Separation of carotenoid glycosides on magnesium oxide

270

NOTES

CI-IROM. 5927

Separation

of caroteneid

glycasides on magnesium

oxide

Secondary and tertiary carotenoid glycosidcs had not been detected in certain bacteria and blue-green algae until a few years ago (for a review, see LIAAEN JENSENI). These compounds, however, seem to be quite common in several bacterial groups. During our investigations on the carotenoid patterns of myxobacteria, we found several new tertiary carotenoid glycoside esters and carotenoid rhamnosides29 3, Some of these bacteria contained up to four closely related compounds that could not be separated by conventional methods. In this communication, a generally applicable isolation procedure for carotenoid glycosides, including a final chromatographic step on magnesium oxide (MgO), is presented. We have found that MgO (Merck, Darmstadt, G.F.R.) is especially suitable for the separation of peracetylated carotenoid glycosides. Aluminium oxide has been used in the separation of secondary carotenoid glycosidcs from blue-green on this adsorbent, however, was less useful for our algae 495, but chromatography purpose. MgO has been used previously for the separation of carotenoiclsfl-O, silylated carotenoids’ and other lipidslo. The following isolation procedure is used. The crude acetone extract is chromatographed on a silica gel column (length 8 cm, diameter 2 cm) with light petroleumdiethyl ether (I : 2, v/v) in order to remove non-glycosidic carotenoids and the bulk of non-polar lipids. Carotenoid glycosides are eluted with diethyl ether and with diethyl ether containing increasing amounts of acetone. Phospholipids remain on the column under these conditions, as has been checked by phospholipicl analysis according to the method described previously ll. The carotenoid glycosides from myxobacteria are generally esterified at one of the glucose hydroxyl groups with various fatty acids. To obtain homogeneous material in the following isolation steps (e.g., for mass spectrometry) saponification of the esters with ethanolic potassium hydroxide is necessary, An important part of the isolation procedure is peracetylation with acetic anhydride in pyridine. By this means the hydrophilic parts of the molecules, viz. the sugar moieties, become more hydrophobic. The peracetates exhibit much better chromatographic properties than the native glycosides (see also ref. 4). Further purification is accomplished by a second chromatographic step on a silica gel column. The various carotenoid fractions, however, generally contain more than one component. A final chromatographic step on MgO (thin layer or column) results in a complete separation of the individual glycosides. An unidentified lipid contaminant that occasionally accompanies the carotenoid glycosides is also removed. The carotenoid glycosides so far identified from myxobacteria, along with their respective Rp values on MgO thin layers, are summarized in Table I. Light petroleum (boiling range Go-70”) -benzene-methanol (40: IO: 1) is used cas the solvent. Column chromatography on MgO with similar solvent mixtures also gives good results for preparative purposes. In this instance, 50 ‘96 Kieselguhr (Merck, Darmstadt, G.F R,) is added to the MgO to increase the flow-rate. As can be seen in Table I, additional conjugated carbon-carbon double bonds within the molecules decrease the RF values, whereas cyclization increases the RF values. Thus, compounds C, E and I-I, which move as one zone on silica gel, can be easily separated. The relatively high I+ value J. Clr~o,>ralo~r.,68 (1972)

270-272

NOTES

BLE

27=

I

3UCTTJRAL

PORXIULAE

in-layer

AND

chromnto~aphy

‘W

\I

Rp

VALUES

on MgO.

’

OP

PERACETYLATED

Solvent:

light

CAROTENOID

GLYCOSLDES

pctrolcum-benzene-methanol

4

,,

(40 : TO :I).

pemcetate

0.6s

o-glucosyl peracetate

0*55

o-gl ucosyl peracetate

o-45

3

0-rhamnosyl peracetate

O./t1

3

AC-O o-g

lucosyl

0.36

peracetate

o-glucosyl

.

peracetate

0-glucosyl

peracetate

0.23

2

0.20

3 ’

AC-O

-o-glucosyl

O-methyl

peracetate

0.15

pentosyl peracetate

0.54

OH

5

AC-O 0 W

n Dctniled

tlcscriptions

of thcsc s~ructurcs

will lx publishccl

clscwhcrc.

of compound I3 on MgO is unexpected. Different types of acetylated hydroxyls (A, C, D and G) or a carbonyl group (I?) cause different retardations, and the nature of the glycosidic bond also seems to have a considerable influence on the Rp values. For example, compound I, the peracetate of the secondary glycoside of +ketomyxol (isolated from Phctoncnta boryanzcm, blue-green alga ; structure established by PRANCIS et nl.G from OsciZZatovia Zinzosa) exhibits a relatively high RF value relative to tile tertiary carotenoid glycoside peracetates from myxobacteria. ,I

8

J.

Cl~~ontnlo~~.,

68

(1972)

270-272

NOTES

272 These investigations (Grant 1-X 2Go/2) .

were supported

by the Deutsche

Forschungsgemeinschaft

I-IANS HANS

I
REICI-IENRACH

I S. LIAAEN JENSEN, in T. W. GOODWIN (Editor), Aspects of Tev~maoitl Chcwzistvy ag?d Ijiochmistvy, Acaclcmic Press, London and New York, 1971, p, 223. 2 I-I.KLEINIG, I-k.REICHENIMCI-I AND I-I.ACI-IENBACII,Arch. M~lzrobiol.,74 (1970) 223. 3 I-I.KLETNIG. I-I..REICI-IENBACII. H. ~xI~NE?Acx~ AND J. STADLER, Arch. 11~i/wobiol., 78 (~971)

224. 4 S. I-IEIZTZBERG AND S. LLAAEN 5 G. W. FRANCIS, S.HERTZBERG,

JENSEN, AC& KRNDEIZSEN

Chewt. Scn~d., 23:(19G7) 15. AND S. LIAAEN JENSEN, Pl~yloclw~rrislvy,~) (1970)

G29. G T. HIYAMA, R/I.NEHIMURA AND B. CHANCE, A,vaaZ.13iocluvn., 29 (rgGg) 339. 7 IX REICEIENBACH AND I-I.I~LBINIG,&C~. MikvoDioZ., 76 (1~371) 364. 8 0. B. WEEKS AND Ii.J. GARNER, Arch. Biochenz., 121 (IgG7) 35. CJG. NEAMTU, IX:. VICOL AND C. RODEA, Rev. Roum. Biochi~rn., 5 (1gG8) 223. IO I-I. I?. KAUPMANN, H. I<. MANGOLD AND I<. D. IMUICIIERJBE, J. Lipid Rcs., 72 (1971) 506. IL PI. KLEINIG AND U. LEMPE,RT, J. ChYO9w7~Op.. 53 (1970) 595.

First received J.

ChYOmntO/y.,

December

qth,

68 (1972) 270-272

1971;revised manuscript

received

January

24th,

1972