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Home , Ergocristine, Ergocryptine, Ergometrinine, Fumigaclavine C, Lysergol, Penniclavine

Acta Pharm, Suecica 2, 357 (1965)

Thin-layer chromatographic and thin-layer electro- phoretic analysis of .Relations between structure, RM value and electrophoretic mobility in the cle vine series

STIG AGUREll

Department of Pharmacognosy, Kunql, Farmaceuliska Insiitutei, Stockholm, Sweden

SUMMARY A thin-layer chromatographic and electrophoretic study of the ergot alkaloids has been made, to find rapid methods for the separation and identification of the known ergot alkaloids. The mobilities of ergot alkaloids in several useful chromatographic and electrophore- tic systems are recorded. Relations have been observed between structure and R" value in methanol-chloroform on Silica Gel G. A simple, rapid thin-layer electrophoretic technique has been de- vised for separation of ergot alkaloids, and a relation between structure and electrophoretic mobility is evident. Two-dimensional combinations of thin-layer chromatography and thin-layer electro- phoresis and chromatography are described.

Numerous paper chromatographic procedures have been published for separation of the ergot alkaloids and their derivatives. Hofmann (1) and Genest & Farmilio (2) have recently listed these systems. The general advantages of thin-layer chromatography (TLC) over paper partition chromatography are well known: shorter time of equilibration and devel- opment, generally better resolution, smaller amounts of substance rc- quired, and wider choice of reagents. Several reports of TLC of ergot alkaloids have been published. In gene- ral, these investigations (2-6 and others) have dealt 'with limited groups of alkaloids, or with a specific problem involving at most a dozen of the 40 now known naturally occurring ergot alkaloids. Some paper chromate-

.357 graphic systems using Iorrnamide-treated papers have also been adopted for thin-layer chromatographic use (7, 8). In our investigations on the of peptide-type ergot alkaloids (9), a number of alkaloidal metabolites of both the clavine type and the type could be expected. It was, therefore, necessary to collect a number of chromatographic data on known natural ergot alkaloids in suitable thin-layer chromatographic systems, to make it possible, in a rapid and predictable way, to separate and identify ergot alkaloids, deter- mine their purity or establish the association of radioactivity with a certain . Preferably, this method should involve one basic chro- matographic system which, by combination with other systems, would achieve this aim. Since the clavine and the lysergic acid types of ergot alkaloids generally coexist in ergot, and also for other reasons, it would be of general interest to know the distribution of all ergot alkaloids in certain useful thin-layer chromatographic systems. Except for a paper chromatographic investigation by Yamatodani (10), including most ergot alkaloids, no such extensive survey has appeared. It was also decided to investigate whether a rapid, thin-layer electro- phoretic method, suitable for routine use, could be devised. Although it is a common method for analysis of e. g. peptides and proteins; thin- layer electrophoresis has seldom been used for the analysis of alkaloids. Since the clavine-type ergot alkaloids comprise a large group of closely related compounds, they are a suitable group in which to study the rela- tions between structure and chromatographic or electrophoretic mobility. The present study is divided into three parts: 1. Thin-layer chromatography of ergot alkaloids 2. Relations between structure and R" value in the clavine series 3. Thin-layer electrophoresis of ergot alkaloids. Relations between structure and electrophoretic mobility

Experimental The ergot alkaloids- used in this investigation were obtained through the courtesy of other laboratories, or were isolated or synthesized by me, in which case, m. p., UV and IR spectra agreed with published data. Ergo- secaline and molliclavine, for which structures have been formulated (1, 11), were isolated in trace amounts by Abe et al, (11) but were no longer available, nor was isochanoclavine- (I), which was recently isolated as a trace alkaloid from rye ergot (12). Although 4-dimethylallyltrypto- ph an is an efficient precursor of the ergo line skeleton, it has not yet been identified as a normal metabolite (1, 11, 31). Nor- is, so far, recognized only as a microbial metabolite of agroclavine (18), and the structure of has not yet been published. (For for- mulas, see refs. 1, 11-13, 18,24.)

1 Some alkaloids are available from e. g. Koch-Light Labs. Ltd., Colnbrook;Cal- biochem, Los Angeles, and Flulia AG, Buchs.

358 All solvents used were of analytical reagent grade. Chloroform con- tained 1 % ethanol as stabilizer. Mean RF values and, for electrophoresis, relative mobilities (1\1) com- pared with (M = 1.00) were obtained from 5-6 runs of each compound on different plates, and are listed in the tables. The stan- dard deviations were calculated from the individual data.

R" = log(1/RF - 1) (20).

Thin-layer chromatography Glass plates (20X20 cm) were covered with a 0.25 mm thick layer of Silica Gel G or Aluminium oxide G for thin-layer chromatography (E. Merck AG, Darmstadt) by spreading a well-stirred mixture of 30 g of the adsorbent and 60 ml of destilled water with an applicator (C. Desaga GmbH, Heidelberg). After drying in air for one hour, the plates were activated at 1100 for 30 min, and then stored in a desiccator over silica gel for not more than two days. Cellulose-coated plates were prepared similarly, by spreading a homo- genized mixture of 15 g of Cellulose MN 300 for thin-layer chromato- graphy (Machery, Nagel & Co., Duren, D. B. R.) and 90 ml of destilled 0 water, and drying for 10 min at 105 • Solvents were mixed immediately before use, and 100 ml of the solvent poured on the bottom of the filter paper lined tank (7 X 23 X 23 em). The tank was shaken, and then allowed to equilibrate for 15 min at 25 ± 0.50 before the plates were quickly inserted. Alkaloids were dissolved in suitable organic solvents to contain 0.5-0.7 p,g of alkaloid in the applied 2-3 p,l of solvent, and were spotted 1.5 ern from the lower edge of the plate. Fifteen cm above the origin, a frontline was drawn through the silica layer. For two-dimensional chromatography, a sample was spotted in a corner of a Silica Gel G plate 1.5 em from each side. The chromatogram was developed 15 em, first in solvent MC and, after air-drying for 5 min, in the second direction in solvent DC(Fig. 5) .

Solvent systems MC. Silica Gel G plates. Methanol-chloroform (2 : 8 by vol.) (4, 9) DC. Silica Gel G plates. Diethylamine-chloroform (1 : 9) (9) l11CA. Silica Gel G plates. Methanol-chloroform-cone, NH3 (20 : 80 : 0.2) EC. Aluminium oxide G plates. Ethanol-chloroform (4 : 96)(4) CBAc. Aluminium oxide G plates. Chloroform-benzene-glacial acetic acid (45 : 45 : 10) (13 ) CM Ac. Silica Gel G plates. Chloroform-metanol-glacial acetic acid (4 : 3 : 3) FEHD. Cellulose plates were impregnated with a solution of 15 % forma- mide and 1 % cone. NH3 in acetone and, after air-drying for 15 min, were

3591 dried in an ovan at 1000 for 90 sec. Solvent: ethyl acetate-n-h eptane- dimethylforrnarnide (250 : 300 : 1) (7).

Detection of alkaloids and documentation Fluorescent alkaloids (Table 1) were detected under short wavelength (254 111ft) UV light. A 4 % solution of p-dimethylaminobenzaldehyde in conc. HCI was used to locate all alkaloids. The use of other suitable colour reagents has recently been discussed by Reio (16), and the mecha- nism underlying this colour reaction by Durkee & Sirois (17). The dry chromatogram was sprayed with Neatan Xeu (Merck ) , air-dried, and then taken up on adhesive tape.

Thin-layer electrophoresis The high-voltage electrophoresis apparatus and the technique used were similar to those described by Honegger (J ,1). For further details regarding the apparatus, see Samuels son (15). A pyridine-acetic acid buffer (25 1111 pyridine and 7 ml conc. acetic acid in 2 I water, pH 5.6) was used. Alka- loids were spotted 5 ern from the anode side on a Silica Gel G plate, 20)( 20 cm. Since the migration of compounds decreased close to the edges, only the central 12 em of the plate was used. Elyrnoclavine was run as a reference, and electrophoretic mobilities determined relative to ely- moclavine (M = 1.00). The plate was then sprayed with buffer until it became semi-transparent, with a shining surface. A buffer-soaked filter paper wick (Whatman 3 MM) was inserted into each buffer compartment. Contact between the wicks and the thin-layer plate was made with buffer- saturated filter paper strips (Whatrnan No.1). These "were separated from the wicks, dipping into the buffer compartments, hy cellophane, to minimize solution flow (15). On the other two sides of the plate, paper strips were applied to the same thickness as the wicks. On the top, a second glass plate was placed, thus forming a narrow moist chamber. Electrophoresis was carried out at HiOO volts (ca 50 mAl for 45 min, during which time the alkaloids migrated at most 90-100 mm. After 0 drying for 10 min at 100 , the alkaloids were located as previously described.

Combined thin-layer electro plioresis and chromatography In a two-dimensional procedure, thin-layer electrophoresis and thin-layer chromatography 'were combined. The sample mixture was spotted i) em from the anode side and 4 cm from the lower edge of a Silica Gel G plate, and sorne known references, e. g. agroclavine and elyrnoclavinc, were spotted 4 em from the upper edge of the plate. After applying buffer pH 5.6, the plate was subjected to electrophoresis for 45 min. The plate was then carefully dried with a fan for a f ew minutes. A frontline was drawn through the coating 5 em from the upper edge, suitable references were applied, and the chromatogram was developed in diethylamine-chloroform (DC) in the second direction. For details, cf. Fig. 6.

.360 Results and discussion Thin-layer chromatography The three chromatographic systems utilizing Silica Gel G plates with methanol-chloroform (MC), diethylamine-chloroform (DC) or methanol- chloroform-ammonia (MCA) and the system utilizing Aluminium oxide G with ethanol-chloroform as solvent (EC), were selected from some 100 tested solvent systems. A previously used system with ethyl acetate-etha- nol-dimcthylformamide was not included, because of a tendency to form a second solvent front (9, 24). The R,. values and standard deviations obtained in four chromato- graphic one-dimensional systems, as well as R"" values in system MC, are listed in Table 1.For convenient use of the chromatographic data when selecting a suitable solvent for a specific separation purpose, or for identification purposes, the distribution of the alkaloids in the chromato- graphic systems is shown in Figs. 1-4. The distribution pattern of alka- loids in the two-dimensional system is presented in Fig. 5. The standard deviations found in the chromatographic systems were reasonably small, Methods to obtain reproducible RF values in TLC have been investigated bye. g. Dallas (19), and techniques were devised to control the factors affecting Rp values in a given system, of which the most important is the relative humidity. Since the suggested methods, designed to obtain identical conditions in different laboratories, were fairly time-consuming, I instead adopted another recommended proce- dnre (20). Here, the agreement between the mobilities of suitable dye- stuffs relative to the solvent front arc checked. The R., values of a com- mercially available dyestuff mixture (Test mixture, Desaga) are recorded in Table 1 and Figs. 1-4. Moreover, several of the alkaloids are com- mercially available", and R values relative to a known standard, can thus be computed.

Identification of alkaloids The methanol-chloroform (MC) system has previously been used for the separation of ergot alkaloids (4, 5, 9).As a rule, it was used as the basic system, since the alkaloids distribute themselves fairly evenly in it. An identification process may be simplified by elution of the individual alka- loids of the mixture separated on a MC thin-layer plate (6).The presence of an alkaloid, chromatographically indicated in the MC system, is thcn checked in other systems, which differentiate between the suspected alkaloid in question and any other alkaloid which, in MC, has an RF value within ± 0.05. Thus, a mixture of ergot alkaloids may, with the excep- tions discussed below, be resolved and identified in MC, or by a combina- tion of two or more of the four major thin-layer chromatographic systems (MC, DC, MCA and ECl. It is recommended that reference alkaloids be used for comparison. In addition to this qualitative analysis, the thin- layer chromatographic technique can be used for quantitative studies (5, 6).

361 Tab 1e 1 RF values of ergot alkaloids in systems MC, DC, iltJCA and EC. R.II values in system MC. TLC RF x 100 Alkaloid Fluoresc. MCDC MCA EC R", in MC Agroclavinc1. 39±1 51 ±1 67± 1 63±1 0.194 Clzanoclavine- (I) 5±O 11±0 12±1 3±0 1.279 Chanoclavinc- (II) 6±0 7±1 10±1 3±1 1.195 Costaclavinc 5±0 66±2 24±1 56±2 1.27!l a-Dihydrolysergol2 8±0 9±0 22±1 18±1 1.061 DIA-Dimcthylallyl- 0 0 0 0 Elymoclavine 17±1 11±1 34±1 29±1 0.689 + 75±2 41 ±2 87±0 71±0 -0.477 Ergocorninine + 82±1 56±1 90±1 71±1 -0.659 Ergoaistine + 75±2 37±1 87±1 72±1 -0.477 Ergocristininc + 83±2 57±1 91±t 72±1 -0.689 + 74±2 38±1 88±0 71±0 -0.454 Ergocryptininc + 81±2 57±2 !l0±1 72±0 -0.630 + 27±1 2±0 40±1 21 ±O 0.432 Ergomelrinine + 39±1 20±0 66±0 46±4 0.194 + 63±1 10±0 73±1 58±1 -0.231 Ergosinine + 76±l 45±1 89 ±O 66±1 -0.501 Et'goslinc + 72±2 18±0 80±0 G3±1 -0.410 Ergoslinine + 81±O 56±1 88±1 64±1 -0.630 + 65±1 9±1 75±0 57±0 -0.269 Ergotaminine + 78±2 44 ±1 90±1 71±1 -0.550 Fesluclavine 23±1 48±2 51±1 60±0 0.525 Fumigaclavine A 57±1 60±2 78±1 71±1 -0.122 Fumigaclavine B 13±0 45±2 39±2 58±1 0.826 Fumigaclavine C 72±1 78±1 91±1 75±1 -0.410 6-Methyl-A 8,9-ergo]enc- 8-carboxylic acid" 0 0 0 0 Lysergene + 58±1 44± 1 74±1 (i3±t -0.140 D-Lysergic acid + 0 0 0 0 D-Lysergic acid amide + 22±1 3±0 34 ±t 25±1 0.550 D-Isolysergic acid amide + 51±1 22±1 68±1 39±0 -0.017 D-Lysergic acid methyl carbinolamide + 36±1 5±0 43±1 26±1 0..250 Lysergine + 42±t 44 ±2 69±1 63±1 0.140 + 16±1 9±0 35±1 28±1 0.720 Isolysergol + 28±1 28±1 (i1±1 52±1 0.410 D-Lysergyl-L-ualine- methylester + 70±1 43±1 80±0 65±1 -0.368 Nor-agrocIavinc 16±1 30± 1 34 ±1 23±1 0.720 + 21±1 7±0 35±0 5±0 0,.575 Isopenniclavine + 41 ±O 10±0 56±1 8±0 0..158 PyrocIavine 20±1 59±2 63±1 68±0 0.602 Seloclavine + 48±1 31±1 66±1 56±! 0.035 Isosetoclavine + 54±1 38±1 68±t 60±1 -0.070 Tesl4 87±2 82±2 91±1 76±1 -0.82li

1 The italicized letters denote the abbreviations used in Figs. 1-6. 2 D -Dihydrolysergol-(I). 3 »A8,9-Lysergic acid». 4 Test Mixture, Desaga (all compounds move as one spot in these systems). RF·100 I I ! I 901----+----j--- Silica Gel G. I @ Me Methanol 2 -t---+----+I:- J!\@~@ __ ___l 80 I Chloroform 8 I. €ill;! T i I I @@@@ I 70 f------j---_f_--__+---f-- .-. - €l ~H) i ©~ 60 f----f----t-----+---+----+@e

I @e 50 I---~f__----j--_f_--__+-@ +---+---+----r---~ ! 40 f-----+---I---+-- «e @@--+---i-i--+!---I-----1 e I I! 30 f-----+---I----+- €il D 20 f----+-----'-- ®@ @ -@+---r---f---~~ --+----+---~ @@@

1 0 f----+- @ I I o ole (j ! o w ~ ~

Fig. 1. Distribution of ergot alkaloids in solvent system MG. Abbreviations as in Table 1. .

An alkaloid may be further identified by chromatography of its chemi- cal conversion products. Reactions that can be carried out with small amounts of substance are, e. g. hydrogenation, isomerization, dehydra- tion (24) and basic and acid hydrolysis (2, 9) . Regarding the distribution of R" values of ergot alkaloids in the basic solvent system diethylamine-chloroform (DC), it is noteworthy that alka- loids containing a C-methyl group e. g. agroclavine, nor-agroclavine, , pyroclavine, costaclavine and fumigaclavine A and B, here have higher R" values than in MC, whereas other alkaloids generally have R" in DC < R]. in MC. An increase in the polarity of the DC system by addition of 5 % ethanol

will increase the R" values, and give better resolution (RL, 0.0-0.7) of the alkaloids having R" 0.0-0.4 in DC. The two just discussed systems MC and DC may be combined success- fully into a two-dimensional system (Fig. 5), which I have used for, e. g. the rapid analysis of microbial metabolites of ergot alkaloids, alkaloid fractions and determination of purity.R" values of the alkaloids run in

363 I I I I ~- Silica Gel G. Diethylamine 1 I DC @- I Chloroform 9 i I I@ I @ I 60 I @~@@@@ I I 50 I ~ i ® ~@@@@@ 40 I @ I e@ e I I I I 30 ®~@ I I I @@ i i 20 @ ! I I 1 0 ®@~@~ID@ I @~Hl I ! o .•."'''''@@ I I

Fig. 2. Distribuiion. of ergot alkaloids in solvent system DC. Abbreviations as in Table 1. the second direction (DC) will here be somewhat higher (Fig. 5) than when run in DC only (Fig. 2). Addition of ammonia to the methanol-chloroform system (MCA) in- creases the RF values compared with MC, and may give better resolution of alkaloids with R]' < ca. 0.50 in MC. There is also a change in the R,! value sequence of the alkaloids. The thin-layer chromatographic system EC, using aluminium oxide plates and ethanol-chloroform as solvent, gives a separation somewhat similar to DC. It can be pointed out that a previously applied paper chro- matographic system, using formamide-treated paper with benzene-pyri- dine as eluant, is both rapid and has high resolving power for clavine and low-molecular alkaloids of lysergic acid type (24).

Special systems Certain specific separation problems are not solved by the above four TLC systems. The perennial problem of separating the alkaloids of the ergotoxine (, ergocryptine, ergocornine) and the ergotinine

364 I I I I [ I Silica Gel G u€iHltl®@®- I ®Q@ MeA Methanol 20 1 80 Chloroform 80 ~@®E.l NH3 Conc. 0.2 ®@® 70

@@66@@@@ 60 I -1, I el I 50 ® I I 40 I @@ i e I ®@®@@ I I 30 i G e 20 I i I f----@ @ i 10 I : I I o IB '" '" i

Fig. 3. Distribution of ergot alkaloids in solvent system IVlCA. Abbrevia- tions as in Table 1.

(ergocristinine, ergocryptinine, ergocorninine) groups was solved paper chromatographically by Pohrn (21) and others (22) by the use of im- pregnated filter papers. The most successful TLC separation of these alkaloids, not using formamide-treated cellulose layers (7, 8), seems to have been achieved by Paul et al. (6) in a two-step process, and others have obtained partial resolution (23). In the systems used here (Figs. 1-4), there is generally separation of the ergotoxine from the ergotinine group, but no separation within the groups, with one exception (ergo- cornine in DC, Fig. 2). For the TLC analysis of these compounds, the slightly modified chromatographic procedure of Hohmann & Rochelmeycr (7) was applied (system FEHD). 'With cellulose plates impregnated with a stationary phase of formamide and ethyl acetate-n-heptane-dnnethyl-, formami.de as solvents, the following Rr values were recorded:

FEHD (R~.X 100): ergocristine 62 ± 2, ergocryptine 76 ± 2, ergo- cornine 68 ± 1, ergocristinine 71 ± 1, ergocryptinine 81 ± 1, ergocor- ninine 76 ± 1.

365 I I --- AI203 G ----- EC Ethanol 4 80 I Chloroform 96 '-"~ 70 f------f------J---- t-r @@@9 ••• @0·· @©@~@@® f--- 60 ©t~Hi®@ @@ 50 ® @ 40 ~I-----t-+ e 30 l\l §@ €l((l I 20 @® ----- f--- 10 f----+© o ~ U@ I I

Fig. 4. Distribution of ergot alkaloids in solvent system EC. Abbreviations as in Table 1.

Elymoclavine and lysergol are also difficult to separate, but the acidic system suggested by Kobel et al. (13)(system CBAc) differentiates well between these two compounds: CBAc rn, X 100): elyrnoclavine 35 ± 1, lysergol 29 ± 1.

The amino acids 4-dimethylallyltryptophan, o-lysergic acid and 6-methyl- -A8,9-ergolene-8-carboxylic acid do not move from the starting line in systems MC, DC, MCA and EC. In a recent biogenetic investigation (25), it was of importance to separate o-lysergic acid from »A 8,9-lyscrgic acid», For this purpose system CMAc was found suitable although, being acidic, it caused some tailing. This can partly be overcome by addition of 10 % formamidc to the solvent:

CMAc (Rr X 100): 4-dimethylallyltryptophan 62 ± 2, n-lysergic acid 26 ± 1, 6-methyl-A 8,9-ergolene-8-carboxylic acid 44 ± 1.

366 Ecorni ..•...Ecristi Etamini ~ Ecor , ~-E t i Ecris" \ (?l '\ cryp I ~@"@, Estini @Estin I m. Esini Ecry <.;z) Ly s vu t ®Fum C ~Etom Esi n 60 @Lysene @Fum A @Isoseto Isolysom @ ®Seto

<® l s open ni ® Lysine 40 c;,. @Emetri @Agro '<4;;;l Lyscaram

@Emet @Isolysol @ __ Lysom ®Festu 20 @Pennl @ Pyro ~Elymo @Norogro 'Lysol ~Fum B @_cx-Di @{§{hono I .•..Chana IT ® Costa o l{Dioltry 20 60 RF ·100 Lysae 40 80 6-8.9-Lysoc n -DC Fig. 5. Two-dimensional thin-lager chromatogram of ergot alkaloids. First solvent MC; second solvent DC.

Relations between structure and R" value in solvent system Me in the clavine series Martin postulated for partition chromatography a linear relation between Ru value and the number of similar groups in a homologous series (26, 27). These postulations were, in fact, confirmed by a number of authors (20, 27). Similar observations have been made in thin-layer chromato- graphy, and it has been found that the contribution of a particular group, »group constant», to the Ru value of a molecule is also dependent on other factors, such as structural and configurational features of the rest of the molecule, and on the solvent mixture (27, 28 and refs. cited therein). An analysis of R'I values of closely related clavine alkaloids in systems MC, DC, EC and MCA indicated that, particularly in system MC, one specific structural change in the molecule produces a characteristic change in the R" value. The difference between two R>r values is t.Rug (20,27), e. g. the change in R" (t.R"g) due to the substitution of a hydr- oxyl group at C-17 for a hydrogen atom, solvent system MC:

R::\I ,x·dihydrolyscl'gol == R::\I r c s tuc l av tno + .6.. RMg

t. RMg = + 0.536 (approx. to + 0.54)

367 The influence on the R" values of certain modifications in the struc- tures of clavine alkaloids in system MC are discussed in the following. In some cases, particularly compounds of the isolysergic acid series, the mean 6. R,," values are based only on the Ru values of two pairs of com- pounds, and consequently must be interpreted with caution. However, when several 6. R~[g values have been obtained for a specific structural change, they have generally agreed well. R~[ values for solvent system MC are listed in Table 1. For the structures of the various compounds, see refs. 1, 12, 24. The C-17 methyl group may be either [3 as in f'estuclavine, and lysergine, or a as in isolysergine and isosetoclavine, or attached to a 8,9-double bond as in agroclavine. The change in R"" value due to the introduction of an additional hydroxyl group on the C-17 methyl group is of similar magnitude (Table 2 a) for compounds with methyl groups being in a [3 position (6. R'Ig = + 0.54-0.58) or attached to a 8,9-double bond (6. R"g = + 0.50), but addition of a hydroxyl group on the C-17 methyl group in a position gave less change (6. R"g = + 0.23-0.25). The different 6. R'Ig values of the epimers are not unexpected (28).

The introduction of an a hydroxyl group at C-8 lowered the R'I value (increased the RF value) compared 'with the parent compound, 6. Rorg = - 0.12-0.15 (Table 2 b). The introduction of the corresponding [3 substituent, as previously, apparently had a greater influence on the Ru value, t.. Rug = - 0.23-0.25. From Table 2 a b it is evident that intro- duction of a hydroxyl group in [3 position caused more change in the R,[ value than the a substitution.

The 6. 9,10-ergolene epimers, where the C-17 substituent at C-8 is as- sumed switched from [3 to a position and the other substituent the reverse way, showed increasing influence on the Ru value with increasing polarity of the substituents, as shown in Table 2 c. The epimers with a C-17 methyl group only (lyserginejisolysergine) showed little change in RM value (substituents CH3, H; 6. R)fg = + 0.02), and the change in R" for the pair sctoclavine/isosetoclavine (substituents CH3, OH; 6. R,rg = - 0.11) thus must be largely attributed to the shift in the hydroxyl rather than in the methyl group. The 6. R"g = - 0.42 for the epimers penniclavine/iso- penniclavine (substituents CH20H, OH) is also roughly equal to the sum of the C-8 OH shift and the C-8 CH20H shift (Table 2 c). A hydrogenation of unsaturated clavine alkaloids (Table 2 d) to com- pounds of the n-dihydrolysergic acid-I series (C-17 (3) gave similar changes in the R,[ values (6. R'Ig = + 0.33-0.39). The increase in R>r value by hydrogenation to the o-dihydroisolysergic acid-I series (C-17 a) was perhaps somewhat greater (c. Rug = + 0.41-0.44). Originally, isolysergine was not included in the calculations, hut its

presumed R" value was calculated as follows. 6.R)f Q C'170l{ (6.R" due to introduction of hydroxyl group at a C-17), was obtained from isoseto- clavinejisopenniclavine (Table 2 a) and 6. R>r m (hydrogenation of double bond) from agroclavinejpyroclavine (Table 2 d).

368 Table 2 Changes in RJI value due to structural modifications of clauine alkaloids (solvent system MC).

Mean Pair of compounds Change in molecule Ll. R,," value a) Introduction of hydroxyl at C-17 Isosetoclavine/Isopenniclavine 8

d) Saturation of S,9- or 9,10-double bonds

Pair of compounds Double Final Ll. R'Ig .Mean bonel position value of C-17 Agroclavine/Festuclavinc 8,9 ,8 +0.33 Elymoclavine/

1 Isolysergine is no naturally occurring compound but was included in the following calculations. R]. X 100 in Me = 41 ± 2, Ru = 0.158.

369 RM Iaolyoerglne = 0.18 and

..6..R:u 2H + R}'I t so t rs e egtne == RM pyr-o c l avIne

Ru i so l ys er-gl ne == 0.19

When synthetically prepared isolysergine was tested, it was found to have an R" value close to the calculated, R,{ = 0.16.

In the same way, one would predict an R" value of ca. 0.84 (RE = 0.13) in system MC for the, so far, not isolated ~-dihydrolysergol (o-dihydro- isolysergol-t l ) :

R:l( P-dihydrolysers;ol == R:\I t sc tvs er-go I + A Ru 2H

R)r p-dih:rdro1ysergol == 0.84 and

R:.u ,8-dihydrolyscrgol == R)I pvro c.t ev tn c + A R1I iX 0-11 OR

Similarly, one can calculate a likely Ru value of 1.38 (R,. = 0.04) for a C-17 hydroxylated derivative of fumigaclavine B. The stereochemistry at C-9 and C-10 of fumigaclavine B is not known, and an analysis of the group constants of this compound cannot yet be done.Some other values such as the t. Rug for the. -methyl group (nor-agroclavine) and the effect of cis-fusion of rings C and D (costaclavine) are presently represented only by single values and, thus, their validity has to be ascertained before these values can be used for the calculation of R>r values of similarly derived compounds. To conclude, it can be stated that a close relation between structure and RM value has been found in the clavine series and, conversely, the chro- matographic data are in agreement with the formulated structures and stereochemistry of the clavine alkaloids. Chromatographic studies in sol- vent system MC may be of value, both in suggesting and confirming the structure of a new clavine-type ergot alkaloid.

Thin-layer electrophoretic separation of ergot alkaloids. Relations between structure and electrophoretic mobility Although electrophoresis is a widely used tool and particularly in bio- chemistry has found a multitude of applications, it has been somewhat sparingly used for the separation of alkaloids. The probable reason is that alkaloids generally separate well using paper chromatography or TLC, and that electrophoresis involves slightly more work. However, paper electrophoresis has been used by Paris et al. (29),Williams et al. {3~), Plieninger et al. (31) and others to separate and characterize alka- loids. Silica gel was used as a stabilizing medium for electrophoresis as early as 1946 by Corisden et al. (32). Fifteen years later, Honegger (14)

.370 ------_._------

used Silica Gel G and other inorganic media to separate amines and amino acids by thin-layer electrophoresis. It was decided to try to devise a thin-layer electrophoretic method, simple, rapid and reproducible enough to be used routinely, together with TLC, for separation of ergot alkaloids. It was found that high- voltage electrophoresis (see Experimental) at 1500 volts on Silica Gel G plates sprayed with an acetic acid-pyridine buffer of pH 5.6 (pH of buffered layer ca. 5.2) satisfactorily fulfilled these requirements. The plates used and the spotting technique are the same as for TLC, and from the application of sample to the location of the compounds requires less than 1 112 hrs, compared to less than 1 hr for TLC. The reprodu- cibility is not quite as good as in TLC (Table 3) but satisfactory. With the exception of the larger peptide alkaloids, which showed some ten- dency to tail, the alkaloids appeared as distinct spots, and the resolution was comparable to that obtained by TLC. A difference in mobility of M = 0.05 was generally sufficient to give a clear separation of two compounds. In a given electrophoretic system, the migration of a compound is governed by the following factors: charge, size, shape, tendency to disso- ciate, and amphoteric behaviour of the molecule. Related compounds with similar groups can then be expected to show similar electrophoretic mobility and, consequently, the electrophoretic mobility of a compound would be of value in its identification. As Table 3 shows, there is a gene- ral agreement between gross structure and electrophoretic migration. In the clavine series, alkaloids having two hydroxyl groups (penni- clavine, isopenniclavine) or one hydroxyl plus a secondary amino group (-(I), chanoclavine-(II)) migrate most rapidly, M = 1.07-- 1.09. All clavine alkaloids with one hydroxyl group have similar mobili- ties (M = 0.93--1.00) regardless of whether the position of this function is C-17 (elymoclavine, lysergol, isolysergol, a-dihydrolysergol), C-8 (seto- clavine, isosetoclavine) or C-9 (fumigaclavine B) and, with some allow- ance for isolysergol, regardless of stereochemistry. In the same way, all derivatives carrying a C-17 methyl group but no hydroxyl or secondary amino function have close mobilities (M = 0.70--0.74). In the lysergic acid series, there is a clear tendency, which was to be expected, for the comparatively high mol. wt. compounds to have low mobilities (M = 0.18--0.27), but as the mol. wt. decreases (e. g. lysergyl valine methylester -- ergometrine --lysergic acid methyl carbinolamide --- lysergic acid amide) the electrophoretic mobility increases. The rather slow migration of the low mol. wt. amino acids (lysergic acid, ».0.8,9-ly- sergic acid», 4-dimethylallyltryptophan) is explained by the fact that although the tertiary amino nitrogen is protonated at this pH, the car- boxyl group exist as an anion. The thin-layer electrophoretic technique devised here may be of value in the separation of mixtures of ergot alkaloids and in the study of unknown compounds. The technique is particularly useful for identifica- tion of ergot alkaloids, since the basic principle of separation is here different from TLC. The usefulness for a certain problem may be deduced from Table 3.

371 · ..__ ..... _------

Table 3 Electrophoretic mobilities of eraoi alkaloids relative to elumoclaoine. Elymoclavine M = 1.00.

Alkaloid M X 100 I Alkaloid JI >( 100

Ergocristine 18±2 Agroclavine 74±3 Ergocristinine 19 ± 3 Ergometrine 72±2 Ergostiue 20 ± 2 80±2 Ergostinine 20 ± 3 Lysergene 79±:1 Ergocornine 23 ± 3 D-Lysergic acid methyl Ergocorninine 22 ± 2 carbinolamide 79±3 Ergocryp tine 23 ± 2 Fumigaclavine A 88±3 Ergocryptinine 23 ± 2 Isolysergol 9312 Ergosine 25 ± 3 Setocla vine 96±2 Ergosinine 26 ± 2 Isosetocla vine 98±2 Ergotamine 26 ± 1 Lysergol 98±1 Ergotaminine 27 ± 2 e-Dihydrolysergol 99±1 n-Lysergic acid 30± 1 Fum.igaclavinc B 99±1 6-Methyl-A 8,9-ergolene- Elymoclavinc 100 8-carboxylic acid- 29 ± 2 n-Lyscrgic acid amide 97±2 DL-4-Dimethylallyltryptophan 30 ± 1 D-Isolysergic acid amide 98±1 n-Lyscrgyl-t-valinc- ChanoeIavine- (1) 107±2 methylester 56±2 Chanoclavine- (II) 108±2 Fumigaclavine C 65±3 Penniclavinc 108±1 Festuclavine 70±:1 Isopenniclavine 109±1 Pvroclavine 70±2 l\'or-agroclavine 112±3 Costacla vine 71 ±1 Lysergine 73±1

1 »A 8,9-Lysergic acid».

Combination of thin-layer electrophoresis atul chromatography Thin-layer electrophoresis and chromatography were combined by Hon- egger (14) for the analysis of amincs and amino acids. Some trials showed that the electrophoretic step should precede the TLC step, in view of the tendency of a compound to migrate 3-8 % farther in the centre of the plate than at the edges and, thus, making direct correlations difficult. The acetic acid-pyridine buffer is volatile, but the sensitivity of the ergot alkaloids makes it hazardous to eliminate the buffer completely, neces- sary to avoid affecting a following chromatographic step in a neutral system, such as methanol-chloroform. However, gentle removal of most of the buffer by subjecting the plate to the airstream from a fan was found to suffice, if the subsequent chromatographic step was carried out in a basic solvent, diethylamine-chloroform (DC). The RF values in the solvent system will be slightly higher than recorded in Table 1. If reference alkaloids are used, this two-dimensional procedure, which can

372 I ! Agro ELymo

I ---~~------~--®----T- ,RF .100~ I ~" DC L I ' 80, Costa I I Costa f- ~ I ,I ~© Ec ri s t i "WW ,I , @60 • I , Agro. I e Etarnini , 40 ~ Se t c I , I I l s o t ys ol ~ I , A.20 I '''' Etam ELymo~~ I I El y m o ~ ~ I Start Cheno II

: R:f. 0 ~ II @> Lysee : I 0 20 40 60 80 100 M'100 1 I pH 5.6. I I I

Fig. 6. Recording of two-dimensional separation of ergot alkaloids. First direction: electrophoresis, 1500 V, 4·5 mA, 4-5 min, acetic acid-pyridine buffer, pH 5.6; second direction: diethylamine-chloroform (system DC). Reference substances for chromatography were applied and front lines through silica layer (- - -) drawn after electrophoretic step. For abbreviations, see Table 1.

easily be reproduced, may be used for analysis just as well as the two- dimensional TLC procedure.The use of this method and reference alka- loids for the separation of ergot alkaloids is demonstrated in Fig. 6 (cf. Experimental).

Acknowledgements I am indebted to Drs. A. Hofmann, Sandoz AG, 1\1.Abe, Takeda Chemical Ind. Ltd., J. F. Spilsbury, Wellcome Research Laboratories and H. Plieninger, Uni- versity of Heidelberg, for samples of alkaloids. The support of Professor Finn Sandberg, Kungl. Farmaceutiska Institutet, and of the Swedish Natural Science

373 Rescarch Council, is gratcfully acknowledged. Miss Gunilla Staaf and Mr. Bernt Staf skilfully carried out most of the technical work in this investigation. Dr. G. Schill gave me valuable advice, and Dr. G. Samuelsson kindly criticized the manuscript.

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Received September 15, 1965.

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