The analysis of brassinosteroids — plant growth-promoting substances

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4 J. M. Hayes, R. Takigiku,

5 6 7 8 9

R. Ocampo, H. J. Callot and P. Albrecht, Nature, 329 (1987) 48-51. I. Gilmour, P. K. Swart and C. T. Pillinger, Org. Geochem., 5 (1984) 665-670. H. H. Chung, W. B. Sheri and P. L. Grizzle, Geochim. Cosmochim. Acta, 45 (1981) 1803-1815. W. H. Stahl, Geochim. Cosmochim. Acta, 42 (1978) 1573-1577. M. Sano, Y. Yotsui, H. Abe and S. Sasaki, Biomed. Mass Spectrom., (1976) l-3. D. E. Matthews and J. M. Hayes, Anal. Chem., 50 (1978) 1465-1473.

10 D. E. Matthews, E. Ben-Galin and D. M. Bier, Anal. Chem., 51(1979) 80-84. 11 H. Craig, Geochim. Cosmochim. Acta, 12 (1957) 133-149. 12 M. A. Northam, in B. M. Thomas et al., Petroleum Geochemistry in Exploration of the Norwegian Shelf, Graham & Trotman, 1985, pp. 93-100. Dr. Malvin Bjoray is managing director of Geolab Nor AIS, Hornebergveien 5, 7038 Trondheim, Norway since 1984; Keith Hall does chromatographic consultancy work in geochemical, medical and chemical R&D; Janine Jumeau is Product Manager for VG Isotech Ltd., Aston Way, Cheshire CWIOOHT, U.K.

The analysis of brassinosteroids growth-promoting substances Nubuo lkekawa Fukushima,Japan Brassinosteroid is the generic name for a new group of steroidal plant growth substances which possess B-ring lactone and two vicinai dials. Microamounts of it are distributed widely in the phtnt kingdom as a mixture of several analogues. Analytical methods for these special steroids by gas chromatography (GC), GC-mass spectrometry and highperformance liquid chromatography as the boronate derivative and their applications are discussed.

Introduction Brassinolide (Fig. l), which was first isolated from rape (Brmsica nupus L.) pollen by USDA scientists in 1979, is a newly discovered plant growth-promoting substance’. It is a steroidal compound with a seven-membered lactonic B-ring and two pairs of vicinal diol groups in the A-ring and the side chain. Since its discovery, extensive studies have been devoted to its chemical synthesis, the screening of brassinolide and related substances in plants, and the possible application of these steroids in agriculture. To date, more than 20 brassinolide analogues, or brassinosteroids as they are now called, have been isolated and characterized in plants, mainly by Japanese scientists. Brassinosteroids generally show plant growthpromoting activities such as cell elongation and cell division, resulting in curvature, swelling and splitting of the internode in the bean second-internode assay2, and inclination of rice lamina3. In particular, the rice-lamina inclination assay, originally developed by Maeda4, is specific and highly sensitive for brassinosteroids. Brassinosteroids also exhibit a 0165-9936/90/$03.00.

plant

broad range of biological activities commonly observed in many of the known plant hormones including auxin, gibberellin and cytokinin. Recently we have synthesized a number of brassinosteroids and assayed them for their biological activity in order to investigate structure-activity relationships. We have also developed analytical methods for brassinosteroids based on gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) techniques, which are very suitable for the analysis of such mixtures. Application of our methods has resulted in the identification of new brassinosteroids in plants, and as a consequence, through the studies of our group and others, it is now apparent that brassinosteroids are ubiquitously distributed in the plant kingdom. In this article I would like to describe these analytical methods and their application to the screening of brassinosteroids. Structure and occurrence of brassinosteroids Fig. 2 summarizes the structures of typical brassinosteroids. In 1982, Yokota and co-workers, on the basis of the rice lamina inclination assay, isolated castasterone (2)5 from the insect galls of Casanea crenatu and dolicholide (8)6 from the immature seeds of Dolichos lablab. In the same year, we identified brassinolide and castasterone in three plants, i.e., the immature seeds and sheaths of the Chinese cabbage, Brassicu campestris L. var. pekinensis, the leaves of green tea, Thea sinensis, and the insect galls of the chestnut tree, Cusanea crenatu L. Sieb. et SUCC.~,*.Further analysis of these plant sources revealed the occurrence of brassinone (5), 28-norbrassinolide (4) and 0

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HO,,,,

Fig. 1. Structure of brassinolide (1).

24-ethylbrassinone (7)9. In 1968, Marumo and co-workers reported the isolation of distylium factors A, and B from the leaves of Distylium recemosum Sieb. et Zucc.“. The two factors showed much higher activity than indol-3acetic acid in the rice lamina inclination assay. Our reinvestigation of these materials has revealed that factor A, is a mixture of brassinone (5) and castasterone (2) and factor B is a mixture of brassinolide (1) and 2%norbrassinolide (4)‘l. Extensive studies on active substances in immature seeds have been carried out by Yokota and Takahashi. They have isolated 6-deoxocastasterone (3) and 6-deoxodolichosterone (10) in addition to dolichosterone (9)) homodolichosterone (12) and homodolicholide (11) and three known brassinosteroids

from the immature seeds of Dolichos lablab L. They also indicated the occurrence of more than brassinosteroids in the immature seeds of Phase02 vulgaris L. 13. Among them, 25methyldolichost rone is interesting as it is the first C-25 methylat brassinosteroid14. Other compounds characterizl include the stereoisomers at the 2, 3, and 24-po’ tions of the known brassinosteroids and 2-epi-2 methyldolichosterone-23-O-P_D-glucopyranoside15 Isolation of typhasterol (Zdeoxycastasteron from pollen of cat-tail, Typha Zatifolia L.16 and pine, Pinus thunbergii17 and teasterone (3-epimer ( typhasterol) from the leaves of green tea” has al! been reported. GC and HPLC of brassinosteroid derivatives For GC analysis, trimethylsilyl ether and meth neboronate ester derivatives of brassinosteroids cl be used. The bis-methanoboronate (BMB) deriv tive (16) seems to be the most appropriate becau: of the presence of two vicinal diols in the A-ring ar the side chain. A series of BMB derivatives of bras: nosteroids were separated as sharp well-resolve peaks Fig. 3) with detection limits at nanogram le 1‘. In order to establish a precise analytic e1s11p19, method, [26,27-21-16]brassinolide has been synthl sized for use as an internal standard21. For HPLC, use of a bis-boronate which can 1:

HO ., HO“

SC \

OH R

R=CH,

Brassinolide

R=H

28-Norbrassinolide

R=C2H5

Homobrassinolide

R=H

Dolicholide

R=CH,

Homodolicholide

I

(4)

6-Deoxocastasterone

(3)

Brassinone (5) (Norbrassinosterone) Homocastasterone (7) (24-Ethylbrassinone)

(6)

Dolichosterone

(8)

24-Ethylbrassinolide

Fig. 2. Structure and nomenclature

Castasterone (2) (Brassinosterone)

(1)

(11)

(14)

(Y)

Homodolichosterone

24-Epicastasterone

of typical brassinosteroia’s (SC = side chain).

6-Deoxodolichosterone (12)

(15)

(10)

6-Deoxohomodolichosterone

(13

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Fig. 3. GC analysis of a mixture of his-methanoboronates of brassinolide (1) castasterone (2), norbrassinolide (4), brassinone (S), homobrassinolide (6), and homocastasterone (7). Conditions: glass capillary column coated with OV-I 7,25 m x 0.25 mm I.D., temp. 280°C; helium flow-rate, 0.5 mllmin; split ratio, 1:3; detector, FID.

monitored by a UV or a fluorimetric detector is desirable. The bis-naphthaleneboronates (17) can be measured by a UV detector down to 100 pg”“. The bis-9-phenanthreneboronates (18) can be measured by a fluorimetric detector down to lo-20 pg”“. Separation of bis-9-phenanthreneboronate derivatives of a brassinosteroid mixture is shown in Fig. 423. Although these bis-boronate derivatives of brassinosteroids can be separated by a conventional ODS column, use of a small bore column such as a Shimpack SBC-ODS affords better resolution22. Partially purified brassinosteroid fractions from several plants have been analyzed by HPLC in this way, after derivatization into bis-boronate analogues. bis-1-cyanoisoindole-2-m-phenylboronate The 24,25 and the bis-dansylaminophenylboronate g25 , can be also used for HPLC with a fluorimetric detector. These derivatives allow the detection of pg levels of brassinosteroids. Structures of the bis-boronate derivatives for HPLC analysis are given in Fig. 5. Mass fragmentation of brassinosteroids and their bis-methaneboronate derivatives MS is a powerful tool for the structural determination of steroids; this is also the case for the brassinosteroids. The fragmentation pattern in the electron impact mass spectrometry (EI-MS) of brassinolide is shown in Fig. 620. The molecular ion at m/z 480 is very weak and occasionally is not observed at all. The characteristic fragment ions are m/z 409, 380, 350, 322, 177 and 173. Further ions resulting from these ions by a loss of a methyl group and/or water were also observed. It should be noted that the ions at m/z 380,350 and 322 were accompanied by hydrogen transfer.

I

I

5

10

I 20

I 15

”

Fig. 4. HPLC analysis of a mixture of bisphenanthreneboronates of brassinolide (l), castasterone (2), norbrassinolide (4), brassinone (S), homobrassinolide (6), homocastasterone (T), and dolichosterone (9). Conditions: 5 pm STR ODS-H column, 15 cm x 4.0 mm I.D., mobile phase, acetonitrile-water (9:1), flow-rate, 0.8 mllmin, temp. 45°C; fluorimetric detector (Shimad 24 Model RF535).

In the field desorption mass spectrometry (FDMS) of brassinolide, a strong protonated molecular ion at m/z 481 (M+l), the fragment ions at m/z 379 and 101 due to the C,,-C,, fission, and the fragment ions at m/z 349 and 131 due to the C,-C, fission were observed along with the water-eliminated ions at m/z 463, 445 and 3612’. The chemical ionizationmass spectrometry (CI-MS) OS brassinolide is sim-

16

R=

CH,

17

R=

:I / 03

18

R=

19

R=

Fig. 5. Structures of boronate derivates analysed by HPLC.

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99+1

I 333

MW=480

129

;

MW=462 9 428-l -

0 % MeB

MeB’ ‘o+ 332 MW=528

MW=526

l-BMB

8-BMB

MeB

i: MW=S12

MW=SlO

2-BMB

9-BMB

Fig. 6. Mass fragmentation thaneboronates.

of brassinosteroids

387

; 124

-

and their bisme-

pler than the FD-MS. The protonated molecular ion at m/z 481 (M + 1) was the base peak, and the watereliminated ions at m/z 463 and 445 were observed as prominent peaks2’. Dolicholide (8) and dolichosterone (9), which have a C,,-G, double bond, gave no molecular ions in either FD or EI modes. In the EI-MS of 926 the prominent ions at m/z 363 and 100 indicated that C,,-C,, fission occurred predominantly. The ions of m/z 345 and 327 represent elimination of one and two molecules of water from the ion of m/z 363, respectively. The ion of m/z 333, resulting from the C20-C22 fission, afforded the ion at m/z 315 by elimination of water. Additionally, the C,,-C,, bond fission gave rise to the weak ion at m/z 305, which also produced prominent ions at m/z 287 and 269 (m/z 305 - 18 and m/z 305 - 2 x 18). For these brassinosteroids, fast atom bombardment (FAB-MS was also found to be an effective method of obtaining the molecular ions. Dolicholide (8) gave rise to the ions at m/z 479 (M+l) and 571 [M+1+92 (G, glycerin)] along with the ions at m/z 461 and 443 which represent elimination of one and

two water molecules from the molecular ion of m/z 479, respectively6. In the FAB-MS of dolichosterone (9), the molecular ion at m/z 463 (M+H) and 555 (M+H+G) ions were observed together with the ions at m/z 445 (463 - 18) and 427 (463 - 2 x 18)26. Similarly, the FAB-MS of castasterone (2) gave rise to the molecular ion at m/z 465 (M+l) and 557 [M+1+92 (G)] along with the water-eliminated ions at m/z 447 and 42g5. Therefore, FAB-MS is very effective for determining the molecular ion of the polyhydroxylated brassinosteroids. The fragmentation resulting from the EI-MS of bis-methaneboronate of brassinolide (l-BMB) is shown in Fig. 619. The derivatives of brassinolide (lBMB), homobrassinolide (6-BMB) and norbrassinolide (4-BMB) afforded the respective molecular ions at m/z 528,542 and 514 and common ions at m/z 457 due to the C2,-C24 fission, at 374 due to the C,,-C,, fission, at m/z 345 due to the C,7-C20 fission and at m/z 177 (assignment shown in Fig. 6). Thus, these fragment ions are characteristic for the brassinolide skeleton’1~19~20.The peak at m/z 374 is accompanied by a hydrogen transfer. The ions at m/z 155, 169 and 141 corresponding to the side-chain cleavage of compounds l-BMB, 6-BMB and 4-BMB are base peaks in these spectra. The derivatives afforded characteristic ions for a B-ring lactone at m/z 332, 346 and 318, respectively. These characteristic ions are useful for the structural determination of brassinolide analogues. The EI-MS fragmentation pattern of C-24(28)-unsaturated brassinosteroids such as dolicholide (8BMB) and dolichosterone (PBMB) is different from that of saturated brassinosteroids such as brassinolide (l-BMB) and castasterone (2-BMB) (Fig. 6). In particular, the fragment ions resulting from cleavage of the cyclic boronate moiety of the side chain are different. The fragment ions are observed at m/z 427,403,385,124 and 82 for 8-BMB and at m/z 411, 387, 369, 124 and 82 for 9-BMB as shown in Fig. 6. Another remarkable difference is that hydrogen transfer observed from the C,,-C,, fission in the saturated series is not recorded in the case of the unsaturated series (&BMB and 9-BMB), but two-hydrogen transfer from the C17-C20 fission is observed for the unsaturated series. In the case of CI-MS, the ions corresponding to M+ 1 at 529,543 and 515 are base peaks for brassinolide (I-BMB), homobrassinolide (iBMB) and norbrassinolide (4-BMB), respectively. They also give side-chain cleavage ions at m/z 345 (C1,-C20 fission), and m/z 155,169 and 141, together with ions at m/z 373 (C20-C22 fission). The ions at m/z 345 and 373 are common for the brassinolide skeleton. Similarly, the ions at m/z 329 and 357 are common peaks

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.

m/r 358.2679 A Brassinone-

h-h I

I

8:30

9:oo

I

9:30

lo:oo

10: 30

ll:oo

11:30 min

m/z 374.2626

1 C

Brassinolide-

8:30

9:oo

9:30

lo:oo

10:30

ll:oo

11:30 min

24-Epibrassinolid

i

1O:lS IO:30 IO:45 11:OO 11:15 11:30 11:45 min

Fig. 7. Selective ion monitoring of bismethaneboronates of brassinosteroid fraction from the pollen of the broad bean. (A) Detection of brassinosteroid with &ketone moiety by monitoring at m/z 358.2679. (B) Detection of brassinosteroid with B-ring lactone moiety by monitoring at mlz 374.2628. (C) Expansion of the pertinent peaks. Conditions: VGA 70-S gas chromatograph-mass spectrometer (VG Analytical Ltd.), OV-I capillary column, 12.5 m x 0.2 mm I.D., programmed oven temperature IlO-32o”C, 25”Clmin, injection temp. ?DCOP

for the castasterone (2-BMB) skeleton. These ions are useful for selective ion monitoring for the screening of brassinosteroids. Thus, picogram amounts of brassinosteroids can be detected in plant extract by means of GC-CI-MS. Applications Applications of GC, using bis-methaneboronate derivatives, on the brassinosteroids of the sheaths of Chinese cabbage, the leaves of green tea and the insect gall of the chestnut tree are described above. Similarly, the selective ion monitoring of m/z 499 indicated the presence of brassinone (5) in the leaves (16 ng/kg) and the insect galls (5 pug/kg) of Distylium rucemosum. Scanning m/z 513 demonstrated the occurrence of castasterone in the leaves (133 @kg) and the insect galls (2.5pglkg)“. The ion monitoring of m/z 511 and 513 of the active fraction from the rice plant, Oryza sativa, showed the presence of dolichosterone (13.6 rig/kg) and castasterone (8.4 ng/kg)27. The utility of the method has also been verified by Yokota and a co-worker13,28,29 We have carried out analysis of brassinosteroids in several pollens. Bee pollen of the broad bean, Viciu fubu L. (2 kg) was extracted with methanol, and the chloroform-soluble material was separated by silica gel column chromatography followed by preparative

thin-layer chromatography. The brassinosteroid fraction (cu. 10 mg) was analyzed after derivatization to methanoboronates, by a VGA-70-S instrument with an OV-1 capillary column. Multiple ion monitoring at m/z 358.2679 for B-ketone and m/z 374.2628 for lactone indicated the presence of brassinone (5), castasterone (2), brassinolide (1) and 24epibrassinolide in this pollen, as shown in Fig. 730. This is the first identification of 24-epibrassinolide in plants. As an example of HPLC analysis, Fig. 8 illustrates the chromatogram of the bis-phenanthreneboronate derivatives of brassinosteroid fraction obtained from sunflower (Heliunthus unnuus L.) pollen31. GC-CI-MS analysis indicated that brassinosteroids exist in plants as mixtures of several analogues, and are more highly concentrated in pollen (cu. 100 pug/kg), immature seeds (several ,q/kg) and insect galls (several ,ug/kg) than in other organs (several @kg)Recently, Yokota’s group has reported the ratioimmunoassay of castasterone with combined BSA after conversion to (6-0-carboxymethyl)oxime Nhemi-succinimide32. [4-‘4C]-24-Epibrassinolide has been synthesized by Shionogi’s group for studies on the transformation and metabolism of brassinosteroids in plants33.

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6 T. Yokota, J. Baba and N. Takahashi, Tetrahedron Lett., 2: (1982) 4965. 7 N. Ikekawa, Trends Anal. Chem., 3 (1984) 81. 8 H. Abe, T. Morishima, M. Uchiyama, S. Marumo, K. Mu nakata, S. Takatsuto and N. Ikekawa, Agric. Biol. Chem. 46 (1982) 2609. 9 H. Abe, T. Morishita, M. Uchiyama, S. Takatsuto, N. Ike kawa, M. Ikeda, T. Sasa, T. Kitsuwa and S. Marumo, Expe rientia, 39 (1984) 351.

10 S. Marumo, H. Hattori, H. Abe, Y. Nonoyama and K. Mu nakata, Agric. Biol. Chem., 32 (1968) 528. 11 N. Ikekawa, S. Takatsuto, T. Kitsuwa, H. Saito, T. Morishi ta and H. Abe, .I. Chromatogr., 290 (1984) 289. 12 T. Yokota, M. Morita and N. Takahashi, Agric. Biol Chem., 47 (1983) 6.55. 13 T. Yokota, S. Koba, S. K. Kim, S. Takatsuto,

N. Ikekawa N. Sakakibara, K. Okada, K. Mori and N. Takahashi, Agric Biol. Chem., 51(1987) 1625. 14 S. K. Kim, T. Yokota and N. Takahashi, Agric. Biol. Chem. 51(1987) 2303.

15 T. Yokota, S. K. Kim, Y. Kosaka, Y. Ogino and N. Takaha shi, Proc. Int. Symp. on Conjugate Plant Hormones, Germ November3-7,1980, p. 288. 16 J. A. Schneider, K. Yoshihara, K. Nakanishi and N. Kate

J 0

Tetrahedron Lett., 24 (1983) 3859. I

I

I

I

5

10

15

20

min

Fig. 8. HPLC analysis of the brassinosteroid fraction from the pollen of the sunflower as the bisphenanthreneboronate derivatives. Conditions, see Fig. 4.

17 T. Yokota, M. Arima, N. Takahashi, S. Takatsuto, N. Ike kawa and T. Takematsu, Agric. Biol. Chem., 47 (1983) 2419 18 H. Abe, T. Morishita, M. Uchiyama, S. Takatsuto and N Ikekawa, Agric. Biol. Chem., 48 (1984) 2171. 19 B. Takatsuto, M. Ying, M. Morisaki and N. Ikekawa, J Chromatogr., 239 (1982) 233.

Conclusion Japanese scientists have made notable contributions to research on brassinosteroids in the past few years. Comparative studies of brassinosteroids in as many as 29 families and 46 genuses have been reported to date. Among them castasterone (2) appears to be the most commonly distributed. In these studies the rice-lamina inclination assay and GCMS using the bis-methaneboronate derivative have been effectively employed. Field testing on brassinosteroids [epibrassinolide, brassinolide and (22S,23S)-homobrassinolide] for increasing crop yield has been carried out in various areas and promising results have been obtained for wheat, rice, soybean, corn, and also for several vegetables34T35.It is expected that the brassinosteroids will become important materials for food production in the future. References 1 M. D. Grove, G. F. Spencer, W. K. Rohwedder, N. Mandava, J. F. Worley, J. D. Warthen, Jr., G. L. Steffens, J. L. Flippen-Anderson and J. C. Cook, Jr., Nature, 281 (1979) 216. 2 J. W. Mitchell. N. Mandava, J. W. Jorley and J. R. Plimmer, Nature, 225 (1970) 1065. 3 K. Wada, S. Marumo, N. Ikekawa, M. Morisaki and K. Mori, Plant Cell Physiol., 22 (1981) 323. 4 E. Maeda, Physiol. Plant., 18 (1965) 813. 5 T. Yokota, M. Arima and N. Takahashi, Tetrahedron Lett., 23 (1982) 1275.

20 N. Ikekawa and S. Takatsuto, Mass Spectrosc., 32 (1984) 55 21 S. Takatsuto and N. Ikekawa, Chem. Pharm. Bull., 3 (1986) 4045.

22 K. Gamoh, T. Kitsuwa, S. Takatsuto, Y. Fujimoto and N Ikekawa, Anal. Sci., 4 (1988) 533. 23 K. Gamoh, K. Omote, N. Okamoto and S. Takatsuto, J Chromatogr., 469 (1989) 424.

24 K. Gamoh and S. Takatsuto,

Anal. Chim. Acta, 222 (1989

201.

25 K. Gamoh, N. Okamoto, S. Takatsuto and I. Tejima, Anal Chim. Acta, 228 (1990) 101. 26 J. Baba, T. Yokota and N. Takahashi, Agric. Biol. Chem. 47 (1983) 659.

27 H. Abe, K. Nakamura, T. Morishita, M. Ichiyama, S. Takat suto and N. Ikekawa, Agric. Biol. Chem., 48 (1984) 1103. 28 M. Arima, T. Yokota and N. Takahashi, Phytochem., 2. (1984) 1587.

29 K. Park, H. Saimoto, S. Nakagawa, A. Sakurai, T. Yokota N. Takahashi and K. Syono, Agric. Biol. Chem. 53 (1989 805.

30 N. Ikekawa, F. Nishiyama and Y. Fujimoto,

Chem. Pharm Bull., 36 (1988) 405. 31 S. Takatsuto, T. Yokota, K. Omote, K. Gamoh and N. Ta kahashi, Agric. Biol. Chem., 53 (1989) 2177.

32 T. Yokota, T. Watanabe, Y. Ogino, I. Yamaguchi and N Takahashi, .I. Plant Growth Regulation, 9 (1990) 151. 33 S. Seo, T. Nagasaki, Y. Katsuyama, F. Matsubara, T. Saka ta, M. Yoshioka and Y. Makisumi, .I. Labelled Comp Radiopharm.,

27 (1989) 1383.

34 T. Takematsu and Y. Takeuchi, Proc. Jpn. Acad. Ser. B, 6. (1989) 149.

35 N. Ikekawa and Y. Zhao, unpublished

data.

Professor N. Ikekawa is at Iwaki Meisei University, Iwaki, Fuku shima 970, Japan.