An aliphatic polyaldehyde from Botryococcus braunii (A race)

Phytochemistry, Vol. 32. No. 4, pp. 875 883, 1993 Printedin Great Britain.

AN ALIPHATIC

003 l-9422/93 $6.00 + 0.00 Q 1993 PergamonPress Ltd

POLYALDEHYDE FROM BOTRYOCOCCUS (A RACE)

PIERRE METZGER,

BRAUNII

YVES POUET, RBMY BISCHOFF and ELIETTE CASADEVALL

Laboratoire. de Chimie Bioorganique et Organique Physique, URA CNRS 1381,ENSCP, 11 rue Pierre et Marie Curie, 75231Paris Cedex 05. France (Received

in revisedform 27 July 1992)

Key Word Index-Botryococcus braunii: Chlorophyceae; alga; A race; aliphatic polyaldehyde; biopolymer; biosynthesis; very long chain a,o-dialdehyde; elongation-w-oxidation mechanism; aldolization.

Abstract-An

aliphatic polyaldehyde, a new type of biopolymer, has been isolated from three strains of the green alga for the three tested strains, from M, 10 000 to 4 000 000, with a peak at 123 000 in the case of the Austin strain. The structure, determined from spectroscopic analysis, ozonolysis and feeding experiments with [l-14C] and [10-14C]oleic acid suggested that the polyaldehyde is synthesized from this acid via an n-C,, diunsaturated r,w-dialdehyde by a condensation-polymerization pathway involving an aldolization-dehydration mechanism. Synthesis of the CJ2 monomer from oleic acid occurs via an elongation-o-oxidation pathway. Isolated from chloroform extracts in a yield of ca 445% of the dry biomass, this biopolymer could be implicated in the synthesis of the polymer which forms the chemically resistant part of the outer walls of the alga. Botryococcus braunii (A race). Size exclusion HPLC showed unimodal distribution

INTRODUCTION

The A race of the green alga Botryococcus braunii has been chemically defined by the production of n-alkadienes (1) and trienes [l]. Lipid analysis indicated also, along with classical neutral lipids such as triacylglycerols, fatty acids and sterols [2,3], the presence of non-classical lipids, some times in high amount, such as aliphatic epoxides, n-alkenylphenols, ether lipids, methyl-branched fatty acids and aldehydes and C52-C64 aldehydes from aldol condensation of fatty aldehydes, the botryals (2) [a, 4-61. All these compounds, sterols excepted, were easily extracted from the dry biomass by a short contact time with hexane, thus suggesting a peripheral localization for these lipids, probably the outer walls which ensure cohesion of the colony; this has been demonstrated for the major part of the hydrocarbons [7]. Then, a subsequent extraction of the biomass with polar solvents such as chloroform-methanol or acetone-methanol mixtures, allowed the recovery of internal lipids located in membranes and in cytoplasmic inclusions, uiz., internal hydrocarbons, sterols, pigments and polar lipids [7-g]. Recently, in continuation of our investigations on B. braunii, we isolated from a collection strain (Austin) a new type of biopolymer, an aliphatic polyaldehyde of high M,, from a chloroform extract performed between the hexane and chloroform-methanol extractions of the dry biomass. In the present report we wish to complement the previously published and rather short description of this polyaldehyde [lo]. Structural determination is based F+lvm 32:&H

on spectroscopic data and oxidative degradations. A sizeexclusion chromatographic method has been used to examine the dispersity of the polymer and to determine M,s. The occurrence of polyaldehydes was also examined in two other strains of the A race, originating from lakes situated in France (Coat ar Herno, Brittany) [3] and in Morocco (Oukaimden, Altas) [ 11. Preliminary incorporation experiments of 14C-labelled oleic acid demonstrated the precursor role played by this fatty acid in polymer biosynthesis [lo]. Unfortunately, the radiotracers used in this preliminary work appeared later as being partially degraded and so the results led us to erroneous conclusions on the biosynthetic scheme. New incubations of the algae with [l-“Cj and [10-14C]oleic acid have now furnished clear evidence for the biosynthetic pathway.

RESULTS AhJ DISCUSSION

A crude polyaldehyde fraction from the dried biomass of the Austin strain was obtained, after a preextraction with hexane, by extraction with chloroform. Addition of an equivalent volume of methanol to the chloroform extract gave a pale green precipitate which was further purified by repeated solution in chloroform and subsequent addition of methanol; two repetitions of the process furnished a white material. After complete elimination of solvents an amorphous elastic material was obtained; it could not be redissolved in hexane, toluene, 875

876

P.

METZGER et al.

x = odd, 11-19

2

E conjugated doubk bond in the major scnes Z conjugated double bond m the minor series m: odd, 15-21 n: even, 14-20

OH ,CHO Me-(CH&

-CH

-CH

-

(C&),7-CH

‘(CH&

\ Me.---(CH&

=C

O\ -CH-CH-

(C&&CH=az

-CH

=CH

-(CH&

---Me

4

I

“\

Fatty acyl

chloroform, methylene dichloride, tetrahydrofuran, methanol, acetone or ethyl acetate. In solvents of low polarity, the dry polymer took only the appearance of a translucent gel. Accordingly, during the purification process, the precipitate was handled impregnated with solvents and the polymer finally kept in chloroform solution at -24”. The yield of the final material was 3.9% of the dry biomass with activally growing cultures (mean value from three experiments of 2 weeks in batch air-lift cultures) and 5.1% when the cultures were in the stationary phase of growth (mean value from three experiments of 5 weeks). Elemental analysis revealed the following composition: C: 83.1%, H: 12.3%, 0: 3.9% and an ash content of 0.7%, fairly constant whatever the culture age. The IR spectrum, recorded from a thin film of polymer obtained by evaporation of a chloroform solution, showed absorption for a CH aldehydic bond at 2710cm-‘, associated with a carbonyl absorption affected by tlunsaturation at 1690 cm- ’ and bands for unsaturations at 3000 and 1640 cm-‘. Strong absorptions at 1465 and 720 cm-‘, together with a very weak band at 1380 cm ml suggested that the polymer contained very long aliphatic chains with little branching. To determine its M, distribution, the polymer was analysed by size-exclusion chromatography, on an Ultrastyragel column with tetrahydrofuran as eluent. Two calibration curves were constructed with polystyrene and polybutadiene standards. Taking into account the fact that separation of polymers by this technique is based upon hydrodynamic volumes in solution, i.e. molecular sizes, and that the biopolymer under study is of an aliphatic nature, exhibiting very little branching, as will be further confirmed, it could be considered that the most accurate results would be obtained from calibration with polybutadienes. The chromatogram of the biopolymer

showed an unimodal distribution of M,s from 10000 to 4000000 with a peak at 123 000 (Table 1). The structure was determined mainly by interpretation of ‘H and “CNMR spectra (Table 2) and comparison with those of botryals (2) [2]. As mentioned earlier, botryals (2) are even carbon numbered C,,-C,, aldehydes arising from aldolization of even C,,-C,, fatty aldehydes. The subsequent dehydration of the aldol leads to a carbonyl conjugated unsaturation, showing an E configuration in the major series (cis-alkenyl chains) while a Z-configuration was observed in the minor one (trans-alkenyl chains). The ‘HNMR spectrum of the biopolymer in CDCI, solution showed the presence of the -CH,-CH=C(CHOwH,pattern of the two botryals series; integration of the two singlets of aldehydic protons at 610.10 (Z configuration) and 9.34 (E configuration) indicated a 12: 88 ratio, in favour of the latter. This was confirmed by the relative intensities of the signals on one hand of the vinylic protons of the conjugated double bonds at 66.48 (t, Z configuration) and 6.45 (t, E conhguration) and on the other hand of the methylenic protons tl to the vinylic CH, at 62.55 (dr. Z configuration) and 2.34 (dt, E configuration). The ‘H NMR spectrum also showed partially overlapped signals for methylenic protons a to the quaternary carbon at 62.2-2.12 (E and Z configurations) and of methylenic protons /I to the vinylic CH at 6 1.49, signals for two isolated double bonds with vinylic CH at 65.34 and allylic protons at 62.01 and an intense and broad signal of all the other methylenic protons at 61.26. Finally, in the region of the terminal methyl groups, a very weak triplet was observed at 60.88: integration indicated that less than one methyl group was present per one hundred formyl groups. The 13CNMR spectrum confirmed the presence of E and Z conformers in the polymer (Table 2) with a pre-

Polyaldehyde from Botryococcus braunii

dominance of the former. By comparison with the spectrum of botryals, it must be noted, however, that the vinylic CH of the conjugated double bond was split into two peaks, both in the E (6 at 155.8 and 155.5) and Z (6 at 149.7 and 149.4) configurations. Although no similar pattern was observed either for the neighbouring carbons or in the ‘H domain, this finding could be interpreted as a result of the existence of gauche and truns conformers producing two chemical shift environments. The “C NMR spectrum also displayed signals at 6 130.0 and 129.7 for non-conjugated double bonds, of Z stereochemistries as clearly indicated by the resonances of the allylic carbons at 627.3 and 27.2, and numerous peaks in the 6 range 29.0-29.7 for methylenic carbons of aliphatic chains. A remaining signal at 624.0 was assigned to the methylene carbon CIto the carbon bearing the formyl group in the E configuration as previously observed in the corresponding botryals. No clear resonance ascribTable 1. Size exclusion chromatography

877

able to carbons of aliphatic chain ends could be observed. Ozonolysis of the polymer followed by oxidative cleavage of the polyozonide afforded principally three ndiacids, Cs, C, and C,,, each corresponding to ca 30% of the whole and several minor acids. Among them were identified nonanoic acid (0.7%) and n-C, and n-C,, diacids (1.5% and 2.3%, respectively). Due to the drastic conditions it is probable that the keto-diacid resulting from cleavage of the fragment bearing the formyl group, =C(CHOHCH,),-CH= , was decarboxylated into a diacid (Fig. 1) as observed in the ozonolysis of compounds having an cc&unsaturated carbonyl group [ll], and observed for botryals [2]. At this point of the work the question arises as to whether the polymer is formed by a repetitive condensation of a monomer unit or there exists a random distribution of C,, C, and C,, subunits bound to each other by carbon-carbon double bonds, one subunit bearing a

analysis of polyaldehydes from three strains of

B. bramii (A race)

Calibration with polybutadiene standards

Calibration with polystyrene standards ~-

Strain origin

M, peak

M, range

M, peak

M, range

Austin-Texas (Collection) Coat ar Hemo (France) Oukaimden (Morocco)

123 000 139ooo 228000

lOooo-4oooooo Mooo-4oooooo 25000-3500000

230000 240000 430000

1800%8oooooo 3800&8000000 5oooc-7 3ooooo

Table 2. Selected ‘H and 13CNMR data of polyaldehyde CH

CH,

CH,

1.49; in

2.34; t, d (7.2, 7.4) ca 29

6.45; t (7.4) 155.8 155.5

2.55; t, d (7.1, 7.4) ca 29

6.48; t (7.4) 149.7 149.4

C

(CHO)

CH,

E configuration (major)

6 ‘H, multiplicity (J in Hz) 61%

2 configuration (minor) 6 ‘H; multiplicity (J in Hz) 613C

ca 29

1.49; m ca 29

9.34; s 143.8

140.1

2.12-2.22*

195.6

24.0

10.10; s

2.12-2.22*

191.2

co 29

*Overlapping resonances.

Fig. 1. Proposed structure for polyaldehyde 3, and oxidative degradation (+).

878

P.

METZGER et al.

formyl group. The common structural features existing between the polymer and botryals suggested that the a$unsaturated aldehyde function would probably originate from an aldol condensation of two aldehydes followed by dehydration. In botryal synthesis, the condensation of two very long-chain fatty aldehydes of even carbon number exhibiting an w9 unsaturation leads to the formation in each molecular species of an odd and an even alkyl moiety on both sides of the central -CI+C(CHO)unit. On the basis of this model and assuming that the polymer arises from successive aldol condensation of a diunsaturated C,, dialdehyde (Fig. 2), the C, and C, diacids recovered from ozonolysis were indicative of the presence of seven and six methylene units, respectively, on the left and on the right side of the unsaturation conjugated to the formyl group (A): =CH-CH&-CH

= C(CHOHCH,),-CH

=

(A)

Joining the partial structure, A, to the C,, moiety, B =CH-(CH&-CH

=

(B) Occurrence of polyaldehydes in other strains of B. braunii

furnished the repeating unit C for the polymer CHO =d \ (CH,),-CH=CH-(CH,),2-CH

=CH
MW--_(cH~),-cH=cH--_(cH~-co~ I elongation, leowiatmn I

desaturaticm. acidreduction

I OHC-_(CH&---CH

-CH=CH

=CH-(CH&

-(c!H&--CHO

then,aldcdisation of two C3* monomen

c32

~‘_......‘...........~......,

3

m”ILo-

?

TCI-I-_(CH&T-CHO CHO-CH&CH&-CHT I______...________..........~ I

aidolisation CHO ?

2 ~CH-(CH~-CH-CH-(~)6-CH=

R---+X&

=C

/HO ‘(‘W,

I

-R

condensation polymaimtion u (C,, m)

I PolYaldehyde

Fig.

B. braunii.

Preliminary analysis performed on strains of the B and L races of B. braunii (characterized by the production of botryococcenes for the former and of lycopadiene for the latter [lo]) have shown that polymers could be also extracted from these algae. Further investigations, complicated by polynomal distributions as deduced from sizeexclusion chromatography analyses, will be necessary to establish their structures. Biosynthetic pathway

dehydration

-CH

The occurrence of aliphatic polyaldehydes has been examined in two other strains of the A race of B. braunii. The French (Coat ar Herno) and Moroccan (Oukaimden) isolates exhibited a lower content of chloroform extractable polymer: 2.4% and 2.1% of the dry biomass, respectively. They showed IR spectra identical to that of the polyaldehyde from the Austin strain. By size-exclusion chromatography analysis (Table l), similar M, distributions were found for the Coat ar Herno and Austin strains, with M, peaks of 139000 and 123 000, respectively; the mass range for the Morrocan strain appeared more limited and a higher M, peak of 228 000 was observed. These results suggest that aliphatic polyaldehydes may be common polymers of the A race of

<

I

OH

I

Moreover, the occurrence of nonanoic acid among the acids resulting from the oxidative degradation suggested that the polymerization would be ended by the condensation with a monoaldehyde exhibiting an 09 unsaturation, such as a C,, monoaldehyde or the C,, aldehyde derived from oleic acid. Further feeding experiments with 14Clabelled oleic acid should give some support to this assumption. The proposed structure 3 (Fig. 1) for the polyaldehyde is in good agreement with all the above spectroscopic and chemical data if one ignores the formation of II-C, and nCl3 diacids upon oxidative degradation. However, the observation of increasing amounts of C7 and C, 3 diacids in the acid mixture released by polyozonide cleavage in hydrogen peroxide-formic acid on extended heating, together with the appearance under these conditions of nClo, n-C, i and n-C,, diacids, could signify that the minor diacids were most likely formed by subsequent oxidation of the native Cs, C, and C,, diacids.

2. Proposed biosynthetic scheme of polyaldehyde from oleic acid via a C,, dialdehyde monomer.

Incubations with [ 1-14C] and [ 10-‘4C]oleic acid. In the A race of B. braunii, oleic acid which represents more than 80% of the fatty acids [9], is the precursor of a number of lipids including hydrocarbons [ 12, 131. Therefore, it was interesting to examine polyaldehyde biosynthesis by feeding algae with radiolabelled oleic acid. Starting from oleic acid four routes could be envisaged leading to a C,, cr,o-dialdehyde monomer, each route involving an elongation process, probably by incorporation of CZ units derived from malonate, and an w oxidation of the terminal methyl. o-Oxidation of fatty acids usually occurs in higher plants [14, 151 and in microorganisms,

Polyaldehyde from Botryococcus braunii particularly

in those capable

of use alkanes

as the sole

source of carbon

[16]. In microorganisms such as Pseudomonas putida long-chain dicarboxylic acids result from the o-hydroxylation of fatty acids to o-hydroxy fatty acids further oxidized to a.ldehydic fatty acids and to . . cr,o-dlaads. Moreover, C,, and C,, o-aldehydic fatty acids have been found in the monomers released by hydrolysis of plant cutin, the insoluble polymeric structural component of cuticles. The four routes for C,, dialdehyde synthesis may be summarized as follows: (a) oleic acid is elongated before w-oxidation; (b) ooxidation of oleic acid takes place prior to elongation; (c) there is no preferential order between elongation and o-oxidation and (d) oleic acid is degraded into acetate units prior to incorporation. Theoretically, by feeding the algae with [l-“C] and [10-14C]oleic acid, followed by degradation of the poly-

(a)

aldehyde by ozonolysis and determining the radioactivity of the acids, it should be possible to distinguish between two of these pathways, namely (a) and (d). Indeed, following pathway (a) radioactivity must be found in specific acids as shown in Fig. 3: with (d) a randomization of radioactivity can be expected, while pathways (b) and (c) are indistinguishable from each other. As shown in Fig. 4, following pathway (b), 14C9 and 14C1, diacids would be formed from [1-14C]oleic acid, 14Cg, 14C, and 14Cl, diacids from [10-‘4C]oleic acid while pathway (c) [a mixture of (a) and (b)] would generate similar distributions of label in the diacids. Two experiments were conducted in parallel with algae originating from the same preculture; they were incubated with tracers in air-lift systems. In both cases, hydrocarbons, botryals and the polyaldehyde were isolated and purified as specified in the Experimental; the

Me-_(CIi&yCH=CH-(CkI&+H

I

elcmgatial + deso-oxidation

-cm

ox-(cl-l&

I

=cH-_(cH&~cH, -_(cH,)‘-cH=cH-_(M&/-C0$3

scidrrduction

“C32dialdchyde

I

condulsatiollpolymcrizaton

t4c,PlY~dehv&

I %,’

@)

4-w?&

Hc4H

diacii

Me-_(cH~~cH=cH-_(~--~ I

elmgstion + des-shmtic4l ce-oxidatii

~-(~~,~cH=cH-_(~,,--cH=~-_(~~--co~ I

acid redttction

‘%2 BY&

1condcnsntMnpdymeritation C’c1 PolYpMshvde I “$

and

4 -%A H’=W

‘“cgdiscids

Fig. 3. Localization of label in an elongation-o-oxidation

819

mechanism leading from oleic acid to polyaldehyde via the C,, monomer.

880

P. METZGER et al.

(a)

Me-_(CI$),

-CH

=CH

14 -(CH&--C%H

w-oxidation I elongatm

(two possible dimtrons). + desaturation

I OHC-(CH&

-CH

=CH

-(CH,$%H,-(CH2),

-CH=CH-(CH,),

-C02H

+ OHC-(CH,),--cH=cH-(CW~,,--cH=cH-(CH2),~~0~ acid reducuon

I

14C32 dAdehyde

condensauon polymerization

I

c’“C, polysldehyde 0, -H&

HCQH

I 14C,, and ‘“Cg diacids

@)

Me-

(CH& -%!H

Cg diacid is u&be3ed due to the toss of the 14CH0 group by heating the poiyomnide in HZ% -H-H (pig. 1)

=CH

-(CH&--CO,H

o-oxidation i clongatm

(two possible directions), + desatumtion

i OHC-(CH&kH

=CH-(CH2)12-CH=CH-(CH2),--C02H

+ 0HC-(CH,)7--CH=CH-(CH2),2-‘4~H’=CH-(~~-_C4H

I

acid reduction

14C, dialdebyde

I

condensatmn polymerizatm

t4C) polyaldcbyde

4 -H?Q, HCO$ i “k ,4, 14Csand 14C9dmcrds

Fig. 4. Localization of label in an o-oxidation-elongation mechanism leading from oleic acid to polyaldehyde via the C,, monomer. results of the incorporations are listed in Table 3. The incorporation of radioactivity into hydrocarbons and botryals were relatively high, conforming to that previously reported for the former [ 17,l S]. The efficiency of r4C incorporation into hydrocarbons and botryals was not significantly affected by tocation, at C-l or C-10, of the label; similar variations have been previously observed in the labelling of B. braunii hydrocarbons and it was shown that no degradation of 14C oleic acid into acetate units occurred before incorporation. By comparison incorporation into the polyaIdehyde were low; however, this statement must be moderated when specifk activities, the only objective measure of the efficiency of

incorporation, are considered. Specific activities of the polyaldehyde were cu half those of hydrocarbons. The lesser efficiency of [10-14CJo1eic acid incorporation into the polymer when compared with Cl-14CJoleic acid cannot be regarded as potential information on the biosynthetic pathway. This phenomenon could be explained by the nonsynchronism of growth in the two experiments, as suggested by a higher biomass concentration in the culture with [l- 14C]oleic acid; furthermore an inverse result was observed in a duplicate experiment. Ozonolysis was carried out on the 14C-labelled polyaldehyde and on the r4C-labelled botryals for comparison. Radio-GC analysis of acids from polymer oxida-

881

Polyaldehyde from Botryococcus braunii Table 3. Feeding experiments with [l-14C] and [10-‘4C]oleic acid: incorporation into hydrocarbons, botryals and polyaldehyde _____

Hydrocarbons Botryals (2) Polyaldehyde (3)

[1-‘4C]Oleic acid .-____

-

[10-‘4C]01eic acid

% Initial radioactivity

Specific activities (dpm mg- 1)

% Initial radioactivity

Specific activities (dpm mg-‘)

10.1 11.0 (Z:18; E:82)* 1.5

75 630 84 530

9.0 11.6 (z:15; E:85)* 1.1

12 700 94 630

43 170

32 280

*Relative distribution in the Z and E series.

Table 4. Distribution of radioactivity in acids resulting from polyaldehyde ozonolysis (relative %, from radio-GC analysis)

Acids

Incubation with [l-W] oleic acid

Incubation with [lo-‘V] oleic acid

mono-C, di-C, di-Cs di-C d&C: 3

traces 1.5 1.5 6

4.0 46.5 44.0 -

di-Cl4 Unidentified

83.0 8.0

1.5 4.0

tion (Table 4) showed that only a limited randomization of the radioactivity had occurred; according to the recovery of 83% of the radioactivity in n-Cl4 diacid after feeding with [1-14C]oleic acid and of 90% in n-C, and C, diacids (almost equally distributed) after feeding with [lo14C]oleic acid it could be assumed that oleic acid was mostly incorporated oia an elongation-w-oxidation pathway (Fig. 3). With botryals, radio-GC analysis indicated no randomization, all the radioactivity was’ recovered in the Ci6-& diacids when algae were incubated with [1-14C]oleic acid and in nonanoic acid after feeding with the [10-14C] tracer. Thus, as previously observed in the hydrocarbon biosynthesis from oleic acid, no degradation into acetate units occurred prior to elongation likely via fatty acyl derivatives, to very long chain fatty aldehydes, the proximate precursors of botryals. Incubation with [U-14C] botryals. To examine a possible involvement of botryal metabolism in the formation of the polyaldehyde, a feeding experiment with 14Clabelled botryals was attempted. [U-‘4Clbotryals were obtained in parallel work from a B. braunii strain cultivated over a three week period in a medium containing [1-14C]acetate. Due to the problem of dispersion of such heavy lipids in the culture medium, the technique used for the incubation consisted of spraying a wet cell paste of algae, placed on agarized medium in a Petri dish, with an hexane solution of [U-14CJbotryals. Following this technique which has given interesting results in the study of other biosynthetic pathways in B. braunii [6], a close

Table 5. Feeding experiment with [U-14C]botryals % Initial radioactivity Polyaldehyde (3) Alkenyl-O-botryalyl ethers (4)

Specific activity @Pm mg-‘)

0.008

8

0.240

115

contact can be envisaged between the oily colonies and the fatty precursor. After incubation for 13 days, only

0.008% of the radioactivity was recovered in the purified polyaldehyde (Table 5), but 0.24% was found in a mixture of ether lipids derived from botryals and alkadienes, the alkenyl-0-botryalyl ethers (4), which form, with some other related compounds, the novel class of ether lipids (botryococcoid ethers) recently identified in B. braunii [6]. Although some difficulties for reaching the site of the polyaldehyde synthesis could not be excluded, a comparison of the specific activities between polyaldehyde and ether lipid (4), showed that exogeneous botryals were preferably involved, probably via metabolism into an epoxide derivative [6], in the biosynthesis of alkenyl-obotryalyl ethers (4) than in that of the polyaldehyde. CONCLUSIONS Both structural considerations and results from feeding experiments with [1-14Cj and [10-14C]oleic acidand [U14C]botryals give support to a biosynthetic relationship between aldehydes in B. braunii as presented in Fig. 5,

with oleic acid as a direct precursor for each type of compound. Very long-chain fatty acyl derivatives contribute in B. braunii to the formation of complex lipids constituted by several homologous series; in this respect polyaldehyde synthesis seems to be an exception, only a c32 cr,o-dialdehyde would be implicated in the condensation-polymerization process leading to a high M, homopolymer. What could be the function of such a polymer in B. braunii! No definitive answer can be given in the present paper; however, results from work in progress in our laboratory suggest that the polyaldehyde could be implicated in the formation of an insoluble and chemically

P. METZGER et al.

882 very long cllam rally

acid derivatives

(o-9 unsaturation)

decarboXYlatiOll alkadienes* 1 condensationof die

elongation Oleic acid w _

delived epxides

/ -

acid reduction

Very long chain fatty akiehydes (o-9 unsaulraton)

Alkfflyl

- 0 - houydyl

elks4

aldokation * dehydration

bovyds 2

o-oxidation

n-C32a.o

\

\

c i$XZive (o-9 and o-23 .nsawations)“Y’ reduction

diidehyde

CondcIlWtioIl

polymerization

F’olyaidehyde 3

Fig. 5. Proposed metabolism of aldehydes and alkadienes from oleic acid in B. braunii (*alkadienes are also involved m the metabolism of some other ether lipids [6, 131).

biopolymer of outer walls termed PRB [19, 201 perhaps via reticulation or (and) via further condensation with botryococcoid ethers. PRB from the A race is a highly aliphatic insoluble polymer whose structure is based on a network of very long polymethylenic chains, probably cross-linked by ether bridges. A survey of the literature shows that B. bra&i polyaldehyde has no equivalent in the plant kingdom, and thus it could be considered as species specific. However, it must be noticed that numerous studies on algal or plant lipids are often performed after either chloroform methanol extraction or saponification of the biomass, both methods being unsuitable for the extraction of aliphatic polyaldehydes. From a general chemical point of view, the polymerization of aldehydes in B. braunii is fundamentally different from that observed in the synthesis of polymer from either lower or higher aldehydes, dialdehydes or also GI$unsaturated aldehydes; with the exception of acrolein, aldehyde polymerization generally furnishes polyketals resistant

WI. EXPERIMENTAL General. IR film on KBr cell. NMR, CDCl,, TMS as int. standard; ‘H: 250 MHz, r3C: 62.5 MHz. CHCl, was stabilized with 0.6% EtOH. [l-r4C]oleic acid and [lo“C]oleic acid (1.66-2.22 GBq mmoll’) were purchased from Dositek, Orsay, France. The strains investigated originated from the culture Collection at Austin, Texas (UTEX 572), from Coat ar Herno lake in Brittany (France) [3] and from a small pool in Oukaimden Valley in Atlas (Morocco) Cl]; they were cultured under air-lift conditions (with air-enriched by 1% CO,) as previously described Cl]. Extraction and isolation of polyaldehyde. Typically a 1.4 1, 21-day-old culture (Austin strain) was filtered through a 10 pm Monyl cloth, the concentrate frozen in liquid N, and lyophilized. The dry biomass (5.2 g) was successively extracted, at room temp., twice with 200 ml hexane for 1 hr. once with 500 ml of CHCl, for 18 hr. The CHCI, extract was then coned under red. pres. to a residual vol. of ca 50 ml. Addition of an equivalent vol. of MeOH afforded an elastic, pale green ppt.; this was

into 50 ml CHCl, and redissolved by agitation. Repetition of the dissolution--precipitation process twice gave a white gum which, was finally dissolved in 50ml CHCl, and kept at -24” for further analyses. The polymer yield, 0.265 g (5.1% of dry biomass) was estimated by weighing an aliquot of residue obtained after evapn of solvent. Size exclusion HPLC. Analyses were performed on an instrument fitted with a differential refractometer, thermostated at 35”. The column was Ultrastyragel (300 x 7.8 mm), thermostated at 20”, linear functional range 500-8.106, THF as mobil phase, 1 mlmin-’ flow rate. Calibrations were performed with standards of polystyrenes from Polymer Laboratories and of polybutadienes from Scientific Polymer Products. Precision on retention vols was estimated to be +0.04 ml. Ozonolysis. A CHCl, soln (3 ml) of polymer (16 mg) added with few drops of MeOH was ozonized at - 15” for 5 min (30 mg 0, l- ’ air; 40 1hr- ‘). Excess 0, and CHCl, were removed at room temp. in a stream of N, and the ozonide decomposed by refluxing with 30% H,O, (0.5 ml) and HCO,H (1 ml) for 15 min. The acid mixt. was dild with H,O and continuously extracted with Et,0 for 24 hr. After evapn of solvent, acids were esterified with CH,N, -Et,O. Esters were determined by GC-MS (SE 52 capillary column, temp. prog. from 140 to 260” at 5” min-‘) and comparison with authentic standards. Incubations with [l-‘“Cl and [10-14C]oleic acid. Two cultures of 500ml (Austin strain), each initiated with a same amount of inoculum, were fed with 2MBq [l“C]oleic acid and 2MBq [10-‘4C]oleic acid (as free fatty acids dispersed with 0.1 ml Tween 20). After 2 weeks growth in air-lift batch systems, the algae were taken for analysis and extracted with hexane and CHCl, as described above. Hydrocarbons and botryals were isolated from the combined hexane extracts by CC and TLC fractionations over silica gel [Z] and the polyaldehyde purified from CHCl, exts as previously mentioned. The radioactivity was determined by liquid scintillation counting using 0.4% butyl-PBD in toluene. The “C-labelled botryals and polyaldehyde were submitted to ozonolysis and the resulting acids analysed as Me esters by radioGC, using a SE 30 column (temp. prog. 120 to 260” at 4” min-I). quickly transferred

Polyaldehyde from Botryococcus braunii Incubation with [U-‘4C] botryals. [U-14C]Botryals were obtained in a parallel work from the Coat ar Herno strain fed with [l-14C]NaOAc [6]. Incubation of the Austin strain was performed as previously described [6], viz. a wet cell paste of algae, placed in a Petri dish on agarized medium was spread with 2 ml of a hexane solution of [U-14C]botryals (173.104 dpm, sp. act. 157 400 dpm mg- ‘). After a 13 day period under a lightdark cycle (14 hr illumination per day, 100 PE mm2 set- ‘) at room temp., the contents of the Petri dishes were freeze-dried. The dry materials were subsequently extd with hexane and then with CHCI,. Fractionation of the hexane ext by silica gel CC and analysis of the fr. containing the alkenyl-0-botryalyl ethers by radio-TLC was as recently described [6]. The polyaldehyde from the CHCI, extract was isolated, purified and examined for radioactivity by scintillation counting.

8. Grung, M., Metzger, P. and Liaaen-Jensen,

883

S. (1989)

Biochem. Syst. Ecol. 17, 263.

9. Metzger, P., Allard, B., Casadevall. E., Berkaloff, C. and Couth, A. (1990) J. Phycol. 26, 258. 10. Metzger, P., Largeau, C. and Casadevall, E. (1991) in Progress in the Chemistry of Organic Natural Products Vol. 57 (Herz, W., Grisebach, H., Kirby, G. W.

and Tamm, C., eds.), p.1. Springer Wien. 11. Belew, J. S. (1969) in Oxidation Vol. 1 (Augustine, R. L., ed.), p. 259. Marcel Dekker, New York. 12. Ternplier, J., Largeau, C. and Casadevall, E. (1984) Phytochemistry 23, 1017.

13. Ternplier, J., Diesendorf, C., Largeau, C. and Casadevall, E. (1992) Phytochemistry 31, 113. 14. Kolattukudy, P. E. (1972) Biochem. Biophys. Res. Comm. 49, 1040. 15. Kolattukudy, P. E. (1981) Ann. Rev. Plant Physiol32, 539.

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