- Email: [email protected]
Analysis of European oak bark beetle (Scolytus intricatus) extracts using hyphenated and chiral chromatography techniques
Talanta 59 (2003) 107 /114 www.elsevier.com/locate/talanta
Analysis of European oak bark beetle (Scolytus intricatus) extracts using hyphenated and chiral chromatography techniques Pavlı´na Vrkocˇova´ , Blanka Kalinova´, Irena Valterova´, Bohumı´r Koutek Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo na´meˇstı´ 2, 166 10 Praha 6, Czech Republic Received 28 March 2002; received in revised form 5 August 2002; accepted 23 August 2002
Abstract Two bioactive compounds, viz. 4-methylheptan-3-ol (I) and 4-methylheptan-3-one (II) have been identified in European oak bark beetle (Scolytus intricatus ) extracts by gas chromatography coupled with mass spectrometric and electroantennographic detector systems. Further examination of these compounds using gas chromatography on chiral stationary phases, as well as a comparison with optically active standards proved the absolute configuration of the identified compounds to be (3R ,4S )-I and (S )-II. The discovery of (3R ,4S )-I and (S )-II as insect-produced compounds in both sexes of S. intricatus constitutes the first reported occurrence in this species. # 2002 Elsevier Science B.V. All rights reserved. Keywords: GC /EAD; Scolytus intricatus ; (S )-4-Methylheptan-3-one; (3R ,4S )-4-Methylheptan-3-ol
1. Introduction Oak wilt, caused by pathogens of genera Ophiostoma , is a severe disease of oak trees that occurs over much of Europe and the United States [1]. Insect vectors play an important role in its above ground transmission by initiating new infection centers. In Europe, activity of the European oak bark beetle (Scolytus intricatus) is
Corresponding author. Tel.: /4202-201-83229; fax: / 4202-243-10177 E-mail address: [email protected] (P. Vrkocˇova´).
considered one of the most important factors responsible for the spread of this disease [1,2]. Synthetic pheromones that mimic the natural substances produced by insects for intra-specific chemical communication might offer a unique approach to S. intricatus management because they affect critical behaviors associated with the insect reproductive processes. Unfortunately, nothing is known about the chemical communication system of S. intricatus at present, although some limited information may be gained from analogies with related elm bark beetles, i.e. Scolytus multistriatus and Scolytus scolytus . It has been demonstrated that secondary mass attack of S. multistriatus results from responses to a phero-
0039-9140/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 0 2 ) 0 0 4 5 6 - 3
108
P. Vrkocˇova´ et al. / Talanta 59 (2003) 107 /114
mone blend consisting of two terpenoids (multistriatin and ( /)-a-cubebene) and 4-methylheptan3-ol (I) (Scheme 1) [3]. The absolute configuration of I was determined as (3S ,4S ) [4]. Similarly, (3S ,4S )-I has been reported as the major component of volatile attractants in S. pygmaeus , S. laevis and S. amygdali [5]. On the other hand, no significant differences in the degree of response to any of the optical isomers of I were found [6] for S. scolytus . It has also been observed [7] that boring S. multistriatus females and S. scolytus males produce 4-methylheptan-3-one (II) (Scheme 1) with absolute configuration (4S ), which was suggested as an intermediate in the biosynthesis of I and/or a pheromone component itself. It should be noted that considerable difficulties are associated with identification of bioactive compounds in Scolytidae because the compounds are often present in ng l 1 (or lower) concentrations, thus making their analysis and quantification very challenging. Elucidation of the structure and function of natural compounds mediating insect intra-specific interactions generally requires utilization of analytical, neurophysiological and biological methods. Among these methods, electroantennography (EAG), a neurophysiological technique that allows measuring and recording an insect’s ability to perceive semiochemicals [8] plays a non-replaceable role. The method has been discussed in detail [8 /10]. Direct coupling of the antennal preparation to a gas chromatograph (GC /EAD) provides a selective and very sensitive biological detector (EAD). We have previously shown [11], by examining hexane extracts of head and thorax (HTP) vs. posterior (PP) parts of the S. intricatus body in an olfactometer, that the attraction is associated exclusively with the PP parts of the beetle in both sexes. Based on this finding, we designed the present experiments with the aim to identify
the main compounds present in hexane extracts of S. intricatus PP parts by combining the GC /EAD technique with GC /MS and chiral chromatographic analyses. The specific objectives of this study were to: (1) detect electrophysiologically active components in the extracts using a coupled gas chromatography /electroantennographic (GC /EAD) detection; and (2) determine the chemical structures, including the stereochemistry, of significant compounds in S. intricatus extracts.
2. Materials and methods 2.1. Insects Infested oak logs were collected during the winter at several localities in central Bohemia and stored at 5 8C. Two or 3 weeks before experiments, the logs were taken to the laboratory and kept in nylon cages until the overwintering larvae finished their development and emerged. Adults were collected daily, sexed (males differ from females by the presence of a tuft of setae on the fronto-clypeal suture of the head), provided with fresh oak twigs and allowed to feed to maturation (1 week). 2.2. Beetle extracts Beetles that finished maturation feeding were dissected to anterior (head and prothorax, HTP) and posterior (mesothorax, methathorax and abdomen, PP) parts. The PP parts were macerated in pure hexane (10 ml per beetle) for 24 h, subsequently shaken for 30 min and the resulting extracts were transferred into a vial. The extracts from males (100 beetles per extract) and females (100 beetles per extract) were concentrated to 20 ml in an argon stream at room temperature. Two microliters of the solution were used for GC /MS and GC /EAD analysis. 2.3. GC /EAD
Scheme 1. Compounds that occur in some Scolytus species.
The GC /EAD measurements were performed with a HP 5890 (Hewllet-Packard, USA) gas chromatograph equipped with a capillary column
P. Vrkocˇova´ et al. / Talanta 59 (2003) 107 /114
(DB-5ms, 25 m /0.25 mm i.d., film thickness 0.25 mm, J&W Scientific), a flame ionization detector (FID) and a split/splitless injector. The injector was operated in the splitless mode. Helium was used as a carrier gas at 2.5 ml min 1 and nitrogen as make-up gas. From the main nitrogen line, a ‘T’ was inserted and one arm directed to the FID (make-up I) and the other to the EAD (make-up II). At the end of the capillary column a splitter (split ratio 1:1) distributed the effluent from the column to the FID and to the EAG preparation via a transfer line (see Section 2.7). Both connections were made of deactivated fused silica and had the same length and diameter to allow simultaneous monitoring of the FID and EAD responses. At the entrance of the splitter a second inlet of make-up gas (make-up II) was used to reduce residence time in the transfer lines and broadening of the peaks. The transfer line to the EAD was heated to 180 8C (Effluent Conditioning Assembly, Syntech, Hilversum, The Netherlands). The column oven temperature was programed as follows: initial temperature 50 8C for 2 min, increase at 4 8C min 1 up to 280 8C and held for 20 min. The antennal sensitivity and possible changes during experiments were checked by stimulation with 0.5 mg of racemic I applied at the beginning and at the end of each GC /EAD run. 2.4. GC /MS Gas chromatography/mass spectrometry was performed on a Carlo /Erba GC 8000 series gas chromatograph and Fisons MD 800 mass detector (quadrupole mass spectrometer using 70 eV electron impact ionization). Chromatography was carried out on a DB-5ms nonpolar fused silica capillary column (5% phenyl /95% methyl siloxane phase, 30 m /0.25 mm, film thickness 0.25 mm, J&W Scientific). The split/splitless injector was operated in the splitless mode at 200 8C; the split vent was opened after 60 s. Helium was used as a carrier gas at 1 ml min 1 (measured at 50 8C). The oven temperature program was 50 8C for 2 min, then 4 8C min 1 to 200 8C and 20 8C min 1 to 290 8C, and held for 20 min. The mass spectra were compared to the Wiley Registry of
109
Mass Spectral Data, 6th edition. Retention times of the compounds were compared with those of authentic samples of I and II. 2.5. Chiral chromatography The absolute configuration of I and II in hexane extracts was determined on a fused silica capillary column coated with octakis [6-O -methyl-2,3-di-O pentyl]-g-cyclodextrin (60% w/w in OV 1701, 25 m /0.25 mm). The chiral column was placed in the GC/MS instrument described in Section 2.4. The oven temperature was held at 60 8C for 30 min, then 10 8C min 1 to 200 8C, and held for 35 min. The elution order of 4-methylheptan-3-one stereoisomers was determined according to the literature [12]. The absolute configuration of insect-produced alcohol I was determined by comparison of the retention times with authentic standards (see Section 2.6). 2.6. Chemicals Racemic 4-methylheptan-3-one (II) was prepared from commercially available 4-methylheptan-3-ol (I) (Aldrich, Milwaukee, Wisconsin) using a standard oxidation procedure with PDC (pyridinium dichromate) and purified to obtain /95% purity (checked on GC). The particular stereoisomers of I were prepared previously in our laboratory [13]. Optical purity of these stereoisomers as checked by GC on a derivatized g-cyclodextrin column (see Section 2.5) was (listed in elution order): SS : 91.9%, RS : 86.2%, RR : 95.7%, SR : 87.3% (the remainder was other stereoisomers). Pure enantiomers of II were not available. 2.7. EAG For EAG, intact insects were used. Beetles were secured (with head and antennae exposed) in a disposable pipette tip and fixed in a plastic holder. The beetle’s head was locked in place with doublesided sticky tape, the antennae were immobilized on the sticky surface and fixed in place by watersoluble tippex. A glass Ag/AgCl microelectrode filled with insect physiological saline [14] was
110
P. Vrkocˇova´ et al. / Talanta 59 (2003) 107 /114
positioned in the antennal club and an indifferent electrode was placed in the prothorax. Antennal responses were passed through a high impedance preamplifier (10 /), digitalized by Data Acquisition Interface Board (IDAC-02, Syntech, The Netherlands) and fed into a PC. Signals were evaluated using EAG and GC /EAD Signal Recording and Analysis Program (Syntech). To determine EAG dose /response relationship, hexane solutions of 10 11 /10 4 g ml 1 concentrations were prepared from racemic derivatives I and II. Solutions (10 ml) were applied to a filter paper disc (10 mm diameter). After solvent evaporation, the disc was inserted into a Pasteur pipette, which was then sealed by ParafilmTM until used. During stimulation a 0.5 s long air pulse (1 l min 1) was delivered onto the antennal preparation from the outlet of the pipette positioned 1 cm from antenna. Stimuli were injected into clean humidified air blown over the antenna (1 l min 1) starting with low doses. Two to 20 min interval (depending on doses) separated two successive stimulations. Data from six antennal preparations of each sex were evaluated.
3. Results and discussion GC /EAD analyses of S. intricatus female and male PP body extracts on DB-5ms column showed repeatedly two distinct EAD peaks in approximate 20 /30:1 ratios that were detectable by both male and female antennae. Fig. 1 exemplifies the analysis for hexane extracts of females. GC /MS analyses revealed the presence of one diastereomer of I (K.I. /979, mass spectrum (m /z (%)): 59 (100%), 83 (24), 101 (15), 112 (1) and II (K.I. / 941, mass spectrum (m /z (%)): 57 (100%), 71 (95), 86 (84), 99 (20), 128 (4)). The mass spectra of both compounds corresponded to those of authentic racemic standards. To further check the relevance of compounds I and II for olfactory communication in S. intricatus, EAG dose /response curves were constructed for both sexes. Fig. 2 shows a typical dose/response relationship demonstrating an approximately equal antennal sensitivity of males and females to both identified compounds, while I is invariably antennally more active than
II. It is to be noted that one additional GC /EADactive minor component was detected in the PP body extracts of females at the longer retention time end of the scale (K.I. /1156). This compound, however, remains unidentified because of the very small amounts present in the extracts. Because the biological activity of a chiral compound may be critically dependent on its absolute configuration [15], the determination of configuration is a crucial part of structure identification, and of a subsequent understanding of a molecule’s function. Since 4-methyl-3-heptanone (II) possesses one chiral center at C-4 and its reduced form I two chiral centers on C-3 and C-4 adjacent carbon atoms, two enantiomers exist in the former case while four enantiomers are possible in the latter. Looking for a single stationary phase that would be suitable for resolution of all stereoisomers of I and II, we evaluated two chiral cyclodextrin columns. A widely used permethylated b-cyclodextrin column was found to be effective only in resolving the enantiomers of II while diastereomeric pairs of I remained unresolved. On the other hand, a selectively methyland pentyl-group substituted g-cyclodextrin column originally designed for separation of terpenoid alcohols [16], offered a good separation of all enantiomers of I and II. Thus, the absolute configuration of II in hexane extracts of insect posterior body parts was determined by correlating our retention data on the octakis [6-O -methyl2,3-di-O -pentyl]-g-cyclodextrin column with those reported in the literature [12] under exactly the same chromatographic conditions (Fig. 3) as described. This comparison demonstrated that the insect-produced compound present in extracts from both sexes of S. intricatus is the (S )-II isomer. As shown in Fig. 4, the use of g-cyclodextrin column led in the separation of all enantiomers of I. Based on a comparison of retention times and mass spectra of naturally occurring I with those of previously prepared [13] synthetic (3S ,4S )-, (3R ,4S )-, (3R ,4R )- and (3S ,4R )-4methylheptan-3-ol stereoisomers, the absolute configuration of I in hexane extracts of S. intricatus was assigned as (3R ,4S )-I.
P. Vrkocˇova´ et al. / Talanta 59 (2003) 107 /114
111
Fig. 1. Three typical analyses of female body extracts of S. intricatus by GC /EAD. The dashed-line boxes a, b indicate two components repeatedly eliciting antennal responses in different antennal preparations of both sexes (a, 4-methyl-3-heptanone; and b, 4methyl-3-heptanol).
While the ketone (S )-II has been reported as a compound usually accompanying (3S ,4S )-I in volatile attractants of several Scolytus species, the (3R ,4S )-I isomer identified from extracts of S. intricatus in this work constitutes a rather exceptional finding. It has been found, however, that male and female S. scolytus produce not only (3S ,4S )-I but also the (3R ,4S )-isomer, although only the former isomer showed attractivity in the field [17,18]. In summary, GC/EAD and GC /MS analyses on chiral chromatographic phases of volatile extracts from S. intricatus revealed three active candidate pheromone components, though only two of them, i.e. (3R ,4S )-I and (S )-II, have been chemically fully identified. Interestingly, all 4methylheptane derivatives identified so far in
Scolytus spp. extracts (including those of S. intricatus ) occur exclusively in the (4S )-configuration. In this respect, our results support the hypothesis that the (S )-II stereoisomer is a common precursor of all Scolytus -produced (3R / S ,4S )-4-methylheptan-3-ols. The differences between the appearance of (3R )- vs. (3S ) configuration in I might then result from different enzymes reducing the prochiral keto group in II either Re or Si-face in particular Scolytus species. Considering, however, that more than forty Scolytus species are recognized in the world while only a few of them have been subjects of studies of chemosensory behavior and, moreover, examples are known (e.g. S. ventralis ) where the beetles appear to be attracted only by host tree-produced attractants [19], further studies will be necessary to
112
P. Vrkocˇova´ et al. / Talanta 59 (2003) 107 /114
Fig. 2. Dose /response curves obtained from male and female antennae of S. intricatus to racemic compounds I and II. Each point represents a mean value of six measurements obtained from six different antennae (vertical bars indicate the standard error of the mean). EAG responses are expressed as % of EAG response to standard stimulus (1 mg of I).
Fig. 3. Chiral GC /MS analysis of a solution of II (A) and hexane extracts of S. intricatus females (B) together with characteristic m /z values (C) confirming the absolute configuration of II-isomer identified in the extracts.
P. Vrkocˇova´ et al. / Talanta 59 (2003) 107 /114
113
Fig. 4. Chiral GC /MS analysis of a solution of I (A) and hexane extracts of S. intricatus females (B) together with characteristic m /z values (C) confirming the absolute configuration of I-isomer identified in the extracts.
ascertain the role of I and II in chemical communication systems of the genus Scolytus.
Acknowledgements The financial support of this work by the Grant Agency of the Czech Republic (grant No. 203/00/ 0219), by COST E16.10 and by research project Z4 055 905 is gratefully acknowledged. We also thank Ondrˇej Blazˇek for electrophysiology measurements and R.E. Doolittle for revision of the manuscript.
References [1] M. Donaglar, R. Schopf, Z. Angew. Entomol. 97 (1984) 153.
[2] M. Von Habermann, R. Schopf, J. Appl. Entomol. 106 (1988) 252. [3] G.N. Lanier, W.E. Gore, G.T. Pearce, J.W. Peacock, R.M. Silverstein, J. Chem. Ecol. 3 (1977) 1. [4] K. Mori, Tetrahedron 33 (1977) 289. [5] S. Ben-Yehuda, T. Tolasch, W. Francke, R. Gries, G. Gries, D. Dunkelblum, Z. Mendel, IOBC wprs Bull. 25 (2002), in press. [6] M.M. Blight, L.J. Wadhams, M.J. Wenham, Insect Biochem. 9 (1979) 525. [7] M.M. Blight, N.C. Henderson, L.J. Wadhams, Insect Biochem. 13 (1983) 27. [8] L.B. Bjostad, in: J.C. Millar, K.F. Haynes (Eds.), Methods in Chemical Ecology (Chapter 9), Kluwer Acad. Publ, Boston, 1998, pp. 339 /375. [9] G.R. Jones, N.J. Oldham, J. Chromatogr. A 843 (1999) 199. [10] E.A. Malo, M. Renou, A. Guerrero, Talanta 52 (2000) 525. [11] O. Hovorka, J. Kindl, P. Vrkocˇova´, B. Kalinova´, M. Hoskovec, J. Doubsky´, B. Koutek, in preparation. [12] R.R. DoNascimento, E.D. Morgan, W.A. Konig, T.M.C. Della Lucia, J. Chem. Ecol. 23 (1997) 1569.
114
P. Vrkocˇova´ et al. / Talanta 59 (2003) 107 /114
[13] J. Ru˚zˇicˇka, B. Koutek, L. Streinz, D. .Sˇaman, L. Lesˇeticky´, Tetrahedron: Asymmetry 10 (1999) 3521. [14] W.L. Roelofs, in: H.E. Hummel, T.A. Miller (Eds.), Techniques in Pheromone Research, Springer-Verlag, New York, 1984, pp. 131 /161. [15] K. Mori, see Ref. [8], Chapter 8, pp. 295 /338. [16] Anonymous, J. High Resolut. Chromatogr. 16 (1993) 312.
[17] M.M. Blight, L.J. Wadhams, M.J. Wenham, C.J. King, Forestry 52 (1979) 83. [18] M.M. Blight, N.J. Fielding, C.J. King, A.P. Ottridge, L.J. Wadhams, M.J. Wenham, J. Chem. Ecol. 9 (1983) 67. [19] J.E. Macias-Samano, J.H. Borden, R. Gries, D.H. Pierce, Jr., G. Gries, G.S. King, J. Chem. Ecol. 24 (1998) 1049.
Analysis of European oak bark beetle (Scolytus intricatus) extracts using hyphenated and chiral chromatography techniques Pavlı´na Vrkocˇova´ , Blanka Kalinova´, Irena Valterova´, Bohumı´r Koutek Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo na´meˇstı´ 2, 166 10 Praha 6, Czech Republic Received 28 March 2002; received in revised form 5 August 2002; accepted 23 August 2002
Abstract Two bioactive compounds, viz. 4-methylheptan-3-ol (I) and 4-methylheptan-3-one (II) have been identified in European oak bark beetle (Scolytus intricatus ) extracts by gas chromatography coupled with mass spectrometric and electroantennographic detector systems. Further examination of these compounds using gas chromatography on chiral stationary phases, as well as a comparison with optically active standards proved the absolute configuration of the identified compounds to be (3R ,4S )-I and (S )-II. The discovery of (3R ,4S )-I and (S )-II as insect-produced compounds in both sexes of S. intricatus constitutes the first reported occurrence in this species. # 2002 Elsevier Science B.V. All rights reserved. Keywords: GC /EAD; Scolytus intricatus ; (S )-4-Methylheptan-3-one; (3R ,4S )-4-Methylheptan-3-ol
1. Introduction Oak wilt, caused by pathogens of genera Ophiostoma , is a severe disease of oak trees that occurs over much of Europe and the United States [1]. Insect vectors play an important role in its above ground transmission by initiating new infection centers. In Europe, activity of the European oak bark beetle (Scolytus intricatus) is
Corresponding author. Tel.: /4202-201-83229; fax: / 4202-243-10177 E-mail address: [email protected] (P. Vrkocˇova´).
considered one of the most important factors responsible for the spread of this disease [1,2]. Synthetic pheromones that mimic the natural substances produced by insects for intra-specific chemical communication might offer a unique approach to S. intricatus management because they affect critical behaviors associated with the insect reproductive processes. Unfortunately, nothing is known about the chemical communication system of S. intricatus at present, although some limited information may be gained from analogies with related elm bark beetles, i.e. Scolytus multistriatus and Scolytus scolytus . It has been demonstrated that secondary mass attack of S. multistriatus results from responses to a phero-
0039-9140/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 0 2 ) 0 0 4 5 6 - 3
108
P. Vrkocˇova´ et al. / Talanta 59 (2003) 107 /114
mone blend consisting of two terpenoids (multistriatin and ( /)-a-cubebene) and 4-methylheptan3-ol (I) (Scheme 1) [3]. The absolute configuration of I was determined as (3S ,4S ) [4]. Similarly, (3S ,4S )-I has been reported as the major component of volatile attractants in S. pygmaeus , S. laevis and S. amygdali [5]. On the other hand, no significant differences in the degree of response to any of the optical isomers of I were found [6] for S. scolytus . It has also been observed [7] that boring S. multistriatus females and S. scolytus males produce 4-methylheptan-3-one (II) (Scheme 1) with absolute configuration (4S ), which was suggested as an intermediate in the biosynthesis of I and/or a pheromone component itself. It should be noted that considerable difficulties are associated with identification of bioactive compounds in Scolytidae because the compounds are often present in ng l 1 (or lower) concentrations, thus making their analysis and quantification very challenging. Elucidation of the structure and function of natural compounds mediating insect intra-specific interactions generally requires utilization of analytical, neurophysiological and biological methods. Among these methods, electroantennography (EAG), a neurophysiological technique that allows measuring and recording an insect’s ability to perceive semiochemicals [8] plays a non-replaceable role. The method has been discussed in detail [8 /10]. Direct coupling of the antennal preparation to a gas chromatograph (GC /EAD) provides a selective and very sensitive biological detector (EAD). We have previously shown [11], by examining hexane extracts of head and thorax (HTP) vs. posterior (PP) parts of the S. intricatus body in an olfactometer, that the attraction is associated exclusively with the PP parts of the beetle in both sexes. Based on this finding, we designed the present experiments with the aim to identify
the main compounds present in hexane extracts of S. intricatus PP parts by combining the GC /EAD technique with GC /MS and chiral chromatographic analyses. The specific objectives of this study were to: (1) detect electrophysiologically active components in the extracts using a coupled gas chromatography /electroantennographic (GC /EAD) detection; and (2) determine the chemical structures, including the stereochemistry, of significant compounds in S. intricatus extracts.
2. Materials and methods 2.1. Insects Infested oak logs were collected during the winter at several localities in central Bohemia and stored at 5 8C. Two or 3 weeks before experiments, the logs were taken to the laboratory and kept in nylon cages until the overwintering larvae finished their development and emerged. Adults were collected daily, sexed (males differ from females by the presence of a tuft of setae on the fronto-clypeal suture of the head), provided with fresh oak twigs and allowed to feed to maturation (1 week). 2.2. Beetle extracts Beetles that finished maturation feeding were dissected to anterior (head and prothorax, HTP) and posterior (mesothorax, methathorax and abdomen, PP) parts. The PP parts were macerated in pure hexane (10 ml per beetle) for 24 h, subsequently shaken for 30 min and the resulting extracts were transferred into a vial. The extracts from males (100 beetles per extract) and females (100 beetles per extract) were concentrated to 20 ml in an argon stream at room temperature. Two microliters of the solution were used for GC /MS and GC /EAD analysis. 2.3. GC /EAD
Scheme 1. Compounds that occur in some Scolytus species.
The GC /EAD measurements were performed with a HP 5890 (Hewllet-Packard, USA) gas chromatograph equipped with a capillary column
P. Vrkocˇova´ et al. / Talanta 59 (2003) 107 /114
(DB-5ms, 25 m /0.25 mm i.d., film thickness 0.25 mm, J&W Scientific), a flame ionization detector (FID) and a split/splitless injector. The injector was operated in the splitless mode. Helium was used as a carrier gas at 2.5 ml min 1 and nitrogen as make-up gas. From the main nitrogen line, a ‘T’ was inserted and one arm directed to the FID (make-up I) and the other to the EAD (make-up II). At the end of the capillary column a splitter (split ratio 1:1) distributed the effluent from the column to the FID and to the EAG preparation via a transfer line (see Section 2.7). Both connections were made of deactivated fused silica and had the same length and diameter to allow simultaneous monitoring of the FID and EAD responses. At the entrance of the splitter a second inlet of make-up gas (make-up II) was used to reduce residence time in the transfer lines and broadening of the peaks. The transfer line to the EAD was heated to 180 8C (Effluent Conditioning Assembly, Syntech, Hilversum, The Netherlands). The column oven temperature was programed as follows: initial temperature 50 8C for 2 min, increase at 4 8C min 1 up to 280 8C and held for 20 min. The antennal sensitivity and possible changes during experiments were checked by stimulation with 0.5 mg of racemic I applied at the beginning and at the end of each GC /EAD run. 2.4. GC /MS Gas chromatography/mass spectrometry was performed on a Carlo /Erba GC 8000 series gas chromatograph and Fisons MD 800 mass detector (quadrupole mass spectrometer using 70 eV electron impact ionization). Chromatography was carried out on a DB-5ms nonpolar fused silica capillary column (5% phenyl /95% methyl siloxane phase, 30 m /0.25 mm, film thickness 0.25 mm, J&W Scientific). The split/splitless injector was operated in the splitless mode at 200 8C; the split vent was opened after 60 s. Helium was used as a carrier gas at 1 ml min 1 (measured at 50 8C). The oven temperature program was 50 8C for 2 min, then 4 8C min 1 to 200 8C and 20 8C min 1 to 290 8C, and held for 20 min. The mass spectra were compared to the Wiley Registry of
109
Mass Spectral Data, 6th edition. Retention times of the compounds were compared with those of authentic samples of I and II. 2.5. Chiral chromatography The absolute configuration of I and II in hexane extracts was determined on a fused silica capillary column coated with octakis [6-O -methyl-2,3-di-O pentyl]-g-cyclodextrin (60% w/w in OV 1701, 25 m /0.25 mm). The chiral column was placed in the GC/MS instrument described in Section 2.4. The oven temperature was held at 60 8C for 30 min, then 10 8C min 1 to 200 8C, and held for 35 min. The elution order of 4-methylheptan-3-one stereoisomers was determined according to the literature [12]. The absolute configuration of insect-produced alcohol I was determined by comparison of the retention times with authentic standards (see Section 2.6). 2.6. Chemicals Racemic 4-methylheptan-3-one (II) was prepared from commercially available 4-methylheptan-3-ol (I) (Aldrich, Milwaukee, Wisconsin) using a standard oxidation procedure with PDC (pyridinium dichromate) and purified to obtain /95% purity (checked on GC). The particular stereoisomers of I were prepared previously in our laboratory [13]. Optical purity of these stereoisomers as checked by GC on a derivatized g-cyclodextrin column (see Section 2.5) was (listed in elution order): SS : 91.9%, RS : 86.2%, RR : 95.7%, SR : 87.3% (the remainder was other stereoisomers). Pure enantiomers of II were not available. 2.7. EAG For EAG, intact insects were used. Beetles were secured (with head and antennae exposed) in a disposable pipette tip and fixed in a plastic holder. The beetle’s head was locked in place with doublesided sticky tape, the antennae were immobilized on the sticky surface and fixed in place by watersoluble tippex. A glass Ag/AgCl microelectrode filled with insect physiological saline [14] was
110
P. Vrkocˇova´ et al. / Talanta 59 (2003) 107 /114
positioned in the antennal club and an indifferent electrode was placed in the prothorax. Antennal responses were passed through a high impedance preamplifier (10 /), digitalized by Data Acquisition Interface Board (IDAC-02, Syntech, The Netherlands) and fed into a PC. Signals were evaluated using EAG and GC /EAD Signal Recording and Analysis Program (Syntech). To determine EAG dose /response relationship, hexane solutions of 10 11 /10 4 g ml 1 concentrations were prepared from racemic derivatives I and II. Solutions (10 ml) were applied to a filter paper disc (10 mm diameter). After solvent evaporation, the disc was inserted into a Pasteur pipette, which was then sealed by ParafilmTM until used. During stimulation a 0.5 s long air pulse (1 l min 1) was delivered onto the antennal preparation from the outlet of the pipette positioned 1 cm from antenna. Stimuli were injected into clean humidified air blown over the antenna (1 l min 1) starting with low doses. Two to 20 min interval (depending on doses) separated two successive stimulations. Data from six antennal preparations of each sex were evaluated.
3. Results and discussion GC /EAD analyses of S. intricatus female and male PP body extracts on DB-5ms column showed repeatedly two distinct EAD peaks in approximate 20 /30:1 ratios that were detectable by both male and female antennae. Fig. 1 exemplifies the analysis for hexane extracts of females. GC /MS analyses revealed the presence of one diastereomer of I (K.I. /979, mass spectrum (m /z (%)): 59 (100%), 83 (24), 101 (15), 112 (1) and II (K.I. / 941, mass spectrum (m /z (%)): 57 (100%), 71 (95), 86 (84), 99 (20), 128 (4)). The mass spectra of both compounds corresponded to those of authentic racemic standards. To further check the relevance of compounds I and II for olfactory communication in S. intricatus, EAG dose /response curves were constructed for both sexes. Fig. 2 shows a typical dose/response relationship demonstrating an approximately equal antennal sensitivity of males and females to both identified compounds, while I is invariably antennally more active than
II. It is to be noted that one additional GC /EADactive minor component was detected in the PP body extracts of females at the longer retention time end of the scale (K.I. /1156). This compound, however, remains unidentified because of the very small amounts present in the extracts. Because the biological activity of a chiral compound may be critically dependent on its absolute configuration [15], the determination of configuration is a crucial part of structure identification, and of a subsequent understanding of a molecule’s function. Since 4-methyl-3-heptanone (II) possesses one chiral center at C-4 and its reduced form I two chiral centers on C-3 and C-4 adjacent carbon atoms, two enantiomers exist in the former case while four enantiomers are possible in the latter. Looking for a single stationary phase that would be suitable for resolution of all stereoisomers of I and II, we evaluated two chiral cyclodextrin columns. A widely used permethylated b-cyclodextrin column was found to be effective only in resolving the enantiomers of II while diastereomeric pairs of I remained unresolved. On the other hand, a selectively methyland pentyl-group substituted g-cyclodextrin column originally designed for separation of terpenoid alcohols [16], offered a good separation of all enantiomers of I and II. Thus, the absolute configuration of II in hexane extracts of insect posterior body parts was determined by correlating our retention data on the octakis [6-O -methyl2,3-di-O -pentyl]-g-cyclodextrin column with those reported in the literature [12] under exactly the same chromatographic conditions (Fig. 3) as described. This comparison demonstrated that the insect-produced compound present in extracts from both sexes of S. intricatus is the (S )-II isomer. As shown in Fig. 4, the use of g-cyclodextrin column led in the separation of all enantiomers of I. Based on a comparison of retention times and mass spectra of naturally occurring I with those of previously prepared [13] synthetic (3S ,4S )-, (3R ,4S )-, (3R ,4R )- and (3S ,4R )-4methylheptan-3-ol stereoisomers, the absolute configuration of I in hexane extracts of S. intricatus was assigned as (3R ,4S )-I.
P. Vrkocˇova´ et al. / Talanta 59 (2003) 107 /114
111
Fig. 1. Three typical analyses of female body extracts of S. intricatus by GC /EAD. The dashed-line boxes a, b indicate two components repeatedly eliciting antennal responses in different antennal preparations of both sexes (a, 4-methyl-3-heptanone; and b, 4methyl-3-heptanol).
While the ketone (S )-II has been reported as a compound usually accompanying (3S ,4S )-I in volatile attractants of several Scolytus species, the (3R ,4S )-I isomer identified from extracts of S. intricatus in this work constitutes a rather exceptional finding. It has been found, however, that male and female S. scolytus produce not only (3S ,4S )-I but also the (3R ,4S )-isomer, although only the former isomer showed attractivity in the field [17,18]. In summary, GC/EAD and GC /MS analyses on chiral chromatographic phases of volatile extracts from S. intricatus revealed three active candidate pheromone components, though only two of them, i.e. (3R ,4S )-I and (S )-II, have been chemically fully identified. Interestingly, all 4methylheptane derivatives identified so far in
Scolytus spp. extracts (including those of S. intricatus ) occur exclusively in the (4S )-configuration. In this respect, our results support the hypothesis that the (S )-II stereoisomer is a common precursor of all Scolytus -produced (3R / S ,4S )-4-methylheptan-3-ols. The differences between the appearance of (3R )- vs. (3S ) configuration in I might then result from different enzymes reducing the prochiral keto group in II either Re or Si-face in particular Scolytus species. Considering, however, that more than forty Scolytus species are recognized in the world while only a few of them have been subjects of studies of chemosensory behavior and, moreover, examples are known (e.g. S. ventralis ) where the beetles appear to be attracted only by host tree-produced attractants [19], further studies will be necessary to
112
P. Vrkocˇova´ et al. / Talanta 59 (2003) 107 /114
Fig. 2. Dose /response curves obtained from male and female antennae of S. intricatus to racemic compounds I and II. Each point represents a mean value of six measurements obtained from six different antennae (vertical bars indicate the standard error of the mean). EAG responses are expressed as % of EAG response to standard stimulus (1 mg of I).
Fig. 3. Chiral GC /MS analysis of a solution of II (A) and hexane extracts of S. intricatus females (B) together with characteristic m /z values (C) confirming the absolute configuration of II-isomer identified in the extracts.
P. Vrkocˇova´ et al. / Talanta 59 (2003) 107 /114
113
Fig. 4. Chiral GC /MS analysis of a solution of I (A) and hexane extracts of S. intricatus females (B) together with characteristic m /z values (C) confirming the absolute configuration of I-isomer identified in the extracts.
ascertain the role of I and II in chemical communication systems of the genus Scolytus.
Acknowledgements The financial support of this work by the Grant Agency of the Czech Republic (grant No. 203/00/ 0219), by COST E16.10 and by research project Z4 055 905 is gratefully acknowledged. We also thank Ondrˇej Blazˇek for electrophysiology measurements and R.E. Doolittle for revision of the manuscript.
References [1] M. Donaglar, R. Schopf, Z. Angew. Entomol. 97 (1984) 153.
[2] M. Von Habermann, R. Schopf, J. Appl. Entomol. 106 (1988) 252. [3] G.N. Lanier, W.E. Gore, G.T. Pearce, J.W. Peacock, R.M. Silverstein, J. Chem. Ecol. 3 (1977) 1. [4] K. Mori, Tetrahedron 33 (1977) 289. [5] S. Ben-Yehuda, T. Tolasch, W. Francke, R. Gries, G. Gries, D. Dunkelblum, Z. Mendel, IOBC wprs Bull. 25 (2002), in press. [6] M.M. Blight, L.J. Wadhams, M.J. Wenham, Insect Biochem. 9 (1979) 525. [7] M.M. Blight, N.C. Henderson, L.J. Wadhams, Insect Biochem. 13 (1983) 27. [8] L.B. Bjostad, in: J.C. Millar, K.F. Haynes (Eds.), Methods in Chemical Ecology (Chapter 9), Kluwer Acad. Publ, Boston, 1998, pp. 339 /375. [9] G.R. Jones, N.J. Oldham, J. Chromatogr. A 843 (1999) 199. [10] E.A. Malo, M. Renou, A. Guerrero, Talanta 52 (2000) 525. [11] O. Hovorka, J. Kindl, P. Vrkocˇova´, B. Kalinova´, M. Hoskovec, J. Doubsky´, B. Koutek, in preparation. [12] R.R. DoNascimento, E.D. Morgan, W.A. Konig, T.M.C. Della Lucia, J. Chem. Ecol. 23 (1997) 1569.
114
P. Vrkocˇova´ et al. / Talanta 59 (2003) 107 /114
[13] J. Ru˚zˇicˇka, B. Koutek, L. Streinz, D. .Sˇaman, L. Lesˇeticky´, Tetrahedron: Asymmetry 10 (1999) 3521. [14] W.L. Roelofs, in: H.E. Hummel, T.A. Miller (Eds.), Techniques in Pheromone Research, Springer-Verlag, New York, 1984, pp. 131 /161. [15] K. Mori, see Ref. [8], Chapter 8, pp. 295 /338. [16] Anonymous, J. High Resolut. Chromatogr. 16 (1993) 312.
[17] M.M. Blight, L.J. Wadhams, M.J. Wenham, C.J. King, Forestry 52 (1979) 83. [18] M.M. Blight, N.J. Fielding, C.J. King, A.P. Ottridge, L.J. Wadhams, M.J. Wenham, J. Chem. Ecol. 9 (1983) 67. [19] J.E. Macias-Samano, J.H. Borden, R. Gries, D.H. Pierce, Jr., G. Gries, G.S. King, J. Chem. Ecol. 24 (1998) 1049.