[23] Ecdysone conjugates: Isolation and identification

[23]

ECDYSONECONJUGATES

411

[23] E c d y s o n e C o n j u g a t e s : I s o l a t i o n a n d I d e n t i f i c a t i o n By C. HETRU, B. LUU, and J. A. HOFFMANN Following the isolation ~ and structural elucidation 2,3 of ecdysone, a series of investigations started on the biosynthetic and catabolic pathways of this steroid hormone. In 1970, Heinrich and Hoffmeister4 proposed that in the blowfly Calliphora erythrocephala, ecdysone was inactivated as a glycoside conjugate (incorporation studies with [~4C]glucose). Several subsequent studies on a variety of insect species showed that enzymatic hydrolysis of highly polar ecdysteroid yielded free ecdysone and/or 20hydroxyecdysone which led to the assumption that conjugation was a major catabolic pathway of ecdysteroids (review in KoolmanS). On the basis of incorporation experiments of labeled molecules into ecdysteroid conjugates and of hydrolytic studies with defined enzymes, it was concluded that ecdysteroids were inactivated as sulfate, glucoside, glucuronide, or phosphate conjugates (references in review cited above). It is only very recently that an ecdysteroid conjugate was isolated from fecal material of Locus ta migratoria 6 and Schistocerca gregaria 7 and identified by physical methods as a 2-phosphate 3-acetate of 20-hydroxyecdysone. By similar methods, Malphighian tubules-digestive tract complexes were shown in L. migratoria to convert 20-hydroxyecdysone to a 22-phosphate 2- (or) 3-acetate conjugate. 8 Independently from the studies on inactivation of ecdysteroids during postembryonic development, Ohnishi and co-workers 9 had noted in 1977 that 2-deoxyecdysone was present in ovaries of Bombyx mori pupae as a polar compound presumed to be a conjugate. The observation that ova1 A. Butenandt and P. Karlson, Z. Naturforsch., B: Anorg. Chem., Org. Chem., Biochem.. Biophys., Biol, 9B, 389 (1954). 2 p. Karlson, H. Hoffmeister, H. Hummel, P. Hocks, and G. Spiteller, Chem. Ber. 98, 2394 (1965). 3 R. Huber and W. Hoppe, Chem. Bet. 98, 2403 (1965). 4 G. Heinrich and H. Hoffmeister, Z. Naturforsch., B: Anorg. Chem., Org. Chem., Biochem., Biophys., Biol. 25B, 358 (1970). 5 j. Koolman, Insect Biochem. 12, 225 (1982). 6 j. F. Modde, Thesis, Universit6 Pierre et Marie Curie, Paris (1983) (submitted as as article for J. Invertebr. Reprod. Dev.) 7 R. E. Isaac and H. H. Rees, Commun. Int. CNRS Syrup. lnvertebr. Horm., 1983 (1983). G. Tsoupras, B. Luu, C. Hetru, ]. F. Muller, and J. A. Hoffmann, C.R. Hebd. Seances Acad. Sci., Set. D 296, 77 (1983). 9 E. Ohnishi, T. Mizuno, F. Chatani, N. Ikekawa, and S. Sakurai, Science 197, 66 (1977).

METHODS IN ENZYMOLOGY, VOL. 1 ! 1

Copyright © 1985 by Academic Press, luc, All rights of reproduction in any form reserved. ISBN 0-12-182011-4

412

STEROL METABOLISM

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ties contain large quantities o f ecdysteroids predominently in conjugated form has since been confirmed in a variety o f species which include S. gregaria, 1o-13 L . migratoria, 14,15 and Galleria mellonella.16 The observations that these conjugated ecdysteroids are synthesized inside the vitellogenic ovaries, ~7,18 accumulate in the oocytes, and are obviously hydrolyzed during embryogenesis 11,14 have prompted studies toward the isolation and identification of these compounds, and several structures have recently been reported. J2,13,19-22 The present chapter will essentially deal with the methods of purification and structure determination of ecdysteroid conjugates. Extraction and Purification F o r the extraction of ecdysteroid conjugates the various authors mostly use polar solvents. Pure methanol, 2z various proportions of methan o l - w a t e r mixtures/3,2j or even pure water 6,23 are r e c o m m e n d e d in the literature. As a rule, the extraction b y polar solvents is followed by partition destined to remove the majority of the lipophilic substances: either m e t h a n o l - w a t e r / h e x a n e partitions 13,2L24or water/chloroform partitions 6,23 can be used. The ecdysteroid conjugates recovered in the polar phase of these partitions can subsequently be submitted to liquid chromatography, either on a silicic acid column 6,~3 or on a reverse-phase Ca column. 21 It has been r e c o m m e n d e d 24 to partition the residues from the first partition step between water-saturated n-butanol and water: the free ecdysteroids are then 10I. D. Wilson and E. D. Morgan, Insect Physiol. 24, 751 (1978). 11L. N. Dinan and H. H. Rees, Insect. Physiol. 27, 51 (1981). ~2R. E. Isaac, M. E. Rose, H. H. Rees, and T. W. Goodwin, Chem. Commun. p. 249 (1982). 13R. E. Isaac, M. E. Rose, H. H. Rees, and T. W. Goodwin, Biochem. J. 213, 533 (1983). 14j. A. Hoffmann, M. Lagueux, C. Hetru, M. Charier, and F. Goltzen6, in "Progress in Ecdysone Research" (J. A. Hoffmann, ed.). Elsevier, Amsterdam, 1980. 1~M. Lagueux, C. Sail, and J. A. Hoffmann, Am. Zool. 21, 751 (1981). 16T. H. Hsiao and C. Hsiao, Insect Physiol. 25, 45 (1979). 17M. Lagueux, M. Him, and J. A. Hoffmann, Insect Physiol. 23, 109 (1977). 1~F. Goltzenr, M. Lagueux, M. Charlet, and J. A. Hoffmann, Hoppe-Seyler's Z. Physiol. Chem. 359, 1427 (1978). 19R. E. Isaac, H. H. Rees, and T. W. Goodwin, Chem. Commun. p. 594 (1981). 20G. Tsoupras, C. Hetru, B. Luu, M. Lagueux, E. Constantin, and J. A. Hoffmann, Tetrahedron Lett. p. 2045 (1982). 2~G. Tsoupras, C. Hetru, B. Luu, E. Constantin, M. Lagueux, and J. A. Hoffmann, Tetrahedron 39, 1789 (1983). 22G. Tsoupras, B. Luu, and J. A. Hoffmann, Science 220, 507 (1983). 23R. Lafont, J. L. Pennetier, M. Andrianjafintrimo, J. Claret, J. F. Modde, and C. Blais, J. Chromatogr. 236, 137 (1982). 24 S. Scalia and E. D. Morgan, J. Chromatogr. 238, 457 (1982).

[23]

ECDYSONE CONJUGATES

413

TABLE I PURIFICATIONOF ECDYSTEROIDCONJUGATESBY HPLC: TECHNIQUESUSEDIN THE RECENTLITERATURE Stationary phase

Mobile phase

ODS, 5~m Spherisorb (Clwyd, U.K.)

Linear gradient (12 rain) from 25 to 50% methanol in 0.4 M ammonium acetate buffer (pH 7) in the continuous presence in the mobile phase of 0.003 M tetrabutylammonium hydroxide ODS, Zorbax Linear gradient from 8 to 40% acetonitrile in 0.02 M (Dupont) Tris/perchloric acid buffer (pH 7.5) or in 0.05 M ODS, Ultrasphere sodium citrate buffer (pH 6.1 or 3) (Beckman) ODS, 5t~m, Linear gradient from 12 to 44% acetonitrile in 0.2 M Lichrosorb, Tris-HCl buffer (pH 7.4) (Merck) ODS-3, Partisil Isocratic elution (32 min) with 35% methanol in 0.2 M (Whatman) sodium acetate buffer (pH 5.5) Linear gradient (30 rain) from 10 to 70% methanol in 0.02 M sodium citrate buffer (pH 6.5) SAX, Partisil Isocratic elution with 0. I M ammonium acetate (Whatman)

Reference 24

6

25 13

13

recovered in the butanol phase, whereas the polar conjugates are present in the aqueous phase. All investigations on ecdysteroid conjugates use H P L C for further purification, either on a preparative or on an analytical scale, Table 125 gives the details on the stationary and mobile phases and the modes of elution used in recent studies. We have summarized in Fig. 1 a complete procedure for extraction and purification which we r e c o m m e n d at present for the extraction of polar ecdysteroid conjugates. Identification Procedures The identification of ecdysteroid conjugates is usually by a combination of spectroscopic techniques: infrared and ultraviolet absorption, mass spectrometry, and 1H, ~3C, 31p nuclear magnetic resonance.

Infrared S p e c t r o s c o p y Infrared spectroscopy can be useful in the study of ecdysteroid conjugates. In the study of Tsoupras et al. 2~ three bands of absorption are 25G. Tsoupras, Thesis, Universit6 Louis Pasteur, Strasbourg (1982).

414

STEROL METABOLISM I

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Homogenizationin L methanol/water 50v/5,0vF

I

,

CenCrifugation

reprocessing 3 times

(lO00g, I0 min)

I

Supernatant 4,

1

Partition

]

,Methanol-water/hexaneI

. Pellet

]

Methanol-water phase reduced (low pressure or N2)

1

Hexane phase discarded

C-8reverse-phase

Free ecdysteroids 4

liquid

Ichromatography; linear |gradient from pure water ]to 50% aqueous methanol; 160 min; 4 ml/min Polar ecdysteroids (RIA before and after hydrolysis)

Elution and RIA I silicage] thin-layer before and a f t e r - m - ~ chromatography hydrolysis ]chloroform/n~thanol 6Ov/4Ov

/

C-18 reverse-phase HPLC (preparative); exponential gradient from 20% to 60% aqueous methanol; 20 min; 4 ml/min

Pure polar • ecdysteroid conjugates

FIG. 1. Proposed extraction and purificationprocedures for polar ecdysteroid conjugates from insects or other invertebrate systems.

given for several ecdysteroid conjugates: two correspond to the ecdysteroid moiety [IR(KBr, cm-l): 1640 for the C-~-O bond and 3400 for the O - - H bonds]; a third band at 1050-1100 is indicative of the presence of a P - - O - - C bond.

Ultraviolet Spectroscopy The presence of an (x,fl-ethylenic ketone in ecdysteroids explains the strong absorption at 250 nm in ecdysteroid conjugates.~3,zl

[23]

ECDYSONECONJUGATES

415

TABLE 1I CHARACTERISTIC ~H NMR DATA OF SOME RELEVANT ECDYSTEROIDS AND ECDYSTEROID CONJUGATES

Chemical shifts of methylgroups Compound Ecdysone 2-Deoxyecdysone 20-Hydroxyecdysone 22-Phosphate of ecdysone 22-Phosphate of 20-hydroxyecdysone 22-Phosphate of ecdysone 22-Phosphate of 2-deoxyecdysone 22-Adenosine monophosphate of ecdysone

C-18 C-19 C-21 C-26 C-27

Solvent

0.74 0.74 1.19 0.70 0.92

1,07 1.05 1.05 0.96 0.98

Reference

1.28 1,28 1.54 0.92 1.32

1.38 1.38 1.38 1.20 1.21

1.38 1.38 1.38 1.20 1.21

CsD5N CsDsN CsD~N DzO D20

25 25 25 25 26

0.75 0.97 0.98 0.73 0.96 0.97

1.18 1.18

1,19 1.18

CD3OD CD3OD

12 12

0.72

1.20

1.20

D20

21

0,96 0.92

Nuclear Magnetic Resonance (NMR) Nuclear magnetic resonance is a powerful tool for the identification of ecdysteroid conjugates. As a rule, tH NMR and t3C NMR spectra are first taken of the intact pure ecdysteroid conjugate, after which the conjugate is subjected to enzymatic or chemical hydrolysis (see below, Table IV). The free ecdysteroid is then purified and analyzed by ~H and ~3C NMR spectroscopy. The comparison between the spectra of the free identified ecdysteroid and the intact conjugated ecdysteroid usually allows inferences on the nature of the conjugating moiety. The comparison of chemical shifts of certain signals in ~H and t3C NMR between the free and the conjugated ecdysteroid is useful in assigning the position at which the linkage between the conjugating moiety and the ecdysteroid has occurred. Table I126gives a certain number of characteristic chemical shifts in ~H NMR of relevant ecdysteroids and ecdysteroid conjugates. In Table III the ~3C NMR signals of the 27 carbon atoms of several ecdysteroids are also presented and the multiplicity of the signals is indicated. These tables illustrate, for instance, that in the JH NMR spectra of the 22-phosphate of ecdysone, the signal of the C-21 methyl group is shifted downfield. In ~3C NMR, the signal of the carbon C-22 of 2-deoxyecdysone is shifted downfield, whereas the signals of C-20 and C-23 are upfield shifted. This sort of observations had led ~3,2~to the conclusion that the conjugating moiety is linked to the ecdysteroids in the C-22 position in these compounds. z6G. Tsoupras, B. Luu, and J. A. Hoffmann,Steroids 40, 551 (1982).

416

[23]

STEaOL METABOLISM

TABLE III 13C NMR DATA OF ECDYSTEROIDS AND

Number of carbon atoms

A

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

37.90(0 68.03(d) 68.03(d) 31.80(0 51.30(d) 203,20(s) 121.50(d) 165.60(s) 34.50(d) 38.60(s) 21.10(t) 31.40(t) 47.50(s) 83.80(s) 32.40(0 25.50(t) 48.30(d) 15.80(q) 24.40(q) 42.90(d) 13.60(q) 73.90(d) 26.60(t) 42.40(0 69.70(s) 30.01(q) 30.20(q)

B 29.48(0 29.07(0 64,06(d) 33.07(0 51.61(d) 203.20(s) 121.30(d) 166.01(s) 34.40(d) 36.96(s) 21.00(t) 31.70(0 48.00(s) 84.01(s) 31.70(t) 25.50(0 48.32(d) 15.80(q) 24.30(q) 42.99(d) 13.60(q) 73.90(d) 26.70(0 42,46(0 69.60(s) 29.98(q) 30.20(q)

CONJUGATES

C 29.50(0 28.20(0 66.04(d) 32.80(0 52.21(d) 210.00(s) 121.00(d) 170.00(s) 35.50(d) 37,60(s) 21.80(t) 32.10(t) -86.70(s) 31.70(0 26.40(t) 48.76(d) 16.57(q) 24.40(q) 40.80(d) 13,60(q) 81.09(d) b 24.90(0 41.24(0 73.02(s) 29.00(q) 29.20(q)

a

D 34.50(0 32,10(0 69.30(d) 35.90(0 57.40(d) 202.20(s) 121.50(d) 166.06(s) 34.20(d) 37.00(s) 21.00(t) 31.60(0 47,80(s) 83.90(s) 31.80(0 35.80(t) 48.50(d) 16.00(q) 24.10(q) 43.20(d) 13.90(q) 74.20(d) 26.80(0 42.70(t) 69.80(s) 30.00(q) 30.20(q)

E 34.00(0 29.70(0 74.60(d) b 33.60(t) 56.90(d) 207.80(s) 121.70(d) 169.60(s) 34.60(d) 37.20(s) 21.00(t) 31.70(t) 48.00(s) 85.90(s) 32.20(0 25.20(t) 48.50(d) 16.40(q) 23.80(q) 42.70(d) 13.50(q) 75.50(d) 26.60(0 41.50(t) 72.70(s) 29.10(q) 28.80(q)

Ecdysone (A), 2-deoxyecdysone (B), 22-phosphate of 2-deoxyecdysone (C), 3-epi 2deoxyecdysone (D), and 3-phosphate of 3-epi-2-deoxyecdysone (E). Spectra of A, B, and D were obtained in [2H~]pyridine, C and E in D20; ~ in parts per million from trimethylsilane (internal standard); s, singlet; d, doublet; t, triplet, q, quadruplet. From Ref. 25. b In proton-noise decoupled spectra, this signal appears as a doublet. 31p N M R is m o s t l y used to confirm the p r e s e n c e o f a p h o s p h a t e g r o u p in s o m e c o n j u g a t e s . 13,z~

Mass Spectrometry M o s t e c d y s t e r o i d c o n j u g a t e s are polar c o m p o u n d s and are not v e r y volatile. I n addition, t h e y are p r o b a b l y unstable u n d e r various conditions. A v a r i e t y o f t e c h n i q u e s o f volatilization and ionization h a v e b e e n em-

t,4

~,~

~ ~

=

~

_~ E ~

0

"=

<

© rj ,,-,,

0

..J to-1

<

r"

r..t4

e-,

~

~

t¢'-~

0 ©

e-

Z © rJ

E

E

~

r~

418

STEROLMETABOLISM

[23]

TABLE V ECDYSTERO1D CONJUGATES ISOLATED AND IDENTIFIED IN INSECTS

Compound

Reference

2-Acetate of ecdysone 2-Acetate of 20-hydroxyecdysone 3-Acetate of ecdysone 3-Acetate of 20-hydroxyecdysone 22-Adenosine monophosphateof 2-deoxyecdysone 22-Adenosine monophosphateof ecdysone 22-N6-(Isopentenyl)adenosinemonophosphateof ecdysone 2-Phosphate-3-acetate of ecdysone 2-Phosphate-3-acetate of 20-hydroxyecdysone 3-Phosphate of ecdysone 3-Phosphate of 3-epi-2-deoxyecdysone 3-Phosphate of 20-hydroxyecdysone 22-Phosphate 3- (or 2-)acetate of 20-hydroxyecdysone 22-Phosphate of 2-deoxyecdysone 22-Phosphate of 2-deoxy-20-hydroxyecdysone 22-Phosphate-2,3-diacetate of ecdysone 22-Phosphate of ecdysone 22-Phosphate of 20-hydroxyecdysone

6 6 6,19 6 20,21 20,21 22 6 6 25 20,21 6 26 12,13,21 13,26 25 12,13,21 13,26

ployed: chemical ionization, ~2m laser ionization,8 gold thread technique, 2~ and fast heating technique. 25 Of special interest is the recently developed technique of fast atom bombardment, which allows the recording of the quasimolecular ion (e.g., for 22-phosphates of ecdysone, 20-hydroxyecdysone, 2-deoxyecdysone, and 2-deoxy-20-hydroxyecdysone, see Isaac et al.13), Hydrolysis Table IV lists several procedures which are commonly used for the enzymatic hydrolysis of ecdysteroid conjugates, as well as the operating conditions. Helix pomatia juice is the most widely used enzymatic mixture in this field; it should be emphasized that some Helix preparations contain ecdysteroids. Ecdysteroid conjugates can also be hydrolyzed chemically, e.g., by a 2 hr treatment with 0.1 N HC1. z5 Concluding Remarks Table V gives a list of ecdysteroid conjugates which have been isolated and subsequently identified by physicochemical methods. Most of these results have been obtained over the last 2 years and there can be no doubt that this list will rapidly increase.

[24]

ECDYSONEOXIDASE

419

With the exception of the acetate esters of ecdysteroids, the identified conjugates are all polar compounds (in fact, mainly phosphates). There is however increasing evidence for the existence of ecdysteroid conjugates of very low polarity: ticks for instance are capable of esterifying ecdysone with fatty acids.27-29 For the extraction of such esters, ethanol can be recommended. Until more information becomes available, partitions as used in Fig. 1 for the purification of polar conjugates, should be employed with great care for the nonpolar conjugates. Esterases or K2CO3 treatment is preferred to the Helix digestive juice for hydrolysis of these products. 27 p. Diehl, J. L. Connat, P. Vuilleme, M. Morici, and J. Bouvier, Commun. Int. CNRS Symp. Inuertebr. Horm., 1983 (1983). 28 K. P. Wigglesworth and H. H. Rees, Commun. Int. CNRS Syrup. lnvertebr. Horm., 1983 (1983). ,,9 p. Diehl and R. Lafont, personal communication.

[24] Ecdysone Oxidase By J. KOOLMAN Ecdysone oxidase (EC 1.1.3.16) catalyzes the reaction shown in Fig. 1.) The enzyme is found m insects. It is probably part of the catabolic system that inactivates the molting hormones? Products of the reaction of ecdysone oxidase are 3-dehydroecdysteroids. These metabolites do not accumulate in vioo but are converted further to 3a-ecdysteroids (3-epiecdysteroids) by action of a 3-dehydroecdysone reductase.3.4 Recent experiments with the butterfly, Pieris brassicae, indicate that another reductase still exists which converts 3-dehydroecdysteroids into 3/3 forms. 5 3aEcdysteroids have been isolated or detected as ecdysone metabolites in various stages of insects 6-9 and represent along with conjugates and ecdysonoic acids major catabolites of ecdysone.t° 1 j. Koolman and P. Karlson, Eur. J. Biochem. 89, 453 (1978). z j. Koolman, in "Actualit6s sur les Hormones d'Invertebres" (M. M. Durchon, ed.), p. 403. CNRS, Paris, 1976. 3 p. Karlson and J. Koolman, Insect Biochem. 3, 409 (1973). 4 R. Lafont and J. Koolman, in "Biosynthesis, Metabolism and Mode of Action of Invertebrate Hormones" (J. A. Hoffman and M. Porchet, eds.), p. 196. Springer-Verlag, Berlin and New York, 1984. -~C. Blais and R. Lafont, Hoppe-Seyler's Z. Physiol. Chem. 365, 809 (1984). 6 j. N. Kaplanis, M. J. Thompson, S. R. Dutky, and W. E. Robbins, Steroids 34, 333 (1979). 7 j. N. Kaplanis, G. F. Weirich, J. A. Svoboda, M. J. Thompson, and W. E. Robbins, in

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