22: 38.5, C-23: 25.4, C-24: 40.6, C-25: 29-0, C-26: 23.0, (2-27: 22.8, CH,CO: 21.2, CHGO: 172.3. 
Blunt, J. W.; Stothers, J. B. Org. Mugii. Res., 1977, 9, p. 439. 
Spectral data of 2: 'H NMR (CD,OD); 6,, = 0.90 (GH, d, J = 7 Hz, CH,-26 and CH,-27), 0.92 (3H, s, CH,-19), 
s, CH,CO-), 2.09 (3H, s, CH,CO-),4.06 (lH, dddd, J =9.5,9.5,7.5 and4.5 Hz, H-G), 4.24(1H, m,H-3)and 
5.27 (LH, m, H-16) ppm. 1-C NMR (CD,OD); 6,= C-1': 96.9*, C-2': 73.2*, C-3': 73.4, C-4': 69.6, C-5': 73.5, 
C-6': 63.3, cH,CO-: 20.6 (~21, 20.8, 21.0 ppm. 
Spectral data of 3: IH NMR (CD,OD); 6, = 0.90 (GH, d, J = 7 Hz, CH,-26 and CH,-27), 0.91 (3H, s, CH,-19), 
dddd, J = 9.5, 9.5,7.5 and 4.5 Hz, H-h), 4.24 (IH, in, H-3) and 5.26 (IH,  in, H-16) ppm. 
Signals appear as doublets in the proton noise decoupled spectrum. 
0.93 (3H, S, CH,-18), 1.00 (3H, d, J = 7  Hz, CH,-21), 1.99 (3H, S, CHACO-), 2.02 (GH, S, CH,CO-), 2.08 (3H, 
0.93(3H,s,CH7-l8), ~.~~(~H,~,J=~HZ,CH,-~~),~.~~(~H,S,CH,CO-),~.~~(~H,S,CH,CO-),~.O~(~H, 
(Received in UK 4 December 1991) 


The first occurrence of Pplyhydroxylated steroids with phosphate conjugation from the starfish Tremaster novaecaledoniae

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