Biochirnica et Biophysica Acta, 1086(1991) 317-325

317

© 1991ElsevierScience PublishersB.V. All rights reserved0005-2760/91/$03.50

BBALIP 53757

Formation of ketols from linolenic acid 13-hydroperoxide via allene oxide. Evidence for two distinct mechanisms

of allene oxide hydrolysis A . N . G r e c h k i n 1, R . A . K u r a m s h i n i, E . Y . S a f o n o v a 1, S.K. L a t ~ 0 o v 2 and A.V. llyasov 2 I Institute of Biology. USSR Academy of Sciences. Kazan ¢,U.S.S.R.) and 2 Aebuzot"s blstitute of Organic and Physical Chemistry, USSR Academy of Sciences, Kazan I U.S.S.R )

(Received 14January 15)91) (Revisedmanuscriptreceived 17June 1991) Key words: Hydroperoxidedehydrase;a-Linolenicacid 13-hydroperoxide;Alleneoxide,hydrolysismechanism: I I-Hydroxy-12-oxo-9(ZLI5(Z)-octadccadienoicacid. novelketol; (Flax seed) Incubations of [ l . t 4 C ] 1 3 - h y d r a p e r o x ~ ' - 9 ( Z ) , l I ( E ) , l S ( Z ) - o c t a d e c a t r i e n o i c acid (13-HPOT) with hydroperoxide de-. hydrase preparations from flax seeds lead to the formation of a novel ketol 2 along with the previously known 12-oxo-13-hydroxy-9(Z),l$(Z)-octadecadienoic (12,13-a-ketol) and 9-hydroxy-12-oxo-10(E),lS(Z)-octadecudienoic (y-ketol) acids. Compound 2 was identified as ll.hydroxy-12-oxo-9(Z),lS(Z)-octadecadienole acid (ll,12-a-ketol) in aceardance with the data of ultraviolet, mass (chemical ionization and electron impact) and t H-NMR spectra. During long.term (30 mlil) |~c~batlons the yields of y-ketol and 11,12-a-ketol increased markedly and the yield of 12,13.a-ketol decreased in response to the pH change from basic (pH 7.4) to acidic (pH $.8) conditions. Short.term (IS s) incubations of 13-HPOT with hydroperoxide dehydrase, terminated by HCI fixation, led to the formation of y-ketol and ketol 2. A similar incubation, followed by NaOH f'Lxatlon, afforded only 12,1~a-ketol. The trapping of allene oxide (a primary product of hydroperoxide dehydrase) with pure methanol gives only compound 4 (12,13-a-ketol methyl ether). Products S (y-ketol methyl ether) and 6 (ll,12-~-ketol n~thyl ether) were formed along with 4 as a result of trapping with acidified methanol. The results obtained indicate that: (a) the formation of 12,13.a-ketol is base-dependent; (b) the formation of y-ketol and ketol 2 is acid-dependent. Two distinct mechanisms of allene oxide hydrolysis are proposed: (I) nucleophilic (S~2 or SN1, O H - is an attacking group) substitution, resulting in formation of 12,13-o-ketol; (2) electrnphilie (S~-Iike) reaction initiated by protonatlon of oxirane, affording .),-ketol and ll,17.-a-ketoL

Introduction The enzyme hydroperoxide isomerase was first found in flax seeds by Zimmerman [I]. The function of this actiw enzyme, widely distributed in the plant kingdom, was considered until recently as the isomerization of

Abbreviations: 13-HPOT. 13.hydroperoxy-°,(ZLIl{ELI5(Z)-oc tadecatrienoic acid: 13-HPOD, 13-hydroperoxy-~Z).ll(Eboctadecadienoic acid: 12,13.,a-ketoL12-oxo-13-hydroxy-~ZLI5(Z)-octadecadienoic acid: y-kctol, 9-hydrox~j-12-oxo-I~ELI5(Z)-octadecadienoic acid; 11.12-a-ketoL ll.hydro~-12-oxo-9(Z),15(Z)-octadecadienoic acid. Correspondence: A.N. Grechkin, Institute of Biology. USSR Academyof Sciences,P.O. Box 30, Kazan,420503°U.S.S.R.

fatty acid hydroperoxides, e.g., of 13-hydroperoxy9(Z),ll(E)-octadecadienoic (13-HPOD) or 13-hydroperoxy-9(Z),i I ( E ) , 1 5 ( Z ) - o c t a d e c a t r i e n o i c (13-HPOT) acid into the corresponding a-ketol and ~-ketol structures [2-4]. In particular, 13-HPOT is transformed into 12-oxo- 13-hydroxy-9(Z), 15( Z )-octadecadienoic (12,13a-ketol) and 9-hydro~-12-oxo-10(E),15(Z)-octadecadienoic (~/-ketol) acids [4]. However, enzymes from flax [5] and corn [6] were recently shown to catalyze not the isomerization, but the dehydration of hydroperoxide precursor, in this way, 13-HPOD and 13-HPOT are transformed into the new metastable metabolites, respectively, 12,13-epoxy-9(Z),ll-octadecadienoic and 1 2 , 1 3 . e p o x y - 9 ( Z ) , l l , 1 5 ( Z ) - o c t a d e c a t r i e n o i c acids (allene oxides) [5,6]. It was shown clearly that a- and 3,-ketols are derived from the spontaneous hydrolysis of

318

Materials and Methods

HPOT solution without additional purification. HPLC analysis has revealed that [I-14C]I3-HPOT was a predominant component in this preparation. Contaminations were the trace amounts of unreacted linolenate and secondary products. For analytical experiments, 74 KBq of [1-14C]linolenate (10.3 MBq/mmol) was incubated with 12 U of lipoxygenase under identical conditions in 10 ml of the same buffer. Products were extracted and [1-t4C]I3HPOT was purified by reverse-phase HPLC as described below. [1- ~4C]I3-HPOD (10.3 MBq/mmol) was prepared from [1-14C]linoleate and purified in the same manner.

Materials

Semi-preparatice incubations

[1-14C]Linolenic acid (2.1 GBq/mmol) and [lJ4C]linoleic acid 12.1 GBq/mmol) were obtained from Amersham International (Amersham, U.K.). Unlabelled linolenic acid and soybean lipoxygenase (6 /~mol/min per rag) were purchased from Fluka Chemic (Buchs, Switzerland). 3-Dodecyldimethylammonium-lpropane sulfonate was obtained from Serva Feinbioehemica (Heidelberg, Germany). Analytical grade solvents and inorganic chemicals as well as sodium borohydride were obtained from Reakhim (Moscow, U.S.S.R.). Before use for HPLC, solvents were distilled in glass and filtered through 0.45/xm Nylon 66 membrane filters (Schleicher & Schuell, Dassel, Germany).

For semi-preparative preparation of compounds 1 and 2, incubations were done in the following way. Fresh enzyme solutions (in 20 ml of buffer) were combined with 74 KBq of [I-t4C]I3-HPOT 11.03 MBq/mmol) preparation (in the same volume of buffer) and incubated (in total volume of 40 ml) at 23°C for 40 rain.

allene oxide [5,6]. Thus, the true function of enzyme is not the isomerization but the dehydration of hydroperoxide. Studying the metabolism of linolenic acid and its 13-hydroperoxide by hydroperoxide dehydrases from wheat seedlings and flax seeds, we have observed distinct pH regulation of a- and 'y-ketol formation. Moreover, we have isolated a novel ketol. These results as well as the proposal about two distinct mechanisms of allene oxide hydrolysis are described in the present paper.

Enzyme preparation Flax seeds (10 g) were ground in an electric coffee mill. The resulting fine powder was extracted twice at -10°C with 250 ml of acetone. The pellet was dried under vacuum and stored at - 20°C. The enzyme solution was prepared from acetone powder just before use. The acetone powder (1 g) was extracted with 10 ml of 1 mM 3-dodecyldimethylammonium-l-propane sulfonate solution in 25 mM Tris-HCI buffer (pH 7.4) (or in M E S / N a O H buffer (pH 5.8), as indicated) at 0°C for 1 h. After the precipitation of pellet by centrifugation at 51100 x g for 2 rain, the resultir.g supernatant was decanted and used as an enzyme preparation for incubation. For short-term incubations the 'concentrated' enzyme solution was prepared. For this purpose, 2 g of seed acetone powder were extracted with 7 ml of 25 mM Tris-HCI buffer (pH 7.4) at 0°C for I h.

Substrate preparations For semi-preparative purposes [I-14C]I3(S)-HPOT (1.03 MBq/mmol) was obtained by incubation of 74 KBq of [1-14C]linolenate (1.03 MBq/mmol) with soybean lipoxygenase (5 rag) for 15 rain in 20 ml of 25 mM Tris-HCl (pH 8.0) under pure oxygen. The reaction mixture was acidified to pH 5.8 with dry MES buffer and used immediately after preparation as [1-14C]13-

Long-term incubations To study the formation of ketols under acidic and alkaline pH values, purified [1-14C]13-HPOT (74 KBq, 10.3 MBq/mmol) preparation in 100 p,I of ethanol was added to enzyme solution in 10 ml of either 25 mM Tris-HCI buffer (pH 7.41, or 25 mM M E S / N a O H buffer (pH 5.8). Incubation was performed for 30 min.

Short-term incubations Incubations were started with the fast injection of 74 KBq of purified [1-14C]I3-HPOT (20.6 MBq/mmol) in 50 /~l of ethanol into 7 ml of cool (0°C) enzyme solution. The reaction was allowed to proceed at 0°C under the extensive mixing within 15 s. Then the reaction mixture was divided into two equal parts. Both parts were simultaneously fixed, one with 0.5 ml of 1 M HCI and the second with 0.5 ml of 1 M NaOH solution (final pH values were about 2.0 and 12.0, respectively). Before extraction, both mixtures were allowed to stand for 5 min at 0°C.

Methanol tropt,ing experiments 74 KBq o| purified [I-14C]I3-HPOD was dissolved in 50 p.I of ethanol. This solution was mixed with 0.5 ml of 25 mM Tris-HCI buffer (pH 7.4). This cold (0°C) substrate solution was rapidly injected into 3.5 ml of enzyme extract (in the same buffer), maintained at 0°C under rapid stirring. After the incubation within 10 s, 75 nd of pure methanol or methanol/acetic acid 100:1 (v/v) mixture were added and the reaction was allowed to proceed for 30 min at ambient temperature. Then the protein pellet was precipitated by centrifugation at 10000 × g. The supernatant was decanted and concen-

319 tra~ed 4-5-fold using a rotatory evaporator. The remainder was extracted three times with equal volumes of diethyl ether.

Incubations with the homogenate of wheat seedlings Wheat seedlings (10 g) were homogenized in 60 ml of 25 mM Tris-HCl (pH 7.4), or in the same volume of 25 mM M E S / N a O H (pH 5.8). The homogenate was filtered through two layers of cheesecloth. The incubation was started with the addition of 74 KBq ef [ltaC]linolenic acid (5.1 MBq/mmol) in 50/zl of ethanol to 15 ml of homogenate filtrate. The incubation was performed within 30 min at 23°C under constant bubbling of oxygen. Reaction was terminated and products were extracted as described below.

Extraction and HPLC separation of products After acidification of the reaction mixture by acetic acid to pH 5.0, the products were extracted 3-fold by equal volumes of ethyl formate (acidic reaction mixtures were extracted directly). Radio-HPLC analyses were done in general as described previously [7,8]. At the first stage, samples were separated by reversedphase radio-HPLC on the Partisil 5 0 D S - 3 (4.6 × 250 ram, Whatman Ltd, Maidstone, U.K.) or Adsorbosphere HS C~s 5 p,m (4.6 x 230 ~,m, Alltech/Applicd Science, State College, PA, U.S.A.) columns using a

1.O

linear gradient from 60:40:0.1 to 96:4:0.1 (v/v) in a solvent mixture of methanol/water/acetic acid for 55 miP, then for a further 25 rain in isocratic conditions with a flow rate 0.4 ml/min (system A). Radioactivity in the eluate was detected by a HPLC radiomonitor model 171 (Beckman instruments, Fullerton, CA, U.S.A.). The peak of a-ketol was collected and purified once more under the same conditions. Finally, the component~ of a-ketnl fraction were separated by normal phase HPLC on two successively connected Sepaton SGX (150x 3.2 ram, 5 /~m)columns, under isocratic conditions, with a solvent mixture hexanc/ isopropanol/acetic acid (99: 1:0.1, v/v), with a flow rate 0.8 ml/min (system B). The products of methanol trapping were purified using the same normal phase column under i ~ r a t i c elution with ,solvent mixture hcxane/isopropanol/acetic acid (99.5 : 0.5 : 0.1, v/v), flow rate 0.8 ml/min (system C).

Spectral studies Ultraviolet spectra (190-370 nm) were recorded on line during HPLC analysis using RSD 2140 diode array detector (LKB, Bromma, Sweden). Chemical ionization (reagent gas - isobutane) mass spectra of purified mctabolitcs were recorded with Hitachi MS0 instrument. High resolution electron impact spectra (70 eV) were recorded either with the same instrument, or with

I "

30:31 L:21

,i .tOO

2 5 D W a v e l e n 9 t h

311D

Iz"

~"

(nm)

Fig, I. The topogram of separation of a-kctol fraction by normal phase HPLC. Chromatographic conditions arc dcscrii~:d in Materials and Methods. The topogram was recalled from stored data of Rapid Spectral Detector. Peak I ('~m,~ 234) nm), 9-oxo-12A3-cpoxy-10(E),15(Z)-t~:tadecadicnoic acid: peak 2 ('~rn~ 211 nm), I l-hydroxy-12.oxo-~Z),15(Z)-octadecadienoic acid ( ! 1,12-e-ketol): peak 3 (Am.~ 214 nm). 12~,~x~13hydroxy-9(Z ), 15(Z )-octadecadienoic (12,13-e-ketol).

320 MKh-1310 high resolution spectrometer. Precise mass values were estimated using low boiling perfluorokerosene as reference standard. All mass spectra records were done using direct sample introduction, without preliminary derivatization. IH-NMR spectra (250 MHz, CDCI.0 were recorded on Bruker WM-250 spectrometer.

acid (7-ketol), 12-oxo-10,15(Z)-phytodienoic acid and the polar product having a retention time of approx. 31 min during HPLC analysis (system A). The last compound was identified as 9-hydroperoxy-12-oxo-13-hydroxy-lO(E),15(Z)-octadecadienoic acid (12,13-a-ketol hydropcroxide). Identification of this compound and the description of its formation by an unusual lipoxygenase reaction are the subjects of another paper published recently [10]. After 2-fold purification by reverse-phase HPLC (system A) a-ketol fraction was analyzed on the normal phase column (system B). As depicted in Fig. 1, this separation reveals metabolites 1 (Am=, at 230 nm) and 2 ( A m a x at 211 nm) along with the 12.13-~-ketol itself (product 3, A,,~ at 214 nm).

Results

Separation and identification of ketols The main products of [1-J4C]HPOT metabolism by flax seed hydroperoxide dehydrase were 12-oxo-13-hydroxy-9(Z),15(Z)-octadecadienoic acid (12,13-a-kctol) and 9-hydroxy-I 2-oxo- 10(E), 15(Z)-octadecadienoic

I:1 o,.o,ot223.,2. (31)

(,,,

.......

.

...... - H = , O "'"'.

~

-H~,O ........ """

OH

COOH

6 3 . 0 6 6 l'~an.,as (29) (69)

11 t.on~li~,~.laa (6:,I

~_H20

~- H=,O

(23)

210.126 (36)

1B1.123 (100)

[C~.tHt?02] +.... "¢lz

.,., [Ct~H~g03 ]+

B

• [c,=H,ooar [cv.~o]

+

i

....

,li i I,

100

.

.

.

i

.

[Ci2H2oO4 ]'1" ......[ H - H=,O]+

I

.,J~, I~, ,

.

.

.

.

,

200

It, ..,L. . , .~.

.

r,,s+ ~:."

,I, aoo

,I .....

m/=

Fig. 2. High resolution electron impact massspectrumof compound 2 (M + at m / z 310.216;CiHH3o04). The schemeof fragmentation(A) showsthe origin of the most abundant and characteristic ions. Sites of chain fragmentationare indicated by dotted lines. The measuredmass values and the correspondingintensities(in round brackets)are shown Massspectrum(B) of the non-derivatized compound2 was recorded as described in Materialsand Methods.Arrowsin spectrumindicate the mainfragmentions.

321 Metabolite 1 was identified by its ultraviolet, mass and ~H-NMR spectra (data not shown) as 9-oxo-12,13epoxy-10(E),15(Z)-octadecadienoic acid. This compound is known as the product of a homolytic rearrangement of hydroperoxide. In our experiments product 1 was formed, probably, as a result of similar but heterolytic rearrangement, because its formation was stimulated under acidic conditions. The following peaks were detected in chemical ionization mass spectrum of compound 2, m / e (relative intensity, %) 311 [ M + HI + (52), 293 [ M + H - H20] + (100). The electron impact mass spectrum of product 2 and the corresponding scheme of fragmentation are presented in Fig. 2. As depicted in Fig. 2, the main site of fragmentation is the bond between C-II and C-12 (vicinal ketol function). The fragment with mass 199.133 (from C-1 to C-11) undergoes extensive dehydration to form the most abundant ion in the spectrum at m / e 181.123. The complementary fragment (from C-12 to C-18) is present mostly in a non-dehydrated form having a mass value of 111.080. One more pair of complementary fragments with masses 83.086 (from C-13 to C-18) and 228.135 (from C-I to C-12) are formed as a result of breakage of the second C - C bond next to carbonyi function. tH-NMR spectrum of compound 2 is presented in Table 1. The most interesting detail of the spectrum is the large chemical shift of the methine proton H-II (B ffi 4.87 ppm). The resonance signal of this proton is delocalized under the influence of a carbonyl group from one side and of a double bond from another side. The similar methine proton of hydroxTicosatetraenoic acids has a chemical shift B ffi 4.2 ppm only [9]. TABLE I SH-NMR spectrum of compound 2 (250 MHz, CDCI))

Proton

H-18a,b,c H-4a,b H-5a,b H-6a,b H-Ta,b H-3a,b H-17a,b H-Sa,b; H-14a,b H-2a,b H-13a H-13b H-I 1 H-10 It-15 H-i6 !"1-9

Chemical shift (ppm) 0.94 1.33 !.33 !.33 1.33 1.64 2.06

Multiplicity

Couplingconstants (Hz)

t m m m m m m

Jt~,m: 7.5

2.24-2.41 2.34 2.43 2.57 4.87 5.18 5.23

m t m m

5.d0 5.76

dt dt

d dd, t-like dt

Jte,l~ ~ 7.5 J2.~= 7.4 J~o.tt = 9.8

Jg.t0 ffi 10.5 /,sa6 ffi 10.6; Jt4~ts~ 7.5 -/s.9= 7.5

TABLE II Transformation of [ I- t~C]linolenate into 13-HPOT and hydroperoxid@ dehydrase products in the homogenate of wheat seedlings under basic and acidic conditions

Incubationswere performedat ambient temperaturewithin 30 rain. Products were extracted and analyzed by the reverse-phaseradioHPLC as described in Materials and Methods. The mean valuesof three experimentsare shown.Standard deviations d',es not exceed 10% of mean value. Compound

Rctentkm time

Radioactivity (% of total) pH 7.5 pH 5.8

51 48 44 39 3l

1.7 6.3 30.5 1.7 2.3

(rain)

13-HPOT I2-OPDA a-ketol y-ketol

AKHP

6.7 5.0 17.3 4.0 4,7

Abbreviations: 18:3, linolenic acid; 13-HPOT, 13-hydroperoxyqqZ).ll(E).lS(Z)-octadecatrienoie acid: I2-OPDA, 12-oxo10.15(Z)phytodienoic acid: AKHP, a-kclol h~Iroperoxide. The obtained data of mass spectrometry, ultraviolet and tH-NMR spectra allow us to identify compound 2 as ! I-hydroxy-12-oxo-9(Z),15(Z)-octadecadienoic acid (ll,12-a-ketol). To our knowledge, this ketol or any similar structure containing 2-hydroxy-3(Z)-buten-lonyl moiety and derived by hydroperoxide dehydrase pathway from other polyenoic acid (e.g., linoleie or arachidonic) have not been found yet. Long-te.,m incubafwns

Our first observations concerning the effect of pH on the formation of separate ketols were obtained during the experiments with young wheat seedlings, which possess high hydropemxide dehydrase activity. As shown in Table II, label inco..rparation into a-ketol decreased after change of pH value of the incubation medium frora 7.5 to 5.8. Simultaneously, the labelling of y-kctol is markedly increased. The same dependence was observed during the experiments on the metabolism of [I-t4C]I3-HPOT and [I-14C]I3-HPOD by hydropcroxide dehydrase preparations from flax seeds (Table lID. Data presented in Tables I! and ill are based on reverse-phase radio-HPLC analysis. Thus, part of radioactivity of mketol fractions, containing 12,13-a-ketol as major component, in *.hese Tables belongs to product 1 and ll,12-a-ketol (2). At any pH the typical proportion of 3,-ketol and 11,12-a-ketol (2) is near 3 : 1. The approximate ratio of 12,13-a-ketoi, y-ketol and ll,12-a-ketol (2) after incubations at pH 5.8 is about 5:3:1. Stimulation of a-ketol hydroperoxide formation under acidic conditions was observed previously [10]. This change is explained by the shift of the positional specificity of wheat and flax iipoxygenases. Under acidic pH

322 TABLE Ill Transformation of II-mC]I3-HPOT and II-t4c]I3-HPOD into hydroperoxide dehydrase products by enzymatic preparation from flax set,tI~ mulet basic and acidic conditions Incubations were performed at ambient temperature within 30 rain. Products were extracted and analyzed by radio-HPLC as described in Materials and Methods. The mean values of three experiments are shown. Standard deviations does not exceed 10% of mean value. Substrate

13-HPOD

Product

Radioactivity (% of total)

12-OPDA a-ketol y-kctol AKHP

19.2 49.8 6.4 4.5

9.2 34.6 17.2 11.7

12-OPA a-kct()l y-ketol AKHP

tr. 30.2 8.4 I 1.5

tr. 25.5 13.9 13.,.6

Abbreviations: 12-OPA, 12-oxo-10-phytenoic acid. Other abbrevialions are the same as in Table 11. values the ability o f lipoxygenases to a t t a c k the C-9 position increases. T h u s , oxygenation o f 12,13-a-ketol at C-9 is m o r e p r o b a b l y u n d e r acidic t h a n u n d e r basic conditions. Distinct p H r e g u l a t i o n of or- a n d -y-ketol f o r m a t i o n may be explained by different m e c h a n i s m s o f ailene oxide hydrolysis. T o prove this p r o p o s a l we have perf o r m e d addi~tonal s h o r t - t e r m experiments, w h i c h are described bc!ow. Short-term incubations Allen,- oxides have a half-life in w a t e r at 0°C a b o u t 1 5 - 3 0 s [5,6]. T h u s , to observe the hydrolysis o f allene oxide t, n d e r s t r o n g acidic a n d basic conditions, we have p e r f o r m e d s h o r t - t e r m experiments. A [ I - t 4 C ] H P O T solutior, in e t h a n o l was injected into a cold (0°C) c o n c e n t r a t e d e n z y m e suspension in 7 ml of 25 m M Tris-HCI buf(er ( p H 7.4). T h e r e a c t i o n w a s allowed to p r o c e e d

"FABLE IV • ormatitm of kctols dttring acidic and basic hydrolysis of allene oxide 74 KBq of purified [IJ4C]I3-HPOT (20.6 MBq/mmol) in 50 /zl of eth;,nol wer- added to 7 ml of cool (0°CI enzyme solution, followed by the extensive nfixing within 15 s. Then the reaction mixture was divided in Iwo equal parts. Both parts were simultaneously fixed, one with ttCI and the second with NaOH solution, final pH values were about 2.O and 12.0. respectively.

ltydrolysis products

Yields (% of original [n'~C]HPOTradioactivity) basic fixation acidic fixation

12,13-tt-ketol y-kctol I 1,12-a-ketol

bA n.d. n.d.

n.d.. not detected.

n.d. 5,9 2.4

for 15 s u n d e r extensive mixing. T h e n the v o l u m e o f the reaction mixture w a s divided into two equal parts. In o n e p a r t reaction was s t o p p e d with N a O H solution a n d iv. the s e c o n d with HCI. A l t h o u g h the yields of ketols a f t e r i n c u b a t i o n with 15 s w e r e small ( a b o u t 10%), it allowed us to d e t e c t a n d quantify s e p a r a t e c o m p o n e n t s . A f t e r basic fixation the only ketol f o r m e d w a s 12,13-a-ketol. N e i t h e r y - k e tol n o r l l,12-a-ketol w a s formed. In contrast, a f t e r acidic fixation the m a i n p r o d u c t o f allene oxide hydrolysis was y-ketol. M e t h a n o l trapping experiments In o r d e r to p r o v e w h e t h e r ketols a r e still derived f r o m allene oxide at different p H values, we have performed experiments on methanol trapping. After the short (10 s) i n c u b a t i o n s o f purified [ 1 - I 4 C ] 1 3 - H P O D p r e p a r a t i o n with flax h y d r o p e r o x i d e d e h y d r a s e at 0*C in Tris-HCI b u f f e r ( p H 7.5), the r e a c t i o n mixture w a s diluted with a n excess a m o u n t o f p u r e m e t h a n o l o r m e t h a n o l / a c e t i c acid ( 1 0 0 : 1 , v / v ) mixtures. T h e n the t r a p p i n g p r o d u c t s w e r e e x t r a c t e d a n d a n a l y z e d by rev e r s e - p h a s e H P L C (system A). A m o r e d e t a i l e d description o f the e x p e r i m e n t a l p r o c e d u r e is given in Materials and Methods.

'

lil .~1/i \

:L'..........

o

1o

.........

--q .o

I /

....... ""

20

in/

!111 I Ill 30

40

50

6 °

llUI

R B t ; e r l t . t o r l ll;lmB t m l r l ) Fig. 3. Methanol trapping of allene oxide formed during the short time (10 s) incubation ('f [I-I4C]I3-HPOD with flax hydroperoxide dehydrase. Reverse-phaseHPLC analysisof trapping products. (A) Radiochromatogram of products of trapping with pure methanoll 4, peak of compound 4; HP, peak of hydroperoxide; insert0 ultraviolet spectrum of product 4, recorded on line by means of the Rapid Spectral Detector. (B) Radiochromatogram of products of trapping with methanol/acetic acid (100:1. v/v) mixture; 5 and 6, compounds 5 and 6. respectively: insert, ultraviolet spectrum of product 5.

322 Some amounts of both 12,13-a-ketol and 7-ketol were formed despite a short incubation time before methanol addition (Fig. 3). Not unexpectedly, 12,13-aketol was the predominant hydrolysis product. Along with ketols, some less polar products, ¢luting close to the peak of 13-HPOD, were formed (Fig. 3). Formation of these non-polar compounds was dependent on methanol addition. As depicted in Fig. 3a, the main product of trapping with pure methanol was compound 4 (Areax at 211 nm). A high resolution electron impact mass spectrum of compound 4 contained the molecular ion M + at role 326.246 [C19H3404] +) as well as the products of water and methanol elimination: [ M H20] + at m/e 308.237 and [ M - C H 3 O H ] + at m/e 294.221. Besides that, the spectrum contained the prominent ions at m/e 213.189 [ M - 1 1 5 + 2 H ] ÷, 115.076 (chain fragment from C-13 to C-18), 83.049 [ 115 - CH 3OH] +. Hydrogen¢ migration, accompanying the cleavage between C-12 and C-13, occurs also with an ~-ketol molecule under the conditions of electron impact (results not presented). Thus, the data of electron impact mass spectrometry allow us to ascribe the

structure of 12-oxo-13-methoxy-9(Z)-octadecenoic acid to compound 4. This result is in full agreement with the existing literature data [5] suggesting that methyl ether of 12,13-a-ketol is the predominant product of methanol trapping of allene oxide derived from 13HPOD. After the interruption of enzymatic reaction by the addition of a mixture of methanol/acetic acid (100:1, v/v) the set of trapping products was strikingly different. In this case the trapping products 5 (,~,m~ at 225 nm) and 6 (Areax at 211 nm) were formed along with 4 (Fig. 3b). The electron impact mass spectrum of compound 5 (M + at m/e 326.246, [C,,,HmO4]+), the main product of trapping with acidified methanol, is depicted in Fig. 4. The most abundant ions in the spectrum of this methoxy derivative, having oxoene chromophor¢, are derived from fragmentation between C-8 and C-9 (particularly [CIIHI902]+), C-9 and C-10, C-I! and C-12, C-12 and C-t3 (Fig.4 a), Thus, compound 5 was identified as 9-methoxy-12-oxo-10(E)-octadecenoic acid (7-ketol methyl ether). The less polar product of acidic methanol trapping,

A

"'"""'"..

OCH.=

~